Vitamin C facilitates direct cardiac reprogramming by inhibiting reactive oxygen species.

BACKGROUND: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction.

BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.

Hierarchical deployment of Tbx3 dictates the identity of hypothalamic KNDy neurons to control puberty onset.

The neuroendocrine system consists of a heterogeneous collection of neuropeptidergic neurons in the brain, among which hypothalamic KNDy neurons represent an indispensable cell subtype controlling puberty onset. Although neural progenitors and neuronal precursors along the cell lineage hierarchy adopt a cascade diversification strategy to generate hypothalamic neuronal heterogeneity, the cellular logic operating within the lineage to specify a subtype of neuroendocrine neurons remains unclear. As human genetic studies have recently established a link between TBX3 mutations and delayed puberty onset, we systematically studied Tbx3-derived neuronal lineage and Tbx3-dependent neuronal specification and found that Tbx3 hierarchically established and maintained the identity of KNDy neurons for triggering puberty. Apart from the well-established lineage-dependent fate determination, we uncovered rules of interlineage interaction and intralineage retention operating through neuronal differentiation in the absence of Tbx3. Moreover, we revealed that human TBX3 mutations disturbed the phase separation of encoded proteins and impaired transcriptional regulation of key neuropeptides, providing a pathological mechanism underlying TBX3-associated puberty disorders.

The comparison between the effect of Glycyrrhizae uralensis (Gan-Cao) and Montelukast on the expression of T-bet and GATA-3 genes in children with allergic asthma.

Pediatric allergic asthma is a chronic disease that affects the lungs and airways. If a child is exposed to certain stimulants such as pollen inhalation, colds, or respiratory infections, the lungs become inflamed and if left untreated can lead to dangerous asthma attacks. One of the most important treatments for this disease is the use of leukotriene modulators, such as montelukast. But recently, due to easier access, cheaper prices and fewer side effects, attention has shifted to non-chemical treatments. Gan-Cao (Glycyrrhizae uralensis), as traditional Chinese medicine, has been proved to have a good therapeutic effect on experimental allergic asthma. But its anti-asthma mechanism is currently unclear. Therefore, the study aimed the comparison between the effect of Gan-Cao and montelukast on the expression of T-bet and GATA-3 genes in children with allergic asthma. For this purpose, fifty children with allergic asthma were divided into two groups. The first group was treated with montelukast for one month. The second group was treated with Gan-Cao root extract. Then the peripheral blood mononuclear cells were isolated, their RNA was extracted, and the relative expression of T-bet and GATA3 transcription factors was evaluated by Real-time PCR. The relationship between them and risk factors for asthma was assessed by relevant statistical tests. The result showed the expression of the GATA3 gene (P = 0.102), T-bet gene (P = 0.888), and the expression ratio of T-bet/GATA-3 genes (P = 0.061) was not significantly different between the two groups. It showed that Gan-Cao can affect the expression of these genes just as much as montelukast. Therefore, this Chinese herb can be used as an alternative or supplement medicine to treat allergic asthma in children.

