Alterations in Plasma Cytokine Levels in Korean Children with Autism Spectrum Disorder.

PURPOSE: Numerous studies have supported the role of the immune dysfunction in the pathogenesis of autism spectrum disorder (ASD); however, to our knowledge, no study has been conducted on plasma cytokine levels in children with ASD in South Korea. In this study, we aimed to analyze the immunological characteristics of Korean children with ASD through plasma cytokine analysis. MATERIALS AND METHODS: Blood samples were collected from 94 ASD children (mean age 7.1; 81 males and 13 females) and 48 typically developing children (TDC) (mean age 7.3; 30 males and 18 females). Plasma was isolated from 1 mL of blood by clarifying with centrifugation at 8000 rpm at 4℃ for 10 min. Cytokines in plasma were measured with LEGENDplex HU Th cytokine panel (BioLegend, 741028) and LEGENDplex HU cytokine panel 2 (BioLegend, 740102). RESULTS: Among 25 cytokines, innate immune cytokine [interleukin (IL)-33] was significantly decreased in ASD children compared with TDC. In acute phase proteins, tumor necrosis factor α (TNF-α) was significantly increased, while IL-6, another inflammation marker, was decreased in ASD children compared with TDC. The cytokines from T cell subsets, including interferon (IFN)-γ, IL-5, IL-13, and IL-17f, were significantly decreased in ASD children compared to TDC. IL-10, a major anti-inflammatory cytokine, and IL-9, which modulates immune cell growth and proliferation, were also significantly decreased in ASD children compared to TDC. CONCLUSION: We confirmed that Korean children with ASD showed altered immune function and unique cytokine expression patterns distinct from TDC.

WNK3 inhibition elicits antitumor immunity by suppressing PD-L1 expression on tumor cells and activating T-cell function.

Immune checkpoint therapies, such as programmed cell death ligand 1 (PD-L1) blockade, have shown remarkable clinical benefit in many cancers by restoring the function of exhausted T cells. Hence, the identification of novel PD-L1 regulators and the development of their inhibition strategies have significant therapeutic advantages. Here, we conducted pooled shRNA screening to identify regulators of membrane PD-L1 levels in lung cancer cells targeting druggable genes and cancer drivers. We identified WNK lysine deficient protein kinase 3 (WNK3) as a novel positive regulator of PD-L1 expression. The kinase-dead WNK3 mutant failed to elevate PD-L1 levels, indicating the involvement of its kinase domain in this function. WNK3 perturbation increased cancer cell death in cancer cell-immune cell coculture conditions and boosted the secretion of cytokines and cytolytic enzymes, promoting antitumor activities in CD4+ and CD8+ T cells. WNK463, a pan-WNK inhibitor, enhanced CD8+ T-cell-mediated antitumor activity and suppressed tumor growth as a monotherapy as well as in combination with a low-dose anti-PD-1 antibody in the MC38 syngeneic mouse model. Furthermore, we demonstrated that the c-JUN N-terminal kinase (JNK)/c-JUN pathway underlies WNK3-mediated transcriptional regulation of PD-L1. Our findings highlight that WNK3 inhibition might serve as a potential therapeutic strategy for cancer immunotherapy through its concurrent impact on cancer cells and immune cells.

Image-guided in situ cancer vaccination with combination of multi-functional nano-adjuvant and an irreversible electroporation technique.

Cancer immunotherapy is a next-generation treatment strategy; however, its side effects limit its clinical translation. Here, a novel combination of a multi-functional nano-adjuvant (M-NA) prepared with an iron oxide/gold core and a cationic polymer shell via multilayer synthesis with CpG oligodeoxynucleotide (CpG-ODN) electrostatically complexed on its surface, and irreversible electroporation (IRE) technique was developed for effective image-guided in situ cancer vaccination. The M-NA can be retained long-term in the dense tumoral extracellular matrix after intratumoral injection and internalized by antigen-presenting cells (APCs). The IRE can induce immunogenic cell death. Indeed, in a mouse tumor model, the M-NA showed longer tumor retention time than free CpG-ODN. Compared with other treatments, the combined treatment significantly inhibited tumor growth with 100% survival rate for ∼60 days. The therapy induced the activation of cytotoxic lymphocytes and the maturation of APCs in vivo. This treatment could be effective in image-guided local cancer immunotherapy.

