Bifidobacterium longum 1714 improves sleep quality and aspects of well-being in healthy adults: a randomized, double-blind, placebo-controlled clinical trial.

Stress and sleep are linked with overall well-being. Bifidobacterium longum 1714 has been shown to influence stress responses and modulate neural responses during social stress, and influence sleep quality during examination stress in healthy adults. Here, we explored the ability of this strain to alter sleep quality in adults using subjective and objective measures. Eighty-nine adults (18-45y) with impaired sleep quality assessed with the Pittsburgh Sleep Quality Index (PSQI) and with a global score ≥ 5 were randomized to receive B. longum 1714 or placebo daily for eight weeks. Assessing the effect of the strain on PSQI global score was the primary objective. Secondary objectives assessed sleep quality and well-being subjectively and sleep parameters using actigraphy objectively. While PSQI global score improved in both groups, B. longum 1714 significantly improved the PSQI component of sleep quality (p < 0.05) and daytime dysfunction due to sleepiness (p < 0.05) after 4 weeks and social functioning (p < 0.05) and energy/vitality (p < 0.05) after 8 weeks, compared to placebo. No significant effect on actigraphy measures were observed. The 1714 strain had a mild effect on sleep, demonstrated by a faster improvement in sleep quality at week 4 compared to placebo, although overall improvements after 8 weeks were similar in both groups. B. longum 1714 improved social functioning and increased energy/vitality in line with previous work that showed the strain modulated neural activity which correlated with enhanced vitality/reduced mental fatigue (ClinicalTrials.gov: NCT04167475).

Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells by WNT Switch Method.

The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe the "WNT Switch" method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious.

Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Dnmt3bas coordinates transcriptional induction and alternative exon inclusion to promote catalytically active Dnmt3b expression.

Embryonic expression of DNMT3B is critical for establishing de novo DNA methylation. This study uncovers the mechanism through which the promoter-associated long non-coding RNA (lncRNA) Dnmt3bas controls the induction and alternative splicing of Dnmt3b during embryonic stem cell (ESC) differentiation. Dnmt3bas recruits the PRC2 (polycomb repressive complex 2) at cis-regulatory elements of the Dnmt3b gene expressed at a basal level. Correspondingly, Dnmt3bas knockdown enhances Dnmt3b transcriptional induction, whereas overexpression of Dnmt3bas dampens it. Dnmt3b induction coincides with exon inclusion, switching the predominant isoform from the inactive Dnmt3b6 to the active Dnmt3b1. Intriguingly, overexpressing Dnmt3bas further enhances the Dnmt3b1:Dnmt3b6 ratio, attributed to its interaction with hnRNPL (heterogeneous nuclear ribonucleoprotein L), a splicing factor that promotes exon inclusion. Our data suggest that Dnmt3bas coordinates alternative splicing and transcriptional induction of Dnmt3b by facilitating the hnRNPL and RNA polymerase II (RNA Pol II) interaction at the Dnmt3b promoter. This dual mechanism precisely regulates the expression of catalytically active DNMT3B, ensuring fidelity and specificity of de novo DNA methylation.

Interactions between symptoms and psychological status in irritable bowel syndrome: An exploratory study of the impact of a probiotic combination.