Dietary zinc supplementation rescues fear-based learning and synaptic function in the Tbr1+/- mouse model of autism spectrum disorders.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a dyad of behavioural symptoms-social and communication deficits and repetitive behaviours. Multiple aetiological genetic and environmental factors have been identified as causing or increasing the likelihood of ASD, including serum zinc deficiency. Our previous studies revealed that dietary zinc supplementation can normalise impaired social behaviours, excessive grooming, and heightened anxiety in a Shank3 mouse model of ASD, as well as the amelioration of synapse dysfunction. Here, we have examined the efficacy and breadth of dietary zinc supplementation as an effective therapeutic strategy utilising a non-Shank-related mouse model of ASD-mice with Tbr1 haploinsufficiency. METHODS: We performed behavioural assays, amygdalar slice whole-cell patch-clamp electrophysiology, and immunohistochemistry to characterise the synaptic mechanisms underlying the ASD-associated behavioural deficits observed in Tbr1+/- mice and the therapeutic potential of dietary zinc supplementation. Two-way analysis of variance (ANOVA) with Sídák's post hoc test and one-way ANOVA with Tukey's post hoc multiple comparisons were performed for statistical analysis. RESULTS: Our data show that dietary zinc supplementation prevents impairments in auditory fear memory and social interaction, but not social novelty, in the Tbr1+/- mice. Tbr1 haploinsufficiency did not induce excessive grooming nor elevate anxiety in mice. At the synaptic level, dietary zinc supplementation reversed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) hypofunction and normalised presynaptic function at thalamic-lateral amygdala (LA) synapses that are crucial for auditory fear memory. In addition, the zinc supplemented diet significantly restored the synaptic puncta density of the GluN1 subunit essential for functional NMDARs as well as SHANK3 expression in both the basal and lateral amygdala (BLA) of Tbr1+/- mice. LIMITATIONS: The therapeutic effect of dietary zinc supplementation observed in rodent models may not reproduce the same effects in human patients. The effect of dietary zinc supplementation on synaptic function in other brain structures affected by Tbr1 haploinsufficiency including olfactory bulb and anterior commissure will also need to be examined. CONCLUSIONS: Our data further the understanding of the molecular mechanisms underlying the effect of dietary zinc supplementation and verify the efficacy and breadth of its application as a potential treatment strategy for ASD.

The Purinergic Receptor P2X4 Promotes Th17 Activation and the Development of Arthritis.

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.

YY1 regulation by miR-124-3p promotes Th17 cell pathogenicity through interaction with T-bet in rheumatoid arthritis.

Th17 cells are involved in rheumatoid arthritis (RA) pathogenesis. Our previous studies have revealed that transcription factor Yin Yang 1 (YY1) plays an important role in the pathogenic mechanisms of RA. However, whether YY1 has any role in Th17 cell pathogenicity and what molecular regulatory mechanism is involved remain poorly understood. Here, we found the proportion of pathogenic Th17 (pTh17) cells was significantly higher in RA than in control individuals and showed a potential relationship with YY1 expression. In addition, we also observed YY1 expression was increased in pTh17, and the pTh17 differentiation was hampered by YY1 knockdown. Consistently, knockdown of YY1 decreased the proportion of pTh17 cells and attenuated joint inflammation in collagen-induced arthritis mice. Mechanistically, YY1 could regulate the pathogenicity of Th17 cells through binding to the promoter region of transcription factor T-bet and interacting with T-bet protein. This function of YY1 for promoting pTh17 differentiation was specific to Th17 cells and not to Th1 cells. Moreover, we found miR-124-3p negatively correlated with YY1 in RA patients, and it could bind to 3'-UTR regions of YY1 to inhibit the posttranscriptional translation of YY1. Altogether, these findings indicate YY1 regulation by miR-124-3p could specifically promote Th17 cell pathogenicity in part through interaction with T-bet, and these findings present promising therapeutic targets in RA.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Eomesodermin in CD4+T cells is essential for Ginkgolide K ameliorating disease progression in experimental autoimmune encephalomyelitis.

Eomesodermin (Eomes), a transcription factor, could suppress the Th17 cell differentiation and proliferation through directly binding to the promoter zone of the Rorc and Il17a gene, meanwhile the expression of Eomes is suppressed when c-Jun directly binds to its promoter zone. Ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) is a diterpene lactone isolated from the leaves of Ginkgo biloba. A previous study indicated that GK could decrease the level of phospho JNK (c-Jun N-terminal kinase). Here, we reported the therapeutic potential of Ginkgolide K (GK) treatment to ameliorate experimental autoimmune encephalomyelitis (EAE) disease progression. Methods: EAE was induced in both wildtype and CD4-Eomes conditional knockout mice. GK was injected intraperitoneally. Disease severity, inflammation, and tissue damage were assessed by clinical evaluation, flow cytometry of mononuclear cells (MNCs), and histopathological evaluation. Dual-luciferase reporter assays were performed to measure Eomes transcription activity in vitro. The potency of GK (IC50) was determined using JNK1 Kinase Enzyme System. Results: We revealed that GK could ameliorate EAE disease progression by the inhibition of the Th17 cells. Further mechanism studies demonstrated that the level of phospho JNK was decreased and the level of Eomes in CD4+T cells was dramatically increased. This therapeutic effect of GK was almost completely interrupted in CD4-Eomes conditional knockout mice. Conclusions: These results provided the therapeutic potential of GK treatment in EAE, and further suggested that Eomes expression in CD4+T cells might be essential in this process.

