O6-methylguanine DNA methyltransferase regulates ß-glucan-induced trained immunity of macrophages via farnesoid X receptor and AMPK.

Trained immunity is the heightened state of innate immune memory that enhances immune response resulting in nonspecific protection. Epigenetic changes and metabolic reprogramming are critical steps that regulate trained immunity. In this study, we reported the involvement of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme of lesion induced by alkylating agents, in regulation the trained immunity induced by ß-glucan (BG). Pharmacological inhibition or silencing of MGMT expression altered LPS stimulated pro-inflammatory cytokine productions in BG-trained bone marrow derived macrophages (BMMs). Targeted deletion of Mgmt in BMMs resulted in reduction of the trained responses both in vitro and in vivo models. The transcriptomic analysis revealed that the dampening trained immunity in MGMT KO BMMs is partially mediated by ATM/FXR/AMPK axis affecting the MAPK/mTOR/HIF1α pathways and the reduction in glycolysis function. Taken together, a failure to resolve a DNA damage may have consequences for innate immune memory.

Coculturing liver cancer cells and monocytes in spheroids conditions monocytes to adopt tumor-associated macrophage phenotypes that favor tumor growth via cholesterol metabolism.

Tumor-infiltrating immune cells and their crosstalk with cancer cells in the tumor microenvironment (TME) play a crucial role in shaping tumor progression and response to therapy. We utilized 3-dimensional liver cancer spheroids incorporating human primary monocytes to investigate the crosstalk between tumor-associated macrophages (TAMs) and Hepatocellular carcinoma (HCC) cells, HepG2 and PLC/PRF/5. Using multiplexed gene expression panels, the critical pathways involved in shaping primary human monocytes to adopt TAMs phenotypes were identified. The specific inhibitor for an identified pathway was used to explore its involvement in polarization of TAMs. In the cocultured spheroids comprising the human HCC cell lines, the infiltrating monocytes resembled protumor M2-like macrophage phenotypes. Gene expression panels of the infiltrating monocytes demonstrated that the upregulated genes were enriched in the cholesterol metabolism pathway. Cholesterol metabolism-related genes were upregulated together with the nuclear receptors, PPARG and LXR. When lysosomal acid lipase (LAL), the key enzyme necessary for the hydrolysis of lipoprotein, was inhibited, infiltrating monocytes in 3-dimensional spheroid coculture showed significantly decreased M2 marker and lipid uptake receptor expression as well as increased cellular lipid content, which indicated that cholesterol metabolism was important for conditioning the TAMs. Moreover, LAL inhibition reduced the spheroid growth and invasiveness of HCC cell lines. Small interfering RNA-mediated LAL silencing in monocytes yielded similar results upon spheroid coculture. These data indicated that liver cancer cells and infiltrating monocytes participate in crosstalk via cholesterol metabolism to condition monocytes toward TAMs, which favors tumor growth and survival, thereby promoting liver cancer progression.

Live-cell imaging Unveils stimulus-specific dynamics of Nrf2 activation in UV-exposed melanoma cells: Implications for antioxidant compound screening.

The transcription factor Nuclear factor e2-related factor 2 (Nrf2) is pivotal in orchestrating cellular antioxidant defense mechanisms, particularly in skin cells exposed to ultraviolet (UV) radiation and electrophilic phytochemicals. To comprehensively investigate Nrf2's role in maintaining cellular redox equilibrium following UV-induced stress, we engineered a novel Nrf2 fusion-based reporter system for real-time, live-cell quantification of Nrf2 activity in human melanoma cells. Utilizing live quantitative imaging, we dissected the kinetic profiles of Nrf2 activation in response to an array of stimuli, including UVA and UVB radiation, as well as a broad spectrum of phytochemicals including ferulic acid, gallic acid, hispidulin, p-coumaric acid, quercetin, resveratrol, tannic acid, and vanillic acid as well as well-known Nrf2 inducers, tert-butylhydroquinone (tBHQ) and sulforaphane (SFN). Intriguingly, we observed distinct dynamical patterns of Nrf2 activity contingent on the specific stimuli applied. Sustained activation of Nrf2 was empirically correlated with the increased antioxidant response element (ARE) activity. Our findings demonstrate the nuanced impact of different phenolic compounds on Nrf2 activity and the utility of our Nrf2-CTΔ16-YFP reporter in characterizing the dynamics of Nrf2 translocation in response to diverse stimuli. In summary, our innovative reporter system not only revealed compounds capable of modulating UVA-induced Nrf2 activity but also showcased its utility as a robust tool for future antioxidant compound screening efforts.

