Sialyl Lewis X Defines an Activated and Functional Regulatory T Cell Subpopulation in Mice.

Attempts have been made to elucidate the functional markers of regulatory T cells (Tregs), CD4+Foxp3+ T cells with an immunosuppressive function. Sialyl Lewis X (sLex), a tetrasaccharide Ag, is involved in leukocyte trafficking as selectin ligands and is a marker of highly differentiated Tregs in humans. However, the importance of sLex in murine Tregs remains unknown. In this study, we report that sLex defines the activated and functional subset of murine Tregs. The contact hypersensitivity model showed that murine Tregs strongly express sLex upon activation, accompanied by functional Treg marker elevation, such as Foxp3, CD25, CD103, CD39, and granzyme B. RNA sequencing analysis revealed sLex-positive (sLex+) Tregs expressed genes involved in Treg function at a higher level than sLex-negative (sLex-) Tregs. Using an in vitro suppression assay, we found that sLex+ Tregs could more efficiently suppress naive CD4+ T cell proliferation than sLex- Tregs. In the murine contact hypersensitivity elicitation model, the topical sLex+ Treg injection into the ears suppressed ear inflammation more efficiently than that of sLex- Tregs. Our results indicate that sLex could serve as a unique surface marker of activated and functional Tregs with immunosuppressive functions in mice.

Functional Peptide-Modified Liposomes for siRNA Delivery to Rat Retinal Pigment Epithelium Cells: Effect of Peptide Sequences.

Most retinal diseases involve the degeneration of choroidal retinal pigment epithelial (RPE) cells. Because of a blood-retina barrier (tight junction formation), RPE cells restrict the entry of hydrophilic macromolecules (e.g., small interfering RNA (siRNA)) through blood stream and eye drops. A cytoplasm-responsive stearylated (STR) peptide, STR-CH2R4H2C (CH2R4) enables stable siRNA complexation, cell permeation, and intracellular dynamics control. We previously demonstrated how CH2R4-modified liposomes promoted siRNA efficacy. We investigated the influence of amino acid sequences of functional peptides on cellular uptake pathways, siRNA transfection efficacy, and the permeation of peptide-modified liposomes in rat RPE-J cells. Four STR-peptides, consisting of arginine (R), cysteine (C), histidine (H), lysine (K) or serine (S), were designed based on CH2R4. We prepared siRNA-loaded, peptide-modified cationic liposomes (CH2R4-, CH2K4-, CH2S4-, SH2R4-, and SH2S4-lipoplexes). CH2R4-, CH2K4-, and SH2R4-lipoplexes induced cellular uptake by macropinocytosis by activating cytoskeletal F-actin, possibly due to cationic amino acids (arginine, lysine). SH2R4-lipoplexes were trapped in endosomes, whereas CH2R4- and CH2K4-lipoplexes enhanced endosomal siRNA release suggesting cysteine contributes to endosomal escape. Although cationic liposome-based, CH2S4- and SH2S4-lipoplexes (not including arginine and lysine) showed lower siRNA transfection efficiency. This difference may be because siRNAs were retained on both peptide moieties and cationic liposomes in CH2R4-, CH2K4- and SH2R4-lipoplexes, whereas in CH2S4- and SH2S4-lipoplexes, siRNAs were loaded to the cationic liposomes, but not on peptides. In three-dimensional spheroids, CH2R4- and CH2K4-modified liposomes promoted permeation through tight junctions. Thus, cationic amino acids and cysteine within peptide sequences of CH2R4 could be effective for siRNA delivery to the retina using functional peptide-modified liposomes.

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina.

Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood-retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.

Therapeutic Effects of an Anti-sialyl Lewis X Antibody in a Murine Model of Allergic Asthma.

Asthma is an allergic disease that causes severe infiltration of leukocytes into the lungs. Leukocyte infiltration is mediated by the binding of sialyl Lewis X (sLex) glycans present on the leukocytes to E-and P-selectins present on the endothelial cells at the sites of inflammation. Here, we found that mouse eosinophils express sLex glycans, and their infiltration into the lungs and proliferation in the bone marrow were significantly suppressed by an anti-sLex monoclonal antibody (mAb) F2 in a murine model of ovalbumin-induced asthma. The percentage of eosinophils in the bronchoalveolar lavage fluid and bone marrow and serum IgE levels decreased significantly in the F2-administered mice. Levels of T helper type 2 (Th2) cytokines and chemokines, involved in IgE class switching and eosinophil proliferation and recruitment, were also decreased in the F2-administered mice. An ex vivo cell rolling assay revealed that sLex glycans mediate the rolling of mouse eosinophils on P-selectin-expressing cells. These results indicate that the mAb F2 exerts therapeutic effects in a murine model of allergen-induced asthma, suggesting that sLex carbohydrate antigen could serve as a novel therapeutic target for allergic asthma.

A new brain-computer interface design using fuzzy ARTMAP.

This paper proposes a new brain-computer interface (BCI) design using fuzzy ARTMAP (FA) neural network, as well as an application of the design. The objective of this BCI-FA design is to classify the best three of the five available mental tasks for each subject using power spectral density (PSD) values of electroencephalogram (EEG) signals. These PSD values are extracted using the Wiener-Khinchine and autoregressive methods. Ten experiments employing different triplets of mental tasks are studied for each subject. The findings show that the average BCI-FA outputs for four subjects gave less than 6% of error using the best triplets of mental tasks identified from the classification performances of FA. This implies that the BCI-FA can be successfully used with a tri-state switching device. As an application, a proposed tri-state Morse code scheme could be utilized to translate the outputs of this BCI-FA design into English letters. In this scheme, the three BCI-FA outputs correspond to a dot and a dash, which are the two basic Morse code alphabets and a space to denote the end (or beginning) of a dot or a dash. The construction of English letters using this tri-state Morse code scheme is determined only by the sequence of mental tasks and is independent of the time duration of each mental task. This is especially useful for constructing letters that are represented as multiple dots or dashes. This combination of BCI-FA design and the tri-state Morse code scheme could be developed as a communication system for paralyzed patients.