Photodegradable Hydrogels for Capture,Detection, and Release of Live Cells

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T‐cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV‐induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95 % purity of CD4 and CD8 T‐cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti‐CD4 and anti‐TNF‐α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence “halo” after immunofluorescent staining and could be retrieved by site‐specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.     

Guinea Pig Skin,a Model for Epidermal Cellular and Molecular Changes Induced by UVR in vivo and in vitro: Effects on Mycobacterium bovis Bacillus Calmette–Guérin Vaccination

Previously, we reported that ultraviolet B‐radiation (UVR) suppressed Bacillus Calmette–Guérin (BCG) vaccine‐induced resistance to Mycobacterium tuberculosis in guinea pigs (GP). Herein, we investigated the cellular and molecular changes within the irradiated GP epidermis and the in vivo effect of supernatants from UV‐irradiated (200 J m^?2) epidermal cells (UV‐sup) on M. bovis BCG vaccination. UVR increased the number of nucleated keratinocytes in the skin, but caused a decrease in the proportions of CD25⁺T cells. In the spleen, UVR resulted in a decrease in the proportions of T‐cell subsets including CD25⁺T cells, and major histocompatibility complex (MHC) class II⁺ and CD14⁺ cells. Similarly, significant up‐regulation of several cytokine mRNAs including IL‐10 was also observed. Furthermore, UV‐sup significantly reduced the MHC class II expression in peritoneal cells and reduced T‐cell proliferation to ConA. The proliferation to purified protein derivative (PPD) was restored to normal levels by anti‐IL‐10 antibody. The UV‐sup when injected into BCG‐vaccinated GP significantly diminished the skin test response and T‐cell proliferation to PPD and up‐regulated the expression of IL‐10, IL‐4, IL‐1β and Foxp3 mRNAs in the lymph node or spleen. Thus, whole body UVR induces profound cellular and molecular changes and injection of UV‐sup from epidermal cells mimics the effect of whole body UVR in BCG‐vaccinated GP.     

Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

In situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1 , whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.     

Direct Structuring of a Biomimetic Anti‐Reflective,Self‐Cleaning Surface for Light Harvesting in Organic Solar Cells

A one‐step method to fabricate a biomimetic dual‐scale hierarchical structure for a transparent anti‐reflective, self‐cleaning layer for organic solar cells is reported. Template‐mediated UV replica molding is used to directly create a multi‐functional surface with an acrylate‐functionalized perfluoropolyether without complicated processing steps. The surface exhibits superhydrophobic properties and self‐cleaning characteristics. In addition, the surface leads to an enhancement of photovoltaic power conversion efficiency by ≈10% as a result of reflection suppression and transmittance enhancement. The method can easily be applied to large area substrates (22 cm × 24 cm) in a cost‐effective manner. Furthermore, the solar cell can withstand harsh outdoor conditions for a long time, without a notable change in the device performance, owing to robust surface layer and non‐fouling properties.

     

Solar-simulated Ultraviolet Irradiation Induces Selective Influx of CD4+ T Lymphocytes in Normal Human Skin

Abstract— The proportion and composition of the human cutaneous CD3+ T lymphocyte population was determined in situ following a single exposure to physiological, erythema-inducing doses of simulated solar radiation, mainly consisting of UV radiation. Biopsies were taken 1, 2 and 7 days after local irradiation of normal volunteers with 1,2 and 4 MED by a xenonarc lamp and immunohistochemistry was performed on cryostat sections. Ultraviolet radiation caused an initial decrease of intraepidermal CD3+ T-cell numbers or even could lead to T-cell depletion 24 and 48 h postirradiation, and this was followed by an infiltration of T cells in the epidermis as determined 1 week after UV exposure. The number of dermal CD3+ T ceDs was increased 24 h after irradiation, reached a maximum at 48 h and subsequently declined at day 7, though remained significantly higher than the unirradiated control Double staining demonstrated that the CD3+ T cells, which immigrated into the (epi)dermis upon UV exposure, coexpressed CD4 but not CD8. Therefore the CD4/CD8 ratio in skin was markedly increased during the first week upon UV exposure. Our time course study shows that UV radiation affects die T-cell population within human skin by depleting the majority of epidermal T cells and initiating a selective influx of CD4+ T cells.     