Impaired functions of human monocyte-derived dendritic cells and induction of regulatory T cells by pathogenic Leptospira.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease.

Sentiment and Topic Modeling Analysis on Twitter Reveals Concerns over Cannabis-Containing Food after Cannabis Legalization in Thailand.

OBJECTIVES: Twitter has been used to express a diverse range of public opinions about cannabis legalization in Thailand. The purpose of this study was to observe changes in sentiments after cannabis legalization and to investigate health-related topics discussed on Twitter. METHODS: Tweets in Thai and English related to cannabis were scraped from Twitter between May 1 and June 13, 2022, during cannabis legalization in Thailand. Sentiment and topic-modeling analyses were used to compare the content of tweets before and after legalization. Health-related topics were manually grouped into categories by their content and rated according to the number of corresponding tweets. RESULTS: We collected 21,242 and 6,493 tweets, respectively, for Thai and English search terms. A sharp increase in the number of tweets related to cannabis legalization was detected at the time of its public announcement. Sentiment analysis in the Thai search group showed a significant change (p < 0.0001) in sentiment distribution after legalization, with increased negative and decreased positive sentiments. A significant change was not found in the English search group (p = 0.4437). Regarding cannabis-containing food as a leading issue, topic-modeling analysis revealed public concerns after legalization in the Thai search group, but not the English one. Topics related to cannabis tourism surfaced only in the English search group. CONCLUSIONS: Since cannabis legalization, the primary health-related concern has been cannabis-containing food. Education and clear regulations on cannabis use are required to strengthen oversight of cannabis in the Thai population, as well as among medical tourists.

Retrospective study of transcriptomic profiling identifies Thai triple-negative breast cancer patients who may benefit from immune checkpoint and PARP inhibitors.

Background: Triple-negative breast cancer (TNBC) is a rare and aggressive breast cancer subtype. Unlike the estrogen receptor-positive subtype, whose recurrence risk can be predicted by gene expression-based signature, TNBC is more heterogeneous, with diverse drug sensitivity levels to standard regimens. This study explored the benefit of gene expression-based profiling for classifying the molecular subtypes of Thai TNBC patients. Methods: The nCounter-based Breast 360 gene expression was used to classify Thai TNBC retrospective cohort subgroups. Their expression profiles were then compared against the previously established TNBC classification system. The differential characteristics of the tumor microenvironment and DNA damage repair signatures across subgroups were also explored. Results: Thai TNBC cohort could be classified into four main subgroups, corresponding to the LAR, BL-2, and M subtypes based on Lehmann's TNBC classification. The PAM50 gene set classified most samples as basal-like subtypes except for Group 1. Group 1 exhibited similar enrichment of the metabolic and hormone response pathways to the LAR subtype. Group 2 shared pathway activation with the BL-2 subtype. Group 3 showed an increase in the EMT pathway, similar to the M subtype. Group 4 showed no correlation with Lehmann's TNBC. The tumor microenvironment (TME) analysis showed high TME cell abundance with increased expression of immune blockade genes in Group 2. Group 4 exhibited low TME cell abundance and reduced immune blockade gene expressions. We also observed distinct signatures of the DNA double-strand break repair genes in Group 1. Conclusions: Our study reported unique characteristics between the four TNBC subgroups and showed the potential use of immune checkpoint and PARP inhibitors in subsets of Thai TNBC patients. Our findings warrant further clinical investigation to validate TNBC's sensitivity to these regimens.

Oxyresveratrol Attenuates Inflammation in Human Keratinocyte via Regulating NF-kB Signaling and Ameliorates Eczematous Lesion in DNCB-Induced Dermatitis Mice.