ETS1 Suppresses Tumorigenesis of Human Breast Cancer via Trans-Activation of Canonical Tumor Suppressor Genes.

ETS1 has shown dichotomous roles as an oncogene and a tumor suppressor gene in diverse cancers, but its functionality in breast cancer tumorigenesis still remains unclear. We utilized the Cancer Genome Atlas (TCGA) database to analyze comprehensive functions of ETS1 in human breast cancer (BRCA) patients by investigating its expression patterns and methylation status in relation to clinical prognosis. ETS1 expression was significantly diminished by hyper-methylation of the ETS1 promoter region in specimens from BRCA patients compared to a healthy control group. Moreover, ETS1 high BRCA patients showed better prognosis and longer survival compared to ETS1 low BRCA patients. Consistent with clinical evidence, comparative transcriptome analysis combined with CRISPR/Cas9 or shRNA based perturbation of ETS1 expression revealed direct as well as indirect mechanisms of ETS1 that hinder tumorigenesis of BRCA cells. Taken together, our study enlightens a novel function of ETS1 as a tumor suppressor in breast cancer cells.

The NF-κB RelA Transcription Factor Is Critical for Regulatory T Cell Activation and Stability.

Regulatory T cells (Tregs) play a major role in immune homeostasis and in the prevention of autoimmune diseases. It has been shown that c-Rel is critical in Treg thymic differentiation, but little is known on the role of NF-κB on mature Treg biology. We thus generated mice with a specific knockout of RelA, a key member of NF-κB, in Tregs. These mice developed a severe autoimmune syndrome with multi-organ immune infiltration and high activation of lymphoid and myeloid cells. Phenotypic and transcriptomic analyses showed that RelA is critical in the acquisition of the effector Treg state independently of surrounding inflammatory environment. Unexpectedly, RelA-deficient Tregs also displayed reduced stability and cells that had lost Foxp3 produced inflammatory cytokines. Overall, we show that RelA is critical for Treg biology as it promotes both the generation of their effector phenotype and the maintenance of their identity.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

Ets1 suppresses atopic dermatitis by suppressing pathogenic T cell responses.

Atopic dermatitis (AD) is a complex inflammatory skin disease mediated by immune cells of both adaptive and innate types. Among them, CD4+ Th cells are one of major players of AD pathogenesis. Although the pathogenic role of Th2 cells has been well characterized, Th17/Th22 cells are also implicated in the pathogenesis of AD. However, the molecular mechanisms underlying pathogenic immune responses in AD remain unclear. We sought to investigate how the defect in the AD susceptibility gene, Ets1, is involved in AD pathogenesis in human and mice and its clinical relevance in disease severity by identifying Ets1 target genes and binding partners. Consistent with the decrease in ETS1 levels in severe AD patients and the experimental AD-like skin inflammation model, T cell-specific Ets1-deficient mice (Ets1ΔdLck) developed severe AD-like symptoms with increased pathogenic Th cell responses. A T cell-intrinsic increase of gp130 expression upon Ets1 deficiency promotes the gp130-mediated IL-6 signaling pathway, thereby leading to the development of severe AD-like symptoms. Functional blocking of gp130 by selective inhibitor SC144 ameliorated the disease pathogenesis by reducing pathogenic Th cell responses. Our results reveal a protective role of Ets1 in restricting pathogenic Th cell responses and suggest a potential therapeutic target for AD treatment.

Upregulation of Ets1 expression by NFATc2 and NFKB1/RELA promotes breast cancer cell invasiveness.