BACKGROUND: Stress is an exacerbator of irritable bowel syndrome (IBS) symptoms, and anxiety and depression are co-morbidities. Bifidobacterium longum strains 1714® and 35642® attenuate stress responses in healthy people and reduce symptoms in IBS, respectively. Here, we explore relationships between the psychological and visceral effects of the two strains (COMBO) in IBS subjects and biomarkers of stress and inflammation. METHODS: We recruited 40 patients with IBS (Rome III) and mild to moderate anxiety (HADS-A) and/or depression (HADS-D) and 57 asymptomatic female controls with low or moderate stress. IBS patients were fed COMBO (1 × 109 cfu/day) for 8 weeks with an 8-week washout. IBS symptoms, psychometric measures, salivary cortisol awakening response (CAR), and plasma inflammatory biomarkers were assessed every 4 weeks. KEY RESULTS: Compared to healthy controls, IBS subjects had a blunted CAR. Treatment with COMBO restored CAR and improved IBS symptoms compared to baseline during the treatment phase. The COMBO reduced HADS-D, HADS-A score, and TNF-α, while sleep quality improved significantly from baseline to the end of the intervention. Surprisingly, these parameters improved further once treatment ended and maintained this improvement by Week 16. CONCLUSIONS AND INFERENCES: These findings suggest that the stress response is a major driver of IBS symptoms. The time course of the beneficial effect of COMBO on IBS symptoms suggests that this is achieved through a restoration of the stress response. In contrast, the time course of the effects of COMBO on anxiety and depression in IBS paralleled an anti-inflammatory effect as indicated by a reduction in circulating levels of TNF-α.

Allergic Rhinitis: A Clinical and Pathophysiological Overview.

Allergic rhinitis (AR) represents a global health concern where it affects approximately 400 million people worldwide. The prevalence of AR has increased over the years along with increased urbanization and environmental pollutants thought to be some of the leading causes of the disease. Understanding the pathophysiology of AR is crucial in the development of novel therapies to treat this incurable disease that often comorbids with other airway diseases. Hence in this mini review, we summarize the well-established yet vital aspects of AR. These include the epidemiology, clinical and laboratory diagnostic criteria, AR in pediatrics, pathophysiology of AR, Th2 responses in the disease, as well as pharmacological and immunomodulating therapies for AR patients.

IL-4/IL-13 Axis in Allergic Rhinitis: Elevated Serum Cytokines Levels and Inverse Association With Tight Junction Molecules Expression.

The IL-4/IL-13 axis is involved in the pathogenesis of allergic rhinitis (AR). In this study, we investigated the serum cytokines levels of IL-4, IL-5, IL-6, and IL-13 in AR patients, and the transcript expression levels of their receptors (i.e. IL4R, IL5RA, IL6R, and IL13RA1) in nasal epithelial cells of AR patients versus non-allergic controls. Nasal epithelial cells and blood samples of non-allergic controls (n = 30) and AR patients (n = 30) were collected to examine mRNA expression and serum cytokines levels, respectively. Bioinformatics analyses of IL-4/IL-13 receptor heterodimer association with tight junction (TJ) and JAK/STAT signaling genes were conducted in a gene expression profiling (GEP) dataset (GSE44037) of AR patients (n = 12) and healthy controls (n = 6). Serum IL-4, IL-5, IL-6 or IL-13 levels, and IL13RA1 transcript expression were significantly higher in AR patients compared with non-allergic controls. IL-4 and IL-13 serum levels were positively correlated with IL13RA1 expression in AR patients but not in non-allergic controls. In the GEP dataset (GSE44037), six TJ (CLDN4, CLDN7, CLDN12, CLDN15, TJP1, and TJP2) genes' expressions were negatively correlated, respectively, with IL-4Rα/IL-13Rα1 heterodimeric receptor expression in AR patients and not in control samples. These six TJ genes contributed to the significant enrichment of tight junction Gene Ontology (GO ID: 0070160). Lastly, STATs DNA binding motif analysis showed that each of these TJ genes contains STATs binding consensus sequence within intronic and intergenic regions. Our results suggest that increased IL-4/IL-13 serum cytokines levels may contribute to decreased TJs expression via IL-4Rα/IL-13Rα1 heterodimeric receptor in nasal epithelium of AR patients.

Higher Wheal Sizes of Dermatophagoides farinae Sensitization Exhibit Worse Nasal Symptoms in Allergic Rhinitis Patients.