Oxyresveratrol (ORV) is one of the novel antioxidants having been extensively studied in recent years. One of the main sources of ORV is Artocarpus lakoocha, which has been used in traditional medicine in Thailand for decades. However, the role of ORV in skin inflammation has not been clearly demonstrated. Therefore, we investigated the anti-inflammatory effects of ORV on dermatitis model. The effect of ORV was examined on human immortalized and primary skin cells exposed to bacterial components including peptidoglycan (PGN) and lipopolysaccharide (LPS) and 2,4-Dinitrochlorobenzene (DNCB)-induced dermatitis mouse model. PGN and LPS were used to induce inflammation on immortalized keratinocytes (HaCaT) and human epidermal keratinocytes (HEKa). We then performed MTT assay, Annexin V and PI assay, cell cycle analysis, real-time PCR, ELISA and Western blot in these in vitro models. H&E staining, immunohistochemistry (IHC) staining with CD3, CD4 and CD8 markers were used to evaluate the effects of ORV in in vivo model of skin inflammation using BALB/c mice. Pretreatment of HaCaT and HEKa cells with ORV inhibited pro-inflammatory cytokine production through inhibition of NF-κB pathway. In DNCB-induced dermatitis mouse model, ORV treatment reduced lesion severity, and skin thickness and numbers of CD3, CD4 and CD8 T cells in the sensitized skin of mice. In conclusion, it has been demonstrated that ORV treatment can ameliorate inflammation in the in vitro models of skin inflammation and in vivo models of dermatitis, suggesting a therapeutic potential of ORV for treatment of skin diseases particularly eczema.

Cyclic GMP-AMP Synthase (cGAS) Deletion Reduces Severity in Bilateral Nephrectomy Mice through Changes in Neutrophil Extracellular Traps and Mitochondrial Respiration.

Uremia-induced systemic inflammation is partly caused by the dissemination of microbial molecules such as lipopolysaccharide and bacterial double-stranded DNA from leaked gut damaged by immune cells in response to the microbial molecules. Cyclic GMP-AMP synthase (cGAS) can recognize fragmented DNA and induce cGAMP synthesis for the activation of the stimulator of interferon genes (STING) pathway. To study the effect of cGAS in uremia-induced systemic inflammation, we performed bilateral nephrectomy (BNx) in wild-type and cGAS knock-out mice and found that the gut leakage and blood uremia from both groups were similar. However, serum cytokines (TNF-α and IL-6) and neutrophil extracellular traps (NETs) decreased significantly in cGAS-/- neutrophils after stimulation with LPS or bacterial cell-free DNA. Transcriptomic analysis of LPS-stimulated cGAS-/- neutrophils also confirmed the down-regulation of neutrophil effector functions. The extracellular flux analysis showed that cGAS-/- neutrophils exhibited a higher respiratory rate than wild-type neutrophils despite having similar mitochondrial abundance and function. Our results suggest that cGAS may control effector functions and the mitochondrial respiration of neutrophils in response to LPS or bacterial DNA.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Comprehensive drug response profiling and pan-omic analysis identified therapeutic candidates and prognostic biomarkers for Asian cholangiocarcinoma.

Cholangiocarcinoma (CCA) is rare cancer with the highest incidence in Eastern and Southeast Asian countries. Advanced CCA patients rely on chemotherapeutic regimens that offer unsatisfied clinical outcomes. We developed a comprehensive drug response profiling to investigate potential new drugs using CCA cell lines from Thai and Japanese patients against 100 approved anti-cancer drugs. We identified two major CCA subgroups that displayed unique molecular pathways from our integrative pan-omic and ligand-induced pathway activation analyses. MEK and Src inhibitors specifically killed the CCA1 subgroup without causing cytotoxicity to the normal cholangiocyte. Next, we developed the CCA45 signature to classify CCA patients based on their transcriptomic data. Our CCA45 signature could accurately predict prognosis, especially for Asian CCA patients. Our study provides a comprehensive public resource for drug repurposing in CCA and introduces analytical strategies for prioritizing cancer therapeutic agents for other rare cancer.

Impact of SARS-CoV-2 infection on the profiles and responses of innate immune cells after recovery.