Therapeutic effects and mechanisms study of Hanchuan Zupa Granule in a Guinea pig model of cough variant asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Hanchuan Zupa Granule (HCZP), a traditional Chinese ethnodrug, has the functions of supressing a cough, resolving phlegm, warming the lungs, and relieving asthma. In clinical practice employing traditional Chinese medicine (TCM), HCZP is commonly used to treat acute colds, cough and abnormal mucous asthma caused by a cold, or "Nai-Zi-Lai" in the Uygur language. Studies have confirmed the use of HCZP to treat cough variant asthma (CVA) and other respiratory diseases. However, the pharmacological mechanisms of HCZP remain unrevealed. AIM OF THE STUDY: To investigate the anti-tussive and anti-asthmatic effects and the possible pharmacological mechanisms of HCZP in the treatment of CVA. MATERIALS AND METHODS: A guinea pig CVA animal model was established by intraperitoneal injection of ovalbumin (OVA) combined with intraperitoneal injection of aluminium hydroxide adjuvant and atomized OVA. Meanwhile, guinea pigs with CVA received oral HCZP (at dosages of 0.571, 0.285 and 0.143 g/kg bodyweight). The number of coughs induced by aerosol capsaicin was recorded, and the airway hyperresponsiveness (AHR) of CVA guinea pigs was detected with the FinePointe series RC system. H&E staining of lung tissues was performed to observe pathological changes. ELISA was used to detect inflammatory cytokines. qRT-PCR and western blotting analyses were used to detect the expression of Th1-specific transcription factor (T-bet), Th2-specific transcription factor (GATA3), and Toll-like receptor 4 (TLR4) signal transduction elements. These methods were performed to assess the protective effects and the potential mechanisms of HCZP on CVA. RESULTS: Great changes were found in the CVA guinea pig model after HCZP treatment. The number of coughs induced by capsaicin in guinea pigs decreased, the body weights of guinea pigs increased, and inflammation of the eosinophilic airway and AHR were reduced simultaneously. These results indicate that HCZP has a significant protective effect on CVA. A pharmacological study of HCZP showed that the levels of interleukin-4 (IL-4) and IL-5 and tumour necrosis factor-α (TNF-α) in serum decreased. The amount of interferon-γ (IFN-γ) increased, mRNA and protein expression of TLR4 and GATA3 weakened, and mRNA and protein expression of T-bet increased. CONCLUSIONS: HCZP ameliorated the symptoms of guinea pigs with CVA induced by OVA by regulating the Th1/Th2 imbalance and TLR4 receptors.

Enhancing WNT Signaling Restores Cortical Neuronal Spine Maturation and Synaptogenesis in Tbr1 Mutants.

Tbr1 is a high-confidence autism spectrum disorder (ASD) gene encoding a transcription factor with distinct pre- and postnatal functions. Postnatally, Tbr1 conditional knockout (CKO) mutants and constitutive heterozygotes have immature dendritic spines and reduced synaptic density. Tbr1 regulates expression of several genes that underlie synaptic defects, including a kinesin (Kif1a) and a WNT-signaling ligand (Wnt7b). Furthermore, Tbr1 mutant corticothalamic neurons have reduced thalamic axonal arborization. LiCl and a GSK3ß inhibitor, two WNT-signaling agonists, robustly rescue the dendritic spines and the synaptic and axonal defects, suggesting that this could have relevance for therapeutic approaches in some forms of ASD.

Central neurogenetic signatures of the visuomotor integration system.