Multiformat T‐Cell‐Engaging Bispecific Antibodies Targeting Human Breast Cancers

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti‐Her2 IgG or Fab site‐specifically conjugated to anti‐CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen‐independent T‐cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.     

Catch and release cell sorting: electrochemical desorption of T-cells from antibody-modified microelectrodes

The development of integrated microsystems capable of interrogation, characterization and sorting of mammalian cells is highly significant for further advancement of point-of-care diagnostics and drug discovery fields. The present study sought to design a novel strategy for releasing antibody-bound cells through electrochemical disruption of the underlying antibody (Ab) layer. A microsystem for selective capture and release of cells consisted of an array of individually addressable gold microelectrodes fabricated on a glass substrate. Poly(ethylene glycol) (PEG) hydrogel photolithography was employed to make the glass regions non-fouling, thus, ensuring selective localization of proteins and cells on the microelectrodes. The gold surfaces were decorated with anti-CD4 Ab molecules using standard alkanethiol self-assembly and carbodiimide coupling approaches. The Ab-functionalized electrodes selectively captured model T-lymphocytes (Molt-3 cells) expressing CD4 antigen while minimal cell adhesion was observed on PEG hydrogel-modified glass substrates. Importantly, application of a reductive potential (-1.2V vs. Ag/AgCl reference electrode) resulted in release of surface-bound T-cells from the electrode surface. Cyclic voltammetry and fluorescence microscopy were employed to verify that the detachment of captured T-cells was indeed due to the electrochemical disruption of the underlying alkanethiol-Ab layer. In the future, the cell sorting approach described here may be combined with microfluidic delivery to enable Ab-mediated capture of T-lymphocytes or other cell types followed by release of select cells for downstream gene expression studies or re-cultivation.     

Differential roles of T-cell subsets in regulation of ultraviolet radiation induced cutaneous photocarcinogenesis

Ultraviolet (UV) radiation, in particular the midwavelength range (UVB; 290-320 nm), is one of the most significant risk factors for the development of nonmelanoma skin cancer. UVB radiation-induced immunosuppression, which occurs in both humans and laboratory animals, contributes to their pathogenesis. However, there are conflicting reports on the relative role of CD4(+) and CD8(+) T cells in UVB induced skin cancer. The purpose of this study was to delineate the contribution of these two cell subpopulations to UVB induced immunosuppression and tumor development using C3H/HeN (WT), CD4 knockout (CD4(-/-) ) and CD8 knockout (CD8(-/-) ) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of number of tumors per group, tumor volume and percentage of mice with tumors, in mice deficient in CD4(+) T cells compared with wild-type mice, whereas significantly greater (P < 0.05) numbers of tumors occurred in CD8(-/-) mice. These results indicate that, CD4(+) T cells promote tumor development while CD8(+) T cells have the opposite effect. Further, we found that CD4(+) T cells from tumor-bearing mice produced interleukin (IL)-4, IL-10, and IL-17 whereas CD8(+) T cells produced interferon-γ. Manipulation of T-cell subpopulations that are induced by UVB radiation could be a means of preventing skin cancers caused by this agent.     

High‐Throughput,Label‐Free Isolation of Cancer Stem Cells on the Basis of Cell Adhesion Capacity

Herein we report a microfluidics method that enriches cancer stem cells (CSCs) or tumor‐initiating cells on the basis of cell adhesion properties. In our on‐chip enrichment system, cancer cells were driven by hydrodynamic forces to flow through microchannels coated with basement membrane extract. Highly adhesive cells were captured by the functionalized microchannels, and less adhesive cells were collected from the outlets. Two heterogeneous breast cancer cell lines (SUM‐149 and SUM‐159) were successfully separated into enriched subpopulations according to their adhesive capacity, and the enrichment of the cancer stem cells was confirmed by flow cytometry biomarker analysis and tumor‐formation assays. Our findings show that the less adhesive phenotype is associated with a higher percentage of CSCs, higher cancer‐cell motility, and higher resistance to chemotherapeutic drugs.     