BACKGROUNDS: SARS-CoV-2 infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic to severe symptoms and death. Most COVID-19 pathogenesis is associated with hyperinflammatory conditions driven primarily by myeloid cell lineages. The long-term effects of SARS-CoV-2 infection post recovery include various symptoms. METHODS: We performed a longitudinal study of the innate immune profiles 1 and 3 months after recovery in the Thai cohort by comparing patients with mild, moderate, and severe clinical symptoms using peripheral blood mononuclear cells (n = 62). RESULTS: Significant increases in the frequencies of monocytes compared to controls and NK cells compared to mild and moderate patients were observed in severe patients 1-3 months post recovery. Increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were observed in all recovered patients, even after 3 months. Increased IL-6 and TNFα levels in monocytes were observed 1 month after recovery in response to lipopolysaccharide (LPS) stimulation, while decreased CD86 and HLA-DR levels were observed regardless of stimulation. A multiplex analysis of serum cytokines performed at 1 month revealed that most innate cytokines, except for TNFα, IL4/IL-13 (Th2) and IFNγ (Th1), were elevated in recovered patients in a severity-dependent manner. Finally, the myelopoiesis cytokines G-CSF and GM-CSF were higher in all patient groups. Increased monocytes and IL-6- and TNFα-producing cells were significantly associated with long COVID-19 symptoms. CONCLUSIONS: These results reveal that COVID-19 infection influences the frequencies and functions of innate immune cells for up to 3 months after recovery, which may potentially lead to some of the long COVID symptoms.

A LILRB1 variant with a decreased ability to phosphorylate SHP-1 leads to autoimmune diseases.

Inborn errors of immunity are known to cause not only immunodeficiencies and allergies but also autoimmunity. Leukocyte immunoglobulin-like receptor B1 (LILRB1) is a receptor on leukocytes playing a role in regulating immune responses. No phenotypes have been reported to be caused by germline mutations in LILRB1. We aimed to identify the causative variant in a three-generation family with nine members suffering from one of the three autoimmune diseases-Graves' disease, Hashimoto's thyroiditis, or systemic lupus erythematosus. Whole-genome linkage study revealed a locus on chromosome 19q13.4 with the maximum LOD score of 2.71. Whole-exome sequencing identified a heterozygous missense variant, c.479G > A (p. G160E) in LILRB1, located within the chromosomal-linked region, in all nine affected members. The variant has never been previously reported. Jurkat cells transfected with the mutant LILRB1, compared with those with the wild-type LILRB1, showed decreased phosphorylation of both LILRB1 and its downstream protein, SHP-1. Flow cytometry was used to study immunophenotype and revealed that LILRB1 was significantly lower on the surface of activated regulatory T lymphocytes (Treg) cells of patients. Single-cell RNA sequencing showed substantially increased M1-like monocytes in peripheral blood mononuclear cells of affected individuals. This study, for the first time, implicates LILRB1 as a new disease gene for autoimmunity.

Analysis of cutaneous bacterial microbiota of Thai patients with seborrheic dermatitis.

Seborrheic dermatitis (SD) is a chronic inflammatory skin condition that occurs in body areas that contain profuse sebaceous glands. Skin microbiota are diverse across ethnic groups and its dysbiosis has been implicated in the pathogenesis of SD. Here, we reported the contribution of cutaneous bacterial microbiota to SD in the Thai population. Healthy individuals and patients with scalp SD were recruited into the study. Normal skin, scalp skin lesion (SL) and non-lesion sites (SNL) samples were collected using a tape stripping method and next-generation sequencing of 16S rRNA for microbiome analysis. Although bacterial diversity in all sample groups was not statistically different, a population of bacteria commonly found on skin of scalp showed signs of dysbiosis. Apart from the reduction of Corynebacterium spp., SD-specific microbiota was dominated by Firmicutes at taxa level and Pseudomonas spp., Staphylococcus spp. and Micrococcus spp. at genus level. The dysbiosis of the skin microbiota in SD was specifically described as an alteration of bacteria populations commonly found on scalp skin, implying that managing and controlling the cutaneous bacterial microbiome can alleviate and prevent SD and pave the way for the development of new SD treatments.