Visuomotor impairments characterize numerous neurological disorders and neurogenetic syndromes, such as autism spectrum disorder (ASD) and Dravet, Fragile X, Prader-Willi, Turner, and Williams syndromes. Despite recent advances in systems neuroscience, the biological basis underlying visuomotor functional impairments associated with these clinical conditions is poorly understood. In this study, we used neuroimaging connectomic approaches to map the visuomotor integration (VMI) system in the human brain and investigated the topology approximation of the VMI network to the Allen Human Brain Atlas, a whole-brain transcriptome-wide atlas of cortical genetic expression. We found the genetic expression of four genes-TBR1, SCN1A, MAGEL2, and CACNB4-to be prominently associated with visuomotor integrators in the human cortex. TBR1 gene transcripts, an ASD gene whose expression is related to neural development of the cortex and the hippocampus, showed a central spatial allocation within the VMI system. Our findings delineate gene expression traits underlying the VMI system in the human cortex, where specific genes, such as TBR1, are likely to play a central role in its neuronal organization, as well as on specific phenotypes of neurogenetic syndromes.

Moringa oleifera treatment increases Tbet expression in CD4+ T cells and remediates immune defects of malnutrition in Plasmodium chabaudi-infected mice.

BACKGROUND: Malaria is a worldwide problem that affects millions of people yearly. In rural areas where anti-malarial drugs are not easily accessible, many people use herbal treatments, such as Moringa oleifera, to treat a variety of diseases and ailments including malaria. While Moringa is reported to possess potent and curative anti-malarial properties, previous studies have mostly been restricted to assessment of parasitaemia. In this study, the effect of Moringa on malaria immunity in a murine model was investigated. METHODS: Using a high dose (60 mg/mouse) for a short time (7 days) or low dose Moringa (30 mg/mouse) for a longer time (3 weeks), cytokine production, and Tbet expression by effector CD4+ T cells (Teff) were determined. Mice were also treated with Moringa after infection (curatively) or before infection (prophylactically) to determine the effect of the plant extract on parasitaemia and immunity. Given that Moringa also possess many nutritional benefits, the contribution of Moringa on malnourished malaria infected mice was determined. Malnutrition was induced by limiting access to food to only 4 h a day for 4 weeks, while control mice had unlimited access to mouse laboratory chow. All data was collected by flow cytometry and analysed using one-Way ANOVA or two tailed Student's t test. RESULTS: Moringa-treated mice had increased numbers of effector CD4+ T cells accompanied by an increase in Tbet expression compared to control untreated mice. Mice that were treated with Moringa curatively also exhibited increased effector CD4+ T cell numbers, IFN-gamma and TNF secretion. Interestingly, the mice that were treated prophylactically had significantly higher Tbet expression. In the absence of adaptive immunity, high parasitaemia was observed in the RAG1 knockout mice. The food limited mice (malnourished) had reduced numbers of CD4+ T cells, TNF proportions, and significantly greater Tbet expression compared to the control group. Supplementation with Moringa in the limited group slightly restored CD4+ T cell activation, IL-2, and IL-10 production. CONCLUSIONS: Taken together, these data suggest that Moringa treatment leads to increased CD4+ T cell activation, Th1 differentiation and production of pro-inflammatory cytokines after malaria infection. Thus, Moringa may be immunologically useful in the treatment of malaria and malnutrition. Further investigations are required to identify the active components in Moringa.

Browning Effects of a Chronic Pterostilbene Supplementation in Mice Fed a High-Fat Diet.

Obesity and related comorbidities are a major health concern. The drugs used to treat these conditions are largely inadequate or dangerous, and a well-researched approach based on nutraceuticals would be highly useful. Pterostilbene (Pt), i.e., 3,5-dimethylresveratrol, has been reported to be effective in animal models of obesity, acting on different metabolic pathways. We investigate here its ability to induce browning of white adipose tissue. Pt (5 µM) was first tested on 3T3-L1 mature adipocytes, and then it was administered (352 µmol/kg/day) to mice fed an obesogenic high-fat diet (HFD) for 30 weeks, starting at weaning. In the cultured adipocytes, the treatment elicited a significant increase of the levels of Uncoupling Protein 1 (UCP1) protein-a key component of thermogenic, energy-dissipating beige/brown adipocytes. In vivo administration antagonized weight increase, more so in males than in females. Analysis of inguinal White Adipose Tissue (WAT) revealed a trend towards browning, with significantly increased transcription of several marker genes (Cidea, Ebf2, Pgc1α, PPARγ, Sirt1, and Tbx1) and an increase in UCP1 protein levels, which, however, did not achieve significance. Given the lack of known side effects of Pt, this study strengthens the candidacy of this natural phenol as an anti-obesity nutraceutical.