Allergic rhinitis (AR) is a global health burden and it manifests in both nasal and non-nasal symptoms. Skin prick test (SPT) is a routine procedure to diagnose AR sensitized to common allergens including house dust mites (HDMs). The degree of sensitivity of a patient toward allergens is determined by the size of the wheal formed by SPT procedure. SPT wheal sizes are influenced by recent anti-histamine usage, however it remains unclear if SPT wheal sizes are also influenced by other factors. In this study, we set out to investigate the association between SPT wheal sizes with the demographical, clinical and environmental characteristics, as well as nasal and non-nasal symptoms severity scores, of AR patients (n = 30) sensitized to common HDMs (i.e., Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis). We showed that SPT wheal sizes of HDM allergens were not associated with clinical, demographical and environmental characteristics examined. Nonetheless, significant correlations were observed between SPT wheal sizes of D. farinae sensitization with worse severity scores of all five nasal symptoms examined (i.e., sneezing, runny nose, itchy nose, congestion and postnasal drip) and four of the six non-nasal symptoms examined (i.e., throat symptoms, ear symptoms, headache and mental function). Such relationships were not observed in SPT wheal sizes of D. pteronyssinus and B. tropicalis sensitization. We suggest that increased SPT wheal sizes for D. farinae sensitization may predict the likelihood of more severe nasal and, to a lesser extent, non-nasal manifestations in AR patients.

Nasal Epithelial Barrier Integrity and Tight Junctions Disruption in Allergic Rhinitis: Overview and Pathogenic Insights.

Allergic rhinitis (AR) is a common disorder affecting up to 40% of the population worldwide and it usually persists throughout life. Nasal epithelial barrier constitutes the first line of defense against invasion of harmful pathogens or aeroallergens. Cell junctions comprising of tight junctions (TJs), adherens junctions, desmosomes and hemidesmosomes form the nasal epithelial barrier. Impairment of TJ molecules plays causative roles in the pathogenesis of AR. In this review, we describe and discuss the components of TJs and their disruption leading to development of AR, as well as regulation of TJs expression by epigenetic changes, neuro-immune interaction, epithelial-derived cytokines (thymic stromal lymphopoietin, IL-25 and IL-33), T helper 2 (Th2) cytokines (IL-4, IL-5, IL-6 and IL-13) and innate lymphoid cells. These growing evidence support the development of novel therapeutic approaches to restore nasal epithelial TJs expression in AR patients.

Reduced occludin and claudin-7 expression is associated with urban locations and exposure to second-hand smoke in allergic rhinitis patients.

The breakdown of nasal epithelial barrier occurs in allergic rhinitis (AR) patients. Impairment of cell junction molecules including tight junctions (TJs) and desmosomes plays causative roles in the pathogenesis of AR. In this study, we investigated the transcript expression levels of TJs including occludin (OCLN), claudin-3 and -7 (CLDN3 and CLDN7), desmoglein 3 (DSG3) and thymic stromal lymphopoietin (TSLP) in AR patients (n = 30) and non-allergic controls (n = 30). Nasal epithelial cells of non-allergic controls and AR patients were collected to examine their mRNA expression levels, and to correlate with clinico-demographical and environmental parameters. We demonstrated that the expression of OCLN (p = 0.009), CLDN3 (p = 0.032) or CLDN7 (p = 0.004) transcript was significantly lower in AR patients compared with non-allergic controls. No significant difference was observed in the expression of DSG3 (p = 0.750) or TSLP (p = 0.991) transcript in AR patients compared with non-allergic controls. A significant association between urban locations and lower OCLN expression (p = 0.010), or exposure to second-hand smoke with lower CLDN7 expression (p = 0.042) was found in AR patients. Interestingly, none of the TJs expression was significantly associated with having pets, frequency of changing bedsheet and housekeeping. These results suggest that defective nasal epithelial barrier in AR patients is attributable to reduced expression of OCLN and CLDN7 associated with urban locations and exposure to second-hand smoke, supporting recent findings that air pollution represents one of the causes of AR.

A profile of TNFR2+ regulatory T cells and CD103+ dendritic cells in the peripheral blood of patients with asthma.