The immediate patch test reaction to fragrance in patients with allergic contact dermatitis to fragrance: A prospective study.

BACKGROUND: Fragrance is one of the common causes of immediate contact reaction. Knowing the prevalence of a reaction in a given population enables prioritization of allergy screening. OBJECTIVE: The purpose of this study was to determine the prevalence of an immediate patch test reaction to fragrance in patients with fragrance allergic contact dermatitis. METHODS: This prospective study enrolled 291 patients who were given standard patch tests for allergic contact dermatitis. Those with positive reactions were then asked to undergo additional patch tests to assess both immediate and delayed reactions to 28 different fragrance substances. RESULTS: Cinnamic aldehyde and cinnamic alcohol were the most frequently encountered substances in positive immediate reactions and standard (delayed) patch test reactions. Immediate patch reactions to benzyl alcohol, sorbic acid, and coumarin were more frequently observed than standard patch test reactions. LIMITATIONS: Because of the small sample size of patients who agreed to continue further patch testing evaluation, a statistical association between patient characteristics and fragrance-positive patch test reactions was difficult to establish. CONCLUSIONS: In this population, cinnamic aldehyde and cinnamic alcohol were the most common fragrance allergens causing both immediate and delayed reactions, whereas reactions to benzyl alcohol, sorbic acid, and coumarin were frequently observed in immediate patch tests.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1ß), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

The chemotherapeutic drug carboplatin affects macrophage responses to LPS and LPS tolerance via epigenetic modifications.

Following re-exposure to lipopolysaccharide (LPS), macrophages exhibit an immunosuppressive state known as LPS tolerance, which is characterized by repressed proinflammatory cytokine production. LPS-induced tolerance in macrophages is mediated in part by epigenetic changes. Carboplatin, an anticancer chemotherapeutic drug, exerts its effect by inhibiting DNA replication and transcription, as well as through epigenetic modifications. Through an unbiased screen, we found that carboplatin rescued TNF-α and IL-6 production in LPS-tolerant macrophages. Transcriptomic analysis and gene set enrichment analyses revealed that p53 was one of the most significantly upregulated hallmarks in both LPS-primed and LPS-tolerant macrophages in the presence of carboplatin, while E2F and G2/M were the most negatively regulated hallmarks. Heterochromatin protein 1 (HP1-α), which is associated with gene silencing, was significantly reduced in carboplatin-treated LPS-tolerant macrophages at the mRNA and protein levels. Dynamic changes in the mRNA level of genes encoding H3K9me3 methyltransferases, setdb2, kdm4d, and suv39h1 were induced in the presence of carboplatin in LPS-tolerant macrophages. Taken together, we provide evidence that carboplatin treatment interferes with proinflammatory cytokine production during the acute LPS response and LPS tolerance in macrophages, possibly via H3K9me3 modification.

Identification and validation of circulating miRNAs as potential new biomarkers for severe liver disease in patients with leptospirosis.

BACKGROUND: Leptospirosis, a global zoonotic infectious disease, has various clinical manifestations ranging from mild self-limiting illness to life-threatening with multi-organ damage, including liver involvement. This study was aimed at identifying circulating microRNAs (miRNAs) as novel biomarkers for predicting severe liver involvement in patients with leptospirosis. METHODS: In a discovery set, 12 serum samples of patients with anicteric and icteric leptospirosis at initial clinical presentation were used for miRNA profiling by a NanoString nCounter miRNA assay. In a validated cohort, top candidate miRNAs were selected and further tested by qRT-PCR in serum samples of 81 and 16 individuals with anicteric and icteric leptospirosis, respectively. RESULTS: The discovery set identified 38 significantly differential expression miRNAs between the two groups. Among these, miR-601 and miR-630 were selected as the top two candidates significantly up-regulated expressed in the icteric group. The enriched KEGG pathway showed that these miRNAs were mainly involved in immune responses and inflammation. In the validated cohort, miR-601 and miR-630 levels were significantly higher in the icteric group compared with the anicteric group. Additionally, these two miRNAs displayed good predictors of subsequent acute liver failure with a high sensitivity of 100%. On regression analysis, elevated miR-601 and miR-630 expression were also predictive of multi-organ failures and poor overall survival. CONCLUSION: Our data indicated that miRNA expression profiles were significantly differentiated between the icteric and anicteric groups. Serum miR-601 and miR-630 at presentation could potentially serve as promising biomarkers for predicting subsequent acute liver failure and overall survival in patients with leptospirosis.