High‐Throughput,Label‐Free Isolation of Cancer Stem Cells on the Basis of Cell Adhesion Capacity

Herein we report a microfluidics method that enriches cancer stem cells (CSCs) or tumor‐initiating cells on the basis of cell adhesion properties. In our on‐chip enrichment system, cancer cells were driven by hydrodynamic forces to flow through microchannels coated with basement membrane extract. Highly adhesive cells were captured by the functionalized microchannels, and less adhesive cells were collected from the outlets. Two heterogeneous breast cancer cell lines (SUM‐149 and SUM‐159) were successfully separated into enriched subpopulations according to their adhesive capacity, and the enrichment of the cancer stem cells was confirmed by flow cytometry biomarker analysis and tumor‐formation assays. Our findings show that the less adhesive phenotype is associated with a higher percentage of CSCs, higher cancer‐cell motility, and higher resistance to chemotherapeutic drugs.     

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low) and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low) T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two- signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.     

UV‐Induced Disulfide Formation and Reduction for Dynamic Photopatterning

UV‐induced disulfide formation (UV‐DF) and disulfide reduction (UV‐DR) reactions for surface functionalization and dynamic photopatterning are presented. Both photochemical reactions allow for the spatially and temporally controlled, reversible transition between thiol‐ and disulfide‐functionalized surfaces. The dynamic photopatterning strategy was demonstrated by the UV‐induced attachment, exchange, and detachment on thiol‐modified substrates.     

A miniature cytometry platform for capture and characterization of T-lymphocytes from human blood

Given the clinical and diagnostic importance of blood analysis, there is considerable interest in developing novel miniature devices for rapid characterization of blood constituents. The present paper describes development of a miniature cytometry platform aimed at analysis of T-lymphocytes from peripheral human blood. Microarrays of T-cell-specific antibodies (Abs), including anti-CD3, -CD4, -CD8 and mouse IgG (negative control) were robotically printed onto glass slides coated with a non-fouling poly(ethylene glycol) (PEG) hydrogel. The glass substrates containing Ab arrays were incubated with 100 μL of red blood cell (RBC)-depleted whole human blood for 15 min and then exposed to a controlled shear of ∼2 dyn cm⁻² for additional 10 min. This process led to the removal of non-specific leukocytes and “development” of patterns of T-cells captured on the Ab spots. The immunofluorescent staining of the surface-bound cells revealed the presence of purified CD4⁺ and CD8⁺ T-cells (purity >94%) on their respective Ab spots. Importantly, the proportions of CD4⁺ and CD8⁺ T-cells captured on the Ab spots correlated closely (R² − 0.9) with flow cytometry analysis of T-cell subsets in blood. Overall, this cytometry platform allowed to rapidly (under 30 min) capture pure T-cell subsets from minimally processed human blood. Significantly, our device provided quantitative information about subset abundance solely based on the location of cells within the microarray. This cytometry platform is envisioned as a miniature immunology tool for determination of T-cell phenotype and will have immediate applications in HIV diagnostics and research.     

Thioether‐Functionalized Quinone‐Based Resorcin[4]arene Cavitands: Electroswitchable Molecular Actuators

The utility of molecular actuators in nanoelectronics requires activation of mechanical motion by electric charge at the interface with conductive surfaces. We functionalized redox‐active resorcin[4]arene‐quinone cavitands with thioethers as surface‐anchoring groups at the lower rim and investigated their propensity to act as electroswitchable actuators that can adopt two different conformations in response to changes in applied potential. Molecular design was assessed by DFT calculations and X‐ray analysis. Electronic properties were experimentally studied in solution and thin films electrochemically, as well as by X‐ray photoelectron spectroscopy on gold substrates. The redox interconversion between the oxidized (quinone, Q ) and the reduced (semiquinone, SQ ) state was monitored by UV‐Vis‐NIR spectroelectrochemistry and EPR spectroscopy. Reduction to the SQ state induces a conformational change, providing the basis for potential voltage‐controlled molecular actuating devices.     