Methylation of H3K27 and H3K4 in key gene promoter regions of thymus in RA mice is involved in the abnormal development and differentiation of iNKT cells.

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.

miR-29 family regulates the puberty onset mediated by a novel Gnrh1 transcription factor TBX21.

Gonadotropin-releasing hormone (GnRH) is the ultimate signal by which the neuroendocrine system controls the puberty onset and fertility in mammals. The pulsatile release of GnRH is regulated by numerous extracellular and intracellular factors, including miRNAs. Here, we report a novel regulation mechanism mediated by miR-29 family. We found that the absence of miR-29s resulted in elevated expression of Gnrh1 in GT1-7 cells. Through in silico and wet analysis, we identified Tbx21, a target gene of miR-29, as the main effector. As a transcription activator, TBX21 stimulates the expression of Gnrh1 directly by binding to its promoter region, and indirectly by activating the expression of Dlx1, another transcription activator of Gnrh1. Stereotactic brain infusion of miR-29 inhibitor into the hypothalamus caused earlier puberty onset in prepubertal female mice than that of intact controls. The female mice with ectopic expression of Tbx21 in the hypothalamus were affected in both puberty onset and fertility, as they had higher level of serum LH and FSH, larger litter size but steeper decline of fertility compared with those of controls. Our results revealed that miR-29-3p and its target Tbx21 played a role in regulating the mammalian puberty onset and reproduction by modulating the Gnrh1 expression.

Inhibition of interferon-γ production and T-bet expression by menthol treatment of human peripheral blood mononuclear cells.

Context: Mentha longifolia (L.) Huds., has shown anti-inflammatory effects. Objective: To evaluate the immunomodulatory effects of menthol, the major constituent of Mentha longifolia on T cells as the main cells affecting the inflammatory responses. Methods: Effect of menthol on: proliferation and viability of the peripheral blood human mononuclear cells (PBMCs) by BrdU and propidium iodide (PI) staining, respectively, interferone (IFN)γ and interleukin (IL)-4 cytokine production in lymphocytes stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate/calcium ionophore (PMA/CI) by ELISA; intracellular staining of CD4+ cells for IFNγ expression by flow cytometry and gene expressions of T heper (Th) cell transcription factors was measured using real time-PCR. Results: Menthol dose-dependently inhibited lymphocytes proliferation from 88.7% at 50 µg/ml to 3.63% at 800 µg/ml (p < .05). According to the results of PI staining, this inhibitory effect was not due to cell death. Menthol dose-dependently decreased IFNγ but not IL-4 production in culture of PHA- and PMA/CI-stimulated lymphocytes to more than 80% at 800 µg/ml. In flow cytometry analysis, menthol reduced the number of IFN-γ-expressing CD4+T cells stimulated either with PHA or PMA/CI. Treatment of PBMCs with 800 µg/ml of menthol decreased levels of T-bet from 14.5 ± 2.26 fold in untreated control to 2.76 ± 1.74 fold (p < .001). Foxp-3 expression decreased to nearly half, but GATA3 did not significantly change. Ratios of T-bet to GATA3 and T-bet to Foxp3 gene expressions were dose-dependently declined. Conclusion: Decreased IFNγ expression plus T-bet down-regulation suggested the inhibitory effect of menthol on Th1 cells differentiation and hence imply its possible therapeutic usefulness in inflammatory diseases.

Histone Acetylation of Immune Regulatory Genes in Human Placenta in Association with Maternal Intake of Olive Oil and Fish Consumption.