Breast cancer is highly aggressive and is the leading cause of cancer-related mortality in women in developed countries. The ETS proto-oncogene 1 (Ets1) has versatile roles during the cellular processes of cancer development. It is often highly expressed in breast cancers and mediates migration and invasion of human breast cancer cells. However, underlying mechanisms of Ets1 gene expression is still ambiguous. Here, we identified a core-regulatory element (CRE) located in the Ets1 promoter region (-540/-80 bp from TSS) that contains elements responsible for associating with NFATs and NF-κBs. Compared with the less metastatic breast cancer cells, metastatic breast cancer cells (MDA-MB-231) show open chromatin configurations in the CRE, which facilitates direct binding of NFATc2 and/or NFKB1/RELA complex to trans-activate Ets1 transcription. Moreover, enhanced level of Nfatc2 and Nfkb1 positively correlated with Ets1 expression in the human breast cancer specimens. Deletion of the CRE region by CRISPR/Cas9 system resulted in significant reduction in Ets1 expression, which led to alterations of Ets1-mediated transcription programs including tumor invasiveness-related genes. Proper regulation of Ets1 gene expression by targeting the NFATc2 and NFKB1/RELA interaction could be a potential therapeutic target for Ets1-mediated metastatic breast cancer.

FoxP3 scanning mutagenesis reveals functional variegation and mild mutations with atypical autoimmune phenotypes.

FoxP3+ regulatory T cells (Tregs) are a central element of immunological tolerance. FoxP3 is the key determining transcription factor of the Treg lineage, interacting with numerous cofactors and transcriptional targets to determine the many facets of Treg function. Its absence leads to devastating lymphoproliferation and autoimmunity in scurfy mutant mice and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. To finely map transcriptionally active regions of the protein, with respect to disease-causing variation, we performed a systematic alanine-scan mutagenesis of FoxP3, assessing mutational impacts on DNA binding and transcriptional activation or repression. The mutations affected transcriptional activation and repression in a variegated manner involving multiple regions of the protein and varying between different transcriptional targets of FoxP3. There appeared to be different modalities for target genes related to classic immunosuppressive function vs. those related to atypical or tissue-Treg functions. Relevance to in vivo Treg biology was established by introducing some of the subtle Foxp3 mutations into the mouse germline by CRISPR-based genome editing. The resulting mice showed Treg populations in normal numbers and exhibited no overt autoimmune manifestations. However, Treg functional defects were revealed upon competition or by system stress, manifest as a strikingly heightened susceptibility to provoked colitis, and conversely by greater resistance to tumors. These observations suggest that some of the missense mutations that segregate in human populations, but do not induce IPEX manifestations, may have unappreciated consequences in other diseases.

Different molecular complexes that mediate transcriptional induction and repression by FoxP3.

FoxP3 conditions the transcriptional signature and functional facets of regulatory T cells (Treg cells). Its mechanism of action, whether as an activator or a repressor, has remained unclear. Here, chromatin analysis showed that FoxP3 bound active enhancer elements, not repressed chromatin, around loci over- or under-expressed in Treg cells. We evaluated the impact of a panel of FoxP3 mutants on its transcriptional activity and interactions with DNA, transcriptional cofactors and chromatin. Computational integration, confirmed by biochemical interaction and size analyses, showed that FoxP3 existed in distinct multimolecular complexes. It was active and primarily an activator when complexed with the transcriptional factors RELA, IKZF2 and KAT5. In contrast, FoxP3 was inactive when complexed with the histone methyltransferase EZH2 and transcription factors YY1 and IKZF3. The latter complex partitioned to a peripheral region of the nucleus, as shown by super-resolution microscopy. Thus, FoxP3 acts in multimodal fashion to directly activate or repress transcription, in a context- and partner-dependent manner, to govern Treg cell phenotypes.

Validity of screening methods for periodontitis using salivary hemoglobin level and self-report questionnaires in people with disabilities.