Facile and Efficient Fabrication of Photoresponsive Microgels via Thiol–Michael Addition

A photoresponsive microgel is designed by the combination of a noncovalent assembly strategy with a covalent cross‐linking method. End‐functionalized poly(ethylene glycol) with azobenzene [(PEG‐(Azo)₂)] was mixed with acrylate‐modified β‐CD (β‐CD‐MAA) to form photoresponsive inclusion complex through host–guest interaction. The above photoresponsive complex was cross‐linked by thiol‐functionalized PEG (PEG‐dithiol) via Michael addition click reaction. The photoreversibility of resulted microgel was studied by TEM, UV–Vis spectroscopy, and ¹H NMR measurements. The characterization results indicated that the reversible size changes of the microgel could be achieved by alternative UV–Vis irradiations with good repeatability.     

Azobenzene‐Functionalized Cage Silsesquioxanes as Inorganic–Organic Hybrid,Photoresponsive, Nanoscale,Building Blocks

Mono‐ and octa‐azobenzene‐functionalized cage silsesquioxanes were easily synthesized by the reaction of 4‐bromoazobenzene with monovinyl‐substituted octasilsesquioxane and cubic octavinylsilsesquioxane through the Heck coupling reaction. Excited‐state energies obtained from time‐dependent density functional theory (TDDFT) and the CAM‐B3LYP functional correlate very well with experimental trans–cis photoisomerization results from UV/Vis spectroscopy. These azobenzene‐functionalized cages exhibit good thermal stability and are fluorescent with maximum emission at approximately 400 nm, making them potential materials for blue‐light emission.     

Functionalized Polystyrene Nanoparticles Trigger Human Dendritic Cell Maturation Resulting in Enhanced CD4+ T Cell Activation

Nanoparticles (NP) represent a promising tool for biomedical applications. Here, sulfonate‐ and phosphonate‐functionalized polystyrene NP are analyzed for their interaction with human monocyte‐derived dendritic cells (DC). Immature dendritic cells (iDC) display a higher time‐ and dose‐dependent uptake of functionalized polystyrene NP compared to mature dendritic cells (mDC). Notably, NP induce an enhanced maturation of iDC but not of mDC (upregulation of stimulatory molecules and cytokines). NP‐triggered maturation results in a significantly enhanced T cell stimulatory capacity (increased CD4⁺ T cell proliferation and IFN‐γ production), indicating a shift to a pronounced Th1 response. Immunomodulatory properties of NP may be a useful strategy for strengthening the efficacy of NP‐based approaches in immunotherapy.

     

Photoregulated Sol‐Gel Transition of Novel Azobenzene‐Functionalized Hydroxypropyl Methylcellulose and Its α‐Cyclodextrin Complexes

Summary: Novel azobenzene‐functionalized hydroxypropyl methylcellulose (AZO‐HPMC) polymers and their α‐cyclodextrin (α‐CD) complexes have been prepared. These polymers show interesting sol‐gel transition behavior in aqueous solutions. In the absence of α‐CD, the gelation temperature increases after UV irradiation, while in the presence of α‐CD, the gelation temperature decreases after UV irradiation. The difference in the gelation temperatures between the trans and cis samples of AZO‐HPMC opens a wide operating window for reversible regulation of the sol‐gel transition behavior by photoirradiation.

The UV‐induced cis/trans isomerism of azobenzene‐functionalized hydroxypropyl methylcellulose and its α‐cyclodextrin complexes.     

