Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

Longitudinal profiling of oligomeric Aß in human nasal discharge reflecting cognitive decline in probable Alzheimer's disease.

Despite clinical evidence indicating a close relationship between olfactory dysfunction and Alzheimer's disease (AD), further investigations are warranted to determine the diagnostic potential of nasal surrogate biomarkers for AD. In this study, we first identified soluble amyloid-ß (Aß), the key biomarker of AD, in patient nasal discharge using proteomic analysis. Then, we profiled the significant differences in Aß oligomers level between patient groups with mild or moderate cognitive decline (n = 39) and an age-matched normal control group (n = 21) by immunoblot analysis and comparing the levels of Aß by a self-standard method with interdigitated microelectrode sensor systems. All subjects received the Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR), and the Global Deterioration Scale (GDS) for grouping. We observed higher levels of Aß oligomers in probable AD subjects with lower MMSE, higher CDR, and higher GDS compared to the normal control group. Moreover, mild and moderate subject groups could be distinguished based on the increased composition of two oligomers, 12-mer Aß*56 and 15-mer AßO, respectively. The longitudinal cohort study confirmed that the cognitive decline of mild AD patients with high nasal discharge Aß*56 levels advanced to the moderate stage within three years. Our clinical evidence strongly supports the view that the presence of oligomeric Aß proteins in nasal discharge is a potential surrogate biomarker of AD and an indicator of cognitive decline progression.

Structure-activity relationship study of thiazolyl-hydroxamate derivatives as selective histone deacetylase 6 inhibitors.

Several human diseases are associated with aberrant epigenetic pathways mediated by histone deacetylases (HDACs), especially HDAC6, a class IIb HDACs, which has emerged as an attractive target for neurodegenerative and autoimmune disease therapeutics. In a previous study, we developed the novel HDAC6-selective inhibitor 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) and showed that it has anti-sepsis activity in vivo. In this study, we conducted structure-activity relationship (SAR) studies to optimize the activity and selectivity of HDAC6, synthesizing its derivatives with various aliphatic linker sizes and cap structures. We identified 6u ((E)-N-hydroxy-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide), which has nanomolar inhibition activity and a 126-fold selectivity for HDAC6 over HDAC1. Through the docking analyses of 6u against HDAC subtypes, we revealed the importance of the optimal aliphatic linker size, as well as the electronic substituent effect and rigidity of the aryl cap group. Thus, we suggest a new rationale for the design of HDAC6-selective inhibitors.

Dielectrophoresis-based filtration effect and detection of amyloid beta in plasma for Alzheimer's disease diagnosis.

The filtration effect improves the impedance change through specific binding of target molecules in plasma, and decreases this change by nonspecific binding of matrix factors in plasma (i.e., matrix effect). A difference in dielectrophoresis (DEP) forces applied to target molecules and matrix factors causes the filtration effect. An optimized DEP force affects target molecules, which remain in the reaction region of an interdigitated microelectrode (IME) sensor. Various matrix factors, which are larger than the target molecules, are influenced by a strong DEP force and are filtered out of the reaction region. To demonstrate the filtration effect, the matrix effect was confirmed in standard plasma and in phosphate-buffered saline, based on the detection of amyloid beta (Aß), an Alzheimer's disease (AD)-associated peptide. The filtration effect was verified using the matrix effect factor (MEF), which was calculated from the impedance change values in different detection environments. In standard plasma, the MEF value decreased by approximately 78.12%, and in buffer with heterogeneous Aß, by approximately 75.43%. Plasma from patients with AD and normal controls (NCs) was analyzed using the value of the impedance change by the filtration effect. The impedance change was enhanced approximately 1.52 ±â€¯0.03-fold in AD plasma, but declined approximately 0.90 ±â€¯0.03-fold in NC plasma. This difference tendency by the filtration effect was the disease evaluation index and used as an important criterion that distinguished between the AD and NC plasma. Plasma-based AD diagnosis may be possible, based on the filtration effect.

Innovative flat-plate solar collector (FPC) with coloured water flowing through a transparent tube.

Of all types of solar collector, the flat-plate collector (FPC) has the lowest performance, but is the most widely used because of its low cost and easy maintenance. To effectively collect solar light in the conventional FPCs, metal tubes with a high thermal conductivity are installed under an absorption plate. However, in this study, in order to take advantage of the sunlight absorption capacity of coloured water flowing through a tube, a transparent tube was installed on the absorbing plate. The resulting new FPC suggested in this study is a direct absorption solar collector (DASC). To investigate its performance as a function of the colours of the working fluid, four colours of water were supplied to the FPC: transparent (pure water), red, violet and black. From the experimental results, the new FPC suggested in this study was found to have about 5% higher performance than those of the conventional types of FPC, which means that the new concept of FPC can profitably replace the conventional FPCs.

Computer-aided identification of new histone deacetylase 6 selective inhibitor with anti-sepsis activity.

Histone deacetylase (HDAC) inhibitors have been recognized as promising approaches to the treatment of various human diseases including cancer, inflammation, neurodegenerative diseases, and metabolic disorders. Several pan-HDAC inhibitors are currently approved only as anticancer drugs. Interestingly, SAHA (vorinostat), one of clinically available pan-HDAC inhibitors, shows an anti-inflammatory effect at concentrations lower than those required for inhibition of tumor cell growth. It was also reported that HDAC6 selective inhibitor tubastatin A has anti-inflammatory and anti-rheumatic effect. In our efforts to develop novel HDAC inhibitors, we rationally designed various HDAC inhibitors based on the structures of two hit compounds identified by virtual screening of chemical database. Among them, 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) was identified as a HDAC6 selective inhibitor (IC50 values of 0.199 µM for HDAC6 versus 13.8 µM for HDAC1), and it did not show significant cytotoxicity against HeLa cells. In vivo biological evaluation of 9a was conducted on a lipopolysaccharide (LPS)-induced mouse model of sepsis. The compound 9a significantly improved 40% survival rate (P = 0.0483), and suppressed the LPS-induced increase of TNF-α and IL-6 mRNA expression in the liver of mice. Our study identified novel HDAC6 selective inhibitor 9a, which may serve as a potential lead for the development of anti-inflammatory or anti-sepsis agents.