Maternal diet modifies epigenetic programming in offspring, a potentially critical factor in the immune dysregulation of modern societies. We previously found that prenatal fish oil supplementation affects neonatal T-cell histone acetylation of genes implicated in adaptive immunity including PRKCZ, IL13, and TBX21. In this study, we measured H3 and H4 histone acetylation levels by chromatin immunoprecipitation in 173 term placentas collected in the prospective birth cohort, ALADDIN, in which information on lifestyle and diet is thoroughly recorded. In anthroposophic families, regular olive oil usage during pregnancy was associated with increased H3 acetylation at FOXP3 (p = 0.004), IL10RA (p = 0.008), and IL7R (p = 0.007) promoters, which remained significant after adjustment by offspring gender. Furthermore, maternal fish consumption was associated with increased H4 acetylation at the CD14 gene in placentas of female offspring (p = 0.009). In conclusion, prenatal olive oil intake can affect placental histone acetylation in immune regulatory genes, confirming previously observed pro-acetylation effects of olive oil polyphenols. The association with fish consumption may implicate ω-3 polyunsaturated fatty acids present in fish oil. Altered histone acetylation in placentas from mothers who regularly include fish or olive oil in their diets could influence immune priming in the newborn.

Artemisinin enhances the anti-tumor immune response in 4T1 breast cancer cells in vitro and in vivo.

BACKGROUND: Breast cancer is a prominent cause of death among women worldwide. Recent studies have demonstrated that artemisinin (ART) displays anti-tumor activity. Using a mouse breast cancer model, we investigated the effects of ART in vitro and in vivo to determine how it influences the anti-tumor immune response. METHODS: We measured the proliferation and apoptosis of 4T1 cells in vitro after ART treatment by MTT assay and FACS. To examine the effects of ART in vivo, tumor volumes and survival rates were measured in 4T1 tumor-bearing mice. FACS was used to determine the frequencies of Tregs, MDSCs, CD4+ IFN-γ+ T cells, and CTLs in the tumors and spleens of the mice. mRNA levels of the transcription factors T-bet and FOXP3 and cytokines IFN-γ, TNF-α, TGF-ß, and IL-10 were also determined by real-time RT-PCR. ELISA was used to measure TGF-ß protein levels in the cell culture supernatants. RESULTS: ART supplementation significantly increased 4T1 cell apoptosis and decreased TGF-ß levels in vitro. ART also impeded tumor growth in 4T1 TB mice and extended their survival. MDSC and Treg frequencies significantly decreased in the 4T1 TB mice after ART treatment while CD4+ IFN-γ+ T cells and CTLs significantly increased. ART significantly increased T-bet, IFN-γ, and TNF-α mRNA levels within the tumor and significantly decreased TGF-ß mRNA levels. CONCLUSION: ART supplementation hindered 4T1 tumor growth in vivo by promoting T cell activation and quelling immunosuppression from Tregs and MDSCs in the tumor.

Effect of ginger extract on expression of GATA3, T-bet and ROR-γt in peripheral blood mononuclear cells of patients with Allergic Asthma.

INTRODUCTION AND OBJECTIVES: Allergic asthma is a chronic inflammatory disorder of the airways. Th1, Th2 and Th17 cells are the main cells involved in the pathophysiology of asthma. The function of these cells is affected by T-bet, GATA3 and RORγt transcription factors (respectively). Therefore, the aim of this study was to evaluate the effect of ginger (officinal Roscoe) extract on the expression of T-bet, GATA-3 and ROR-γ in peripheral blood mononuclear cells (PBMC) of asthmatic patients, in comparison with healthy volunteers as controls. MATERIALS AND METHODS: In this case-control study, a total of 50 individuals including 25 patients with severe, moderate and mild allergic asthma and 25 unrelated healthy controls were involved. The PBMCs were isolated and divided into four groups: negative control, two positive controls (Budesonide and PHA) and ginger-extract treated group. After cell treatment and incubation for 48h, PBMCs were isolated and cDNA was synthesized. Gene expressions of T-bet, GATA3 and ROR-γt were evaluated by Real-time PCR. RESULTS: According to the results of this study, hydroalcoholic extract of ginger could reduce the expression of GATA-3, ROR-γt, and T-bet in PBMCs of asthmatic patients in comparison with untreated PBMCs (P values=0.001, 0.001, and 0.002, respectively). It was also shown that the ginger extract could affect T-bet/GATA-3, T-bet/ROR-γt, and ROR-γt/GATA-3 expression ratios. CONCLUSIONS: This study showed that the use of ginger extract could control asthma and decrease the severity of this disease by affecting the main cells involving the symptoms of asthma in the airways.