BACKGROUND: The aim of this study is to evaluate the validity of screening methods in predicting periodontitis in people with disabilities using the objective salivary hemoglobin level, a subjective self-report questionnaire, and a combined model of the two methods with demographic characteristics. METHODS: The participants were 195 patients with disabilities aged >18 years who were examined using the community periodontal index (CPI), salivary hemoglobin level, and answers to 10 self-report questions (n = 192). Multivariable logistic regression and receiver operating characteristic (ROC) curve analysis were performed to evaluate the validity of the methods and the combined model in predicting the prevalence of ≥CPI 3 (probing depth [PD] ≥4 mm) or CPI 4 (PD ≥6 mm). RESULTS: Overall, 75.9% of the study group (148 of 195) were diagnosed with ≥CPI 3, and 38.5% of the study group (75 of 195) were diagnosed with CPI 4. The areas under the ROC curve (AUCs) of the salivary hemoglobin level were 0.578 (sensitivity of 41% and specificity of 77%) and 0.662 (sensitivity of 53% and specificity of 75%) for predicting the prevalence of ≥CPI 3 and CPI 4, respectively. Multivariable modeling incorporating four different questions for predicting ≥CPI 3 or CPI 4 indicated higher AUCs of 0.710 and 0.732, respectively, yielding higher sensitivity (55% for ≥CPI 3 and 69% for CPI 4) than that of salivary hemoglobin level. The most useful prediction models for ≥CPI 3 or CPI 4 were combined models, which yielded AUCs of 0.773 and 0.807, respectively, with sensitivity values of 70% and specificity values >75%. CONCLUSION: The salivary hemoglobin level, self-report questionnaire, and the combined method demonstrated screening potential that could predict the population prevalence of ≥CPI 3 or CPI 4.

Desensitizing efficacy of nano-carbonate apatite dentifrice and Er,Cr:YSGG laser: a randomized clinical trial.

OBJECTIVE: The aim of this study was to evaluate the desensitizing effects of a dentifrice containing nano-carbonate apatite (n-CAP) and Er,Cr:YSGG laser in the treatment of dentin hypersensitivity. BACKGROUND DATA: Most studies of hypersensitivity treatment have been conducted with different methods of professional treatment and self-care in each study. Moreover, clinical studies that compare self-care and professional treatment have not yet been published. METHODS: Eighty-two patients with dentin hypersensitivity were divided randomly into three groups: (1) a control group with strontium chloride dentifrice (SC), (2) n-CAP dentifrice (n-CAP), and (3) an Er,Cr:YSGG laser (laser) group. The study was conducted for 4 weeks: a treatment period of 2 weeks and a maintenance period of 2 subsequent weeks. The SC and n-CAP groups were instructed to brush their teeth twice a day for 1 min. The laser group visited twice at 1 week intervals for irradiation of the sensitive teeth. The desensitizing effect was evaluated by assessing the tactile sensitivity using the visual analogue scale (VAS), and evaporative air sensitivity was determined using an air blast score (ABS). RESULTS: The n-CAP group and the laser group showed significantly different desensitizing effects in VAS after 4 weeks (69% and 63%, respectively) and a 33% (p<0.05) and 3% (p>0.05) desensitizing effect, respectively, in VAS during the maintenance period. CONCLUSIONS: The n-CAP and the laser were effective in reducing dentin hypersensitivity. The laser had a superior desensitizing effect at the initial stage, whereas the n-CAP maintained its effect for a relatively longer time in clinical situations.

Short-term improvement of masticatory function after implant restoration.