House dust mite allergen suppresses neutrophil apoptosis by cytokine release via PAR2 in normal and allergic lymphocytes.

House dust mite (HDM) is an essential allergen in allergic diseases such as allergic rhinitis and asthma. The pathogenic mechanism of allergy is associated with cytokine release of lymphocytes and constitutive apoptosis of neutrophils. In this study, we examined whether HDM induces cytokine release of lymphocytes and whether the secretion of cytokines is involved in modulation of neutrophil apoptosis. In normal and allergic subjects, extract of Dermatophagoides pteronyssinus (DP) increased IL-6, IL-8, MCP-1, and GM-CSF secretion in a time-dependent manner. This secretion was suppressed by PAR2i, an inhibitor of PAR2, in a dose-dependent manner, as well as by LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, and BAY-11-7085, and an inhibitor of NF-κB. DP induced ERK and NF-κB activation in a time-dependent manner. ERK activation was suppressed by PAR2i, LY294002, and AKTi, and NF-κB activation was blocked by PAR2i, LY294002, AKTi, and PD98059. Supernatants collected from normal and allergic neutrophils after DP treatment inhibited the apoptosis of normal and allergic neutrophils through suppression of caspase 9 and caspase 3 cleavage. DP inhibited neutrophil apoptosis in coculture of normal neutrophils with normal lymphocytes, similar to the anti-apoptotic effects of DP on neutrophils alone. DP more strongly inhibited apoptosis of allergic neutrophils cocultured with allergic lymphocytes than allergic neutrophils without lymphocytes. In summary, DP induces the release of cytokines through the PAR2/PI3K/Akt/ERK/NF-κB pathway, which has anti-apoptotic effects on neutrophils of normal and allergic subjects. These results will facilitate elucidation of the pathogenic mechanism of allergic diseases.

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4.

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.

Inhibitory effect of arazyme on the development of atopic dermatitis-like lesions in BALB/c and Nc/Nga mice.

Arazyme is a metalloprotease released by Aranicola proteolyticus that was shown to inhibit cytokine release in HaCaT and endothelial cells. However, the regulatory effects of arazyme in atopic dermatitis remain to be fully understood. In the present study, the anti­inflammatory effects of arazyme in BALB/c and Nc/Nga mice induced with 2,4­dinitrochlrobenzene (DNCB) were investigated. BALB/c mice were sensitized with DNCB and were subsequently administered arazyme for 4 weeks either orally, dorsally or orally/dorsally. Arazyme administration significantly reduced epidermal thickening and infiltration of inflammatory cells into the dermis compared with the DNCB group. However, serum immunoglobulin E (IgE) levels were not altered by arazyme treatment. Additionally, the level of secretion of interleukins (IL)­4, ­5 and ­13 in the splenocytes of BALB/c mice was elevated following stimulation with concanavalin A, while the increase of IL­4 and IL­13 was inhibited by arazyme. Administration of arazyme (25 mg/kg in phosphate­buffered saline) to Nc/Nga mice that had been sensitized with DNCB for 6 weeks reduced the skin severity score compared with that in the DNCB group and inhibited the histological manifestations of atopic dermatitis­like skin lesions. In addition, the serum IgE levels were reduced in the arazyme­treated NC/Nga mice relative to the DNCB group. Collectively, these results indicated that arazyme attenuates the development of atopic dermatitis­like lesions via lowering the levels of IgE and inflammatory cytokines. The results of the present study will aid in the development of effective therapeutic strategies for the treatment of allergic diseases, including atopic dermatitis.

Regulation of constitutive neutrophil apoptosis due to house dust mite allergen in normal and allergic rhinitis subjects.

House dust mite (HDM) is a primary allergen in allergic rhinitis (AR) and asthma. Neutrophil apoptosis is associated with allergic diseases and innate immunity to infection. The present study examined how HDM affects constitutive neutrophil apoptosis in normal and AR subjects. Total IgE increased in AR subjects when compared to normal subjects, and patients with AR were HDM-specific IgE positive (+), which is specific IgE to Dermatophagoides pteronissinus and Dermatophagoides farinae. In normal and AR subjects, neutrophil apoptosis was inhibited by extract of Dermatophagoides pteronissinus (DP), but not by extract of Dermatophagoides farina (DF). Aprotinin (serine protease inhibitor) and E64 (cysteine protease inhibitor) have no effect on neutrophil apoptosis due to DP. The anti-apoptotic effect of DP was blocked by TLR4i, an inhibitor of TLR4, rottlerin, an inhibitor of PKCδ, PD98059, an inhibitor of ERK, and BAY-11-7085, an inhibitor of NF-κB. DP induced PKCδ, ERK, and NF-κB activation in a time-dependent manner. DP inhibited the cleavage of procaspase 3 and procaspase 9. The expression of IL-6, IL-8, TNF-α, G-CSF, GM-CSF, and CCL2 increased in the supernatant collected from the normal and AR neutrophils after DP treatment and the supernatant inhibited the apoptosis of normal and AR neutrophils. In summary, DP has anti-apoptotic effects on neutrophils of normal and AR subjects through the TLR4/PKCδ/ERK/NF-κB pathway, and this finding may contribute to solution of the pathogenic mechanism of allergic diseases triggered by DP.