The interaction of tolerogenic CD103+ dendritic cells (DCs) with regulatory T (Tregs) cells modulates immune responses by inducing immune tolerance. Hence, we determined the proportion of these cells in the peripheral blood mononuclear cells (PBMC) of asthmatic patients. We observed lower trends of CD11b-CD103+ DCs and CD86 within CD11b-CD103+ DCs, while increased levels of Foxp3 expressing CD25+/-TNFR2+ cells in asthmatics. There was a positive correlation in the expression of Foxp3 within CD3+CD4+CD25+TNFR2+ Tregs and CD11b-CD103+ as well as the expression of CD86 within HLA-DR+CD11c+CD11b-CD103+ DCs. In conclusion, we suggest that the increased levels of Tregs in blood could continuously suppress the T helper 2 (Th2) cells activation in the circulation which is also supported by the increase of anti-inflammatory cytokines IL-10 and TNF. Overall, functional immunoregulation of the regulatory cells, particularly Tregs, exhibit immune suppression and induce immune tolerance linked with the immune activation by the antigen presenting cells (APC).

Tight junction, mucin, and inflammasome-related molecules are differentially expressed in eosinophilic, mixed, and neutrophilic experimental asthma in mice.

BACKGROUND: Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct in vivo comparison has not been performed. METHODS: We developed mouse models representing three different phenotypes of allergic airway inflammation-eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT-PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air-liquid interface were used to study the mechanisms revealed in the in vivo models. RESULTS: By whole-genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome-related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo-1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation. Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1, and IL-1ß was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine-IL-1ß-may transiently impair epithelial barrier function, while IL-1ß and IL-17 increase mucin expressions in primary human bronchial epithelial cells. CONCLUSION: Our findings suggest that differential expression of TJ, mucin, and inflammasome-related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic.

Reduced CD4+ terminally differentiated effector memory T cells in moderate-severe house dust mites sensitized allergic rhinitis patients.

BACKGROUND: Terminally differentiated effector memory (TEMRA) T cells exert potent effector function after activation. The proportions of CD4+ T cell subsets especially memory cells in allergic rhinitis (AR) patients sensitized to house dust mites (HDMs) have not been extensively studied. OBJECTIVE: This study aimed to compare the mean percentages and absolute counts of CD4+ memory T cell subsets between: (i) non-allergic controls and AR patients; (ii) mild AR patients and moderate-severe AR patients. METHODS: Sensitization to Dermatophagoides farinae and Dermatophagoides pteronyssinus were determined in 33 non -allergic controls, 28 mild AR and 29 moderate-severe AR patients. Flow cytometry was used to determine the percentage of CD4+ na?ve (TN; CD45RA+CCR7+), central memory (TCM; CD45RA-CCR7+), effector memory (TEM; CD45RA-CCR7-) and TEMRA (CD45RA+CCR7-) T cells from the peripheral blood. The absolute counts of CD4+ T cell subsets were obtained by dual platform method from flow cytometer and hematology analyzer. RESULTS: There were no significant differences in the mean percentages and absolute counts of CD4+ T cell subsets between non-allergic controls and AR patients sensitized to HDMs. However, there were significant reduction in the mean percentage (p=0.0307) and absolute count (p=0.0309) of CD4+ TEMRA cells in moderate-severe AR patients compared to mild AR patients sensitized to HDMs and 13/24 (54.2%) moderate-severe AR patients sensitized to HDMs had persistent symptoms. CONCLUSION: Reduction in the mean percentage and absolute count of CD4+CD45RA+CCR7- TEMRA cells were observed in moderate-severe AR patients compared to mild AR patients in our population of AR patients sensitized to HDMs.

Inhibitors targeting CDK4/6, PARP and PI3K in breast cancer: a review.