PURPOSE: Dental implants present several advantages over other tooth replacement options. However, there has been little research on masticatory function in relation to implant treatment. Therefore, the aim of the present study was to evaluate the improvement of masticatory function two weeks after implant restoration. METHODS: Masticatory ability was evaluated with the subjective food intake ability (FIA) and objective mixing ability index (MAI) methods. Fifty-four subjects with first and second missing molars completed the study. The subjects were asked to complete a self-reported questionnaire about 30 different food items, and to chew wax samples 10 times both before and two weeks after implant restoration. A total of 108 waxes were analyzed with an image analysis program. RESULTS: Dental implant restoration for lost molar teeth on one side increased the FIA score by 9.0% (P<0.0001). The MAI score also increased, by 14.3% after implant restoration (P<0.0001). Comparison between the good and poor mastication groups, which were subdivided based on the median MAI score before implant restoration, showed that the FIA score of the poor group was enhanced 1.1-fold while its MAI score was enhanced 2.0-fold two weeks after an implant surgery. CONCLUSIONS: Using the FIA and MAI assessment methods, this study showed that masticatory function was improved two weeks after implant restoration. In particular, the enhancement of masticatory function by implant restoration was greater in patients with relatively poor initial mastication than in those with good initial mastication.

Effect of a new combined therapy with nano-carbonate apatite and CO2 laser on dentin hypersensitivity in an in situ model.

OBJECTIVE: The aim of this study was to determine the occluding effects of a combination of dentifrice containing nano-carbonate apatite (n-CAP) and CO2 laser on dentinal tubules, and to evaluate the acid resistance of this combination after 4 days of treatment, by using an in situ model. BACKGROUND DATA: The synergic effect of this combination was demonstrated in an in vitro study. METHODS: This was a two period crossover, single-blind, randomized, four-treatment, split-mouth study. Ten healthy participants wore lower intraoral appliances during the treatment period. Specimens were divided into the following four groups: no treatment (control group), tooth-brushing using 20% n-CAP dentifrice (n-CAP group), CO2 laser irradiation (laser group), and laser irradiation after n-CAP application (combined group). Occluding effects were evaluated on 2 days (days 1 and 2), and then acid challenge was performed using grape juice on 2 days (days 3 and 4). All of the specimen surfaces were evaluated by a scanning electron microscope. RESULTS: The combined group showed a better occluding effect than control group compared with other treatment groups, and this effect was 20% higher than that in the n-CAP group. Also, the combined group had the smallest open dentinal tubular area among all of the treatment groups. CONCLUSIONS: The combined therapy is a promising method for ensuring a long-lasting effect of dentin hypersensitivity treatment in clinical practice.

Synergistic effect of dentinal tubule occlusion by nano-carbonate apatite and CO2 laser in vitro.

OBJECTIVE: The aim of this study was to evaluate the occluding effects of a combination of dentifrice containing nano-carbonate apatite (n-CAP) and a CO2 laser on dentinal tubules, and to assess the acid resistance of the occluded dentinal tubules produced. BACKGROUND DATA: A number of experiments have been conducted recently to relieve the symptoms of dentin hypersensitivity (DH) using a laser in combination with desensitizing products. MATERIALS AND METHODS: One hundred and twenty specimens with exposed dentinal tubules were divided into four groups: the control, n-CAP, laser, and combined groups. Thirty specimens in each group were reassigned into three different conditions: baseline, treatment for occluding dentinal tubules, and acid challenge (pH 4.0 acetate buffer solution for 3 min). At the end of each phase, all specimen surfaces were evaluated by scanning electron microscope (SEM). RESULTS: The combined group had a significantly smaller mean dentinal tubule area than the control group, and the fewest reopened dentinal tubules after acid challenge. CONCLUSIONS: The combined therapy is a promising means of treating DH patients in the clinic.

Combined effects of Er:YAG laser and nano-carbonate apatite dentifrice on dentinal tubule occlusion: in vitro study.