MicroRNA-378a-3p is overexpressed in psoriasis and modulates cell cycle arrest in keratinocytes via targeting BMP2 gene.

Psoriasis is a chronic autoimmune skin disease driven by dysregulations at the cellular, genomic and genetic levels. MicroRNAs are key mediators of gene expression regulation. However, how microRNAs control the pathogenesis of psoriasis is still unclear. Here, we reported a significant up-regulation of miR-378a-3p (miR-378a) in skin biopsies from active psoriatic lesions while it was down-regulated after treatment with methotrexate or narrow-band ultraviolet B phototherapy. Using the keratinocyte in vitro model, we showed that miR-378a disturbed the cell cycle progression, causing cell cycle arrest at G1 phase. Transcriptomic analysis of keratinocytes with miR-378a overexpression and depletion revealed several important biological mechanisms related to inflammation and tight junction. Target mRNA transcript assessed by luciferase assay identified bone morphogenetic protein 2 as a novel target gene of miR-378a. These findings offer a mechanistic model where miR-378a contributes to the pathogenesis of psoriasis.

Novel Analytical Platform For Robust Identification of Cell Migration Inhibitors.

Wound healing assay is a simple and cost-effective in vitro assay for assessing therapeutic impacts on cell migration. Its key limitation is the possible confoundment by other cellular phenotypes, causing misinterpretation of the experimental outcome. In this study, we attempted to address this problem by developing a simple analytical approach for scoring therapeutic influences on both cell migration and cell death, while normalizing the influence of cell growth using Mitomycin C pre-treatment. By carefully mapping the relationship between cell death and wound closure rate, contribution of cell death and cell migration on the observed wound closure delay can be quantitatively separated at all drug dosing. We showed that both intrinsic cell motility difference and extrinsic factors such as cell seeding density can significantly affect final interpretation of therapeutic impacts on cellular phenotypes. Such discrepancy can be rectified by using the actual wound closure time of each treatment condition for the calculation of phenotypic scores. Finally, we demonstrated a screen for strong pharmaceutical inhibitors of cell migration in cholangiocarcinoma cell lines. Our approach enables accurate scoring of both migrastatic and cytotoxic effects, and can be easily implemented for high-throughput drug screening.

Interaction Between Dendritic Cells and Candida krusei ß-Glucan Partially Depends on Dectin-1 and It Promotes High IL-10 Production by T Cells.

Host-Candida interaction has been broadly studied during Candida albicans infection, with a progressive shift in focus toward non-albicans Candida species. C. krusei is an emerging multidrug resistant pathogen causing rising morbidity and mortality worldwide. Therefore, understanding the interplay between the host immune system and C. krusei is critically important. Candia cell wall ß-glucans play significant roles in the induction of host protective immune responses. However, it remains unclear how C. krusei ß-glucan impacts dendritic cell (DC) responses. In this study, we investigated DC maturation and function in response to ß-glucans isolated from the cell walls of C. albicans, C. tropicalis, and C. krusei. These three distinct Candida ß-glucans had differential effects on expression of the DC marker, CD11c, and on DC maturation. Furthermore, bone-marrow derived DCs (BMDCs) showed enhanced cytokine responses characterized by substantial interleukin (IL)-10 production following C. krusei ß-glucan stimulation. BMDCs stimulated with C. krusei ß-glucan augmented IL-10 production by T cells in tandem with increased IL-10 production by BMDCs. Inhibition of dectin-1 ligation demonstrated that the interactions between dectin-1 on DCs and cell wall ß-glucans varied depending on the Candida species. The effects of C. krusei ß-glucan were partially dependent on dectin-1, and this dependence, in part, led to distinct DC responses. Our study provides new insights into immune regulation by C. krusei cell wall components. These data may be of use in the development of new clinical approaches for treatment of patients with C. krusei infection.