Breast cancer is the global leading cause of cancer-related death in women and it represents a major health burden worldwide. One of the promising breast cancer therapeutic avenues is through small molecule inhibitors (SMIs) which have undergone rapid progress with successful clinical trials. Recently, three emerging and vital groups of proteins are targeted by SMIs for breast cancer treatment, namely cyclin-dependent kinase 4 and 6 (CDK4/6), poly (adenosine diphosphate-ribose) polymerase (PARP) and phosphoinositide 3-kinase (PI3K). Several of these inhibitors have been approved for the treatment of breast cancer patients or progressed into late-stage clinical trials. Thus, modeling from these successful clinical trials, as well as their limitations, is pivotal for future development and trials of other inhibitors or therapeutic regimens targeting breast cancer patients. In this review, we discuss eight recently approved or novel SMIs against CDK4/6 (palbociclib, ribociclib and abemaciclib), PARP (olaparib, veliparib and talazoparib), and PI3K (buparlisib and alpelisib). The mechanisms of action, series of clinical trials and limitations are described for each inhibitor.

Ozone exposure induces respiratory barrier biphasic injury and inflammation controlled by IL-33.

BACKGROUND: IL-33 plays a critical role in regulation of tissue homeostasis, injury, and repair. Whether IL-33 regulates neutrophil recruitment and functions independently of airways hyperresponsiveness (AHR) in the setting of ozone-induced lung injury and inflammation is unclear. OBJECTIVE: We sought to examine the role of the IL-33/ST2 axis in lung inflammation on acute ozone exposure in mice. METHODS: ST2- and Il33-deficient, IL-33 citrine reporter, and C57BL/6 (wild-type) mice underwent a single ozone exposure (1 ppm for 1 hour) in all studies. Cell recruitment in lung tissue and the bronchoalveolar space, inflammatory parameters, epithelial barrier damage, and airway hyperresponsiveness (AHR) were determined. RESULTS: We report that a single ozone exposure causes rapid disruption of the epithelial barrier within 1 hour, followed by a second phase of respiratory barrier injury with increased neutrophil recruitment, reactive oxygen species production, AHR, and IL-33 expression in epithelial and myeloid cells in wild-type mice. In the absence of IL-33 or IL-33 receptor/ST2, epithelial cell injury with protein leak and myeloid cell recruitment and inflammation are further increased, whereas the tight junction proteins E-cadherin and zonula occludens 1 and reactive oxygen species expression in neutrophils and AHR are diminished. ST2 neutralization recapitulated the enhanced ozone-induced neutrophilic inflammation. However, myeloid cell depletion using GR-1 antibody reduced ozone-induced lung inflammation, epithelial cell injury, and protein leak, whereas administration of recombinant mouse IL-33 reduced neutrophil recruitment in Il33-deficient mice. CONCLUSION: Data demonstrate that ozone causes an immediate barrier injury that precedes myeloid cell-mediated inflammatory injury under the control of the IL-33/ST2 axis. Thus IL-33/ST2 signaling is critical for maintenance of intact epithelial barrier and inflammation.

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties.

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs.

Novel Biologicals for the Treatment of Allergic Diseases and Asthma.

PURPOSE OF REVIEW: The development of biological therapies has rapidly progressed during the last few years, and major advances were reported for the treatment of allergic diseases, such as atopic dermatitis, allergic rhinitis, urticaria, food allergy, and asthma. Here, we review biologicals targeting the type 2 immune response involving Th2 cells, type 2 innate lymphoid cells, natural killer T cells, mast cells, basophils, and epithelial cells, such as IL-4, IL-5, IL-13, IL-31, tumor necrosis factor alpha (TNF-α), and thymic stromal lymphopoietin (TSLP). RECENT FINDINGS: The biologicals that have been currently approved for asthma are omalizumab targeting IgE and reslizumab and mepolizumab targeting interleukin (IL)-5. Many other monoclonal antibodies are currently in various phases of clinical development. The new biological therapies for allergic diseases will eventually be tailored to the endotypes of these diseases and the identification of novel biomarkers. Further development of novel biologicals for the treatment of allergic diseases and asthma will be possible upon improved understanding of mechanisms of allergic diseases. Accordingly, further refinement of endotypes of allergen-specific and non-specific type 2 immune response and related inflammatory mediators is needed for optimal treatment of allergic diseases.