Photocurrent Response of Surface‐Functionalized Metal Oxides with Well‐Matched Energy Levels: From Nothing to Something

In recent years, an enormous amount of research has been devoted to the study of photosensitive materials from both fundamental and practical viewpoints, due to their wide applications in photocatalytic 1 – 3 and optoelectronic devices, 4 , 5 ultraviolet (UV) photodetectors, 6 – 9 photoswitch microdevices, 10 , 11 light‐emitting diodes, 12 , 13 photovoltaic devices, 14 – 16 and photoelectrochemical cells. 17 Metal oxides, such as ZnO, TiO₂, SnO₂, and NiO have been the most investigated photosensitive materials. 3 , 6 – 8 , 18 – 21 To enhance and take full advantage of their photosensitivity, functionalizing their surface with a polymer that has a high light absorption ability has become one of the widely used methods. 1 – 12 , 22 – 24 For example, Z. L. Wang et al. reported that the UV photocurrent of a ZnO nanobelt‐based sensor was enhanced by close to five orders of magnitude after functionalizing its surface with polystyrene sulfate which has a high UV absorption ability. 25 T. Sasaki et al. reported the assembly of a TiO₂ nanoparticle film with poly(3,4‐ethylenedioxythiophene) and poly(4‐styrene sulfonate) (PEDOT‐PSS) through layer‐by‐layer fabrication in the nanometer scale. The electric conductivity of the TiO₂ composite films could be tuned by UV and visible (Vis) light. 22 Thus, sunlight or photon energy can be used and transformed to electrical energy by UV‐photosensitive metal oxides after their surfaces have been functionalized with a dye that has a high Vis absorption ability. To date, most of the dye‐sensitized solar cells are based on the surface functionalization of UV‐photosensitive metal oxides by dyes. 26 – 28 However, to the best of our knowledge, all of the reports on surface functionalization enhanced only the UV photosensitivity of the metal oxide. In other words, this method has been used exclusively to enhance the UV photocurrent in metal oxides that already have UV‐photosensitive properties, but not to induce UV photocurrent in metal oxides that have no UV‐photosensitive properties. In fact, to the best of our knowledge, there are no surface‐functionalizing reports on inducing UV or Vis photocurrent in metal oxides that have no UV‐ or Vis‐photosensitive properties.     

Oclacitinib,a Janus Kinase Inhibitor,Reduces the Frequency of IL-4- and IL-10-, but Not IFN-γ-, Producing Murine CD4+ and CD8+ T Cells and Counteracts the Induction of Type 1 Regulatory T Cells

The purpose of the present study was to broaden the knowledge and understanding of the effects of oclacitinib (OCL), a Janus kinase inhibitor, on T cells in the context of both the immune mechanisms underlying anti-inflammatory and anti-allergic properties of the drug and its safety. The results indicate that beneficial effects of OCL in the treatment of skin allergic diseases may be partially mediated by the inhibition of IL-4 production in CD4⁺ and CD8⁺ T cells. To a certain extent, the antiproliferative effect of OCL on CD8⁺ T cells may also contribute to its therapeutic effect. The study found that OCL does not affect the proliferation of CD4⁺ T cells or the number of IFN-γ- and IL-17-producing CD4⁺ and CD8⁺ T cells. Moreover, OCL was found to counteract the induction of type 1 regulatory T (Tr1) cells and to act as a strong inhibitor of IL-10 production in both CD4⁺ and CD8⁺ T cells. Thus, these results indicate that beneficial effects of OCL in the treatment of skin allergic diseases are not mediated through: (a) the abolishment of IFN-γ and IL-17-production in CD4⁺ and CD8⁺ T cells; (b) generation of Tr1 cells; (c) inhibition of CD4⁺ T cell proliferation; (d) induction of IL-10 production in CD4⁺ T cells. The results of this study strongly suggest that, with respect to the evaluated parameters, OCL exerts a suppressive effect on Th2- but not Th1-mediated immunity.