OBJECTIVE: The aim of this study was to evaluate the occluding effects of Er:YAG laser and dentifrice containing nano-carbonate apatite (n-CAP) on the dentinal tubules. BACKGROUND DATA: Although there are various treatment methods for dentin hypersensitivity (DH), more effective methods are still being sought. METHODS: Sound human root dentin specimens were etched with 6% citric acid to open the dentinal tubules. The specimens were divided randomly into four groups: no treatment (control group), toothbrushing using 20% n-CAP dentifrice (n-CAP group), Er:YAG laser irradiation (LI group), and LI after n-CAP (combined group). The degree of occlusion of the dentinal tubules and the thickness of the deposition layer were evaluated by scanning electron microscopy and an image analyzer. RESULTS: It was shown that the occlusion effect of the n-CAP group was 87%, that of the combined group was 54%, and that of the LI group was 52%, compared with that of the control group. And the n-CAP particles were observed not only under the irradiated surface and but also above the surface in the combined group. CONCLUSIONS: This combination method has the potential for enhancement of the dentinal tubule occlusion. However, further study is needed to present the combination effects with additional analysis, and the long-term effects.

Immunomodulatory effect of water soluble extract separated from mycelium of Phellinus linteus on experimental atopic dermatitis.

BACKGROUND: Complementary and alternative medicine (CAM) is becoming a popular treatment for modulating diverse immune disorders. Phellinus linteus (P. linteus) as one of the CAMs has been used to modulate cancers, inflammation and allergic activities. However, little evidence has been shown about its underlying mechanism of action by which it exerts a beneficial role in dermatological disease in vivo. In this study, we examined the immunomodulatory effects of P. linteus on experimental atopic dermatitis (AD) and elucidated its action mechanism. METHODS: The immunomodulatory effect of total extract of P. linteus on IgE production by human myeloma U266B1 cells was measured by ELISA. To further identify the effective components, P. linteus was fractionated into methanol soluble, water soluble and boiling water soluble extracts. Each extract was treated to U266B1 cells and primary B cells to compare their inhibitory effects on IgE secretion. To test the in vivo efficacy, experimental atopic dermatitis (AD) was established by alternative treatment of DNCB and house dust mite extract into BALB/c mice. Water soluble extract of P. linteus (WA) or ceramide as a positive control were topically applied to ears of atopic mouse every day for 2 weeks and progression of the disease was estimated by the following criteria: (a) ear thickness, clinical score, (b) serum total IgE, IgG and mite specific IgE level by ELSIA, (c) histological examination of ear tissue by H&E staining and (d) cytokine profile of total ear cells and CD4(+) T cells by real time PCR and ELSIA. RESULTS: Treatment of total extracts of P. linteus to U266B1 inhibited IgE secretion. Among the diverse extracts of P. linteus, water soluble extract of P. linteus (WA) significantly reduced the IgE production in primary B cells and B cell line U266B1. Moreover, treatment of WA reduced AD symptoms such as ear swelling, erythema, and dryness and decreased recruitment of lymphocyte into the inflamed site. Interestingly WA treatment significantly reduced IgE level without affecting IgG levels and also down-regulated the levels of pathogenic cytokines (IL-4, IL-13, IL-12 and IFN-γ) and chemokines (CCL17 and CCL22) involved in AD development. CONCLUSIONS: Our study indicates that protective effect of water soluble extract of P. linteus in atopic dermatitis is mediated by inhibiting IgE production and expression of AD associated pathogenic cytokines as well as chemokines, suggesting the beneficial effect of P. linteus to modulate allergic skin disease.

Interaction of Ets-1 with HDAC1 represses IL-10 expression in Th1 cells.

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4(+) T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1-mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.

Targeted chemoimmunotherapy using drug-loaded aptamer-dendrimer bioconjugates.

We reported an innovative, targeted chemoimmuno drug-delivery system. Although chemoimmunotherapy, as an alternative to or in combination with conventional therapeutic systems, has been in the forefront of recent oncological research, as presently configured, such systems face several major obstacles for efficient clinical application. Here, we establish a novel nano-platform for effective chemoimmunotherapy designed to overcome the drawbacks of conventional cancer therapies, describing a delivery system based on a dendrimer and a single-strand DNA-A9 PSMA (prostate-specific membrane antigen) RNA aptamer hybrid. Employing these vehicles, we demonstrate the promising possibility of this chemoimmuno therapeutic system against prostate cancer in in vivo and in vitro models.