Baicalein alleviates cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway.

BACKGROUND: The experimental autoimmune myocarditis (EAM) model is valuable for investigating myocarditis pathogenesis. M1-type macrophages and CD4+T cells exert key pathogenic effects on EAM initiation and progression. Baicalein (5,6,7-trihydroxyflavone, C15H10O5, BAI), which is derived from the Scutellaria baicalensis root, is a primary bioactive compound with potent anti-inflammatory and antioxidant properties. BAI exerts good therapeutic effects against various autoimmune diseases; however, its effect in EAM has not been thoroughly researched. PURPOSE: This study aimed to explore the possible inhibitory effect of BAI on M1 macrophage polarisation and CD4+T cell differentiation into Th1 cells via modulation of the JAK-STAT1/4 signalling pathway, which reduces the secretion of pro-inflammatory factors, namely, TNF-α and IFN-γ, and consequently inhibits TNF-α- and IFN-γ-triggered apoptosis in cardiomyocytes of the EAM model mice. STUDY DESIGN AND METHODS: Flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (q-PCR), and western blotting were performed to determine whether BAI alleviated M1/Th1-secreted TNF-α- and IFN-γ-induced myocyte death in the EAM model mice through the inhibition of the JAK-STAT1/4 signalling pathway. RESULTS: These results indicate that BAI intervention in mice resulted in mild inflammatory infiltrates. BAI inhibited JAK-STAT1 signalling in macrophages both in vivo and in vitro, which attenuated macrophage polarisation to the M1 type and reduced TNF-α secretion. Additionally, BAI significantly inhibited the differentiation of CD4+T cells to Th1 cells and IFN-γ secretion both in vivo and in vitro by modulating the JAK-STAT1/4 signalling pathway. This ultimately led to decreased TNF-α and IFN-γ levels in cardiac tissues and reduced myocardial cell apoptosis. CONCLUSION: This study demonstrates that BAI alleviates M1/Th1-secreted TNF-α- and IFN-γ-induced cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway.

Cordycepin prevents and ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte infiltration and reducing neuroinflammation.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease characterized by multifocal perivascular infiltration of immune cells in the central nervous system (CNS). Cordycepin (3'-deoxyadenosine), an adenosine analogue initially extracted from the fungus Cordyceps militarisa, is one of the candidates that has multiple actions. We investigated that cordycepin attenuated the activation of LPS-induced mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) through the inhibition of the AKT, ERK, NFκB, and ROS pathways and impaired the migration of BMDCs through the downregulation of adhesion molecules and chemokine receptors in vitro. In experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with cordycepin decreased the expression of trafficking factors in the CNS, inhibited the secretion of inflammatory cytokines (IFN-γ, IL-6, TNF-α, and IL-17), and attenuated disease symptoms. A chemokine array indicated that cordycepin treatment reversed the high levels of CCL6, PARRES2, IL-16, CXCL10, and CCL12 in the brain and spinal cord of EAE mice, consistent with the RNA-seq data. Moreover, cordycepin suppressed the release of neuroinflammatory cytokines by activated microglial cells, macrophages, Th17 cells, Tc1 cells, and Th1 cells in vitro. Furthermore, cordycepin treatment exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that cordycepin treatment may not only prevent the occurrence of MS by inhibiting DC activation and migration but also potentially ameliorates the progression of MS by reducing neuroinflammation, which may provide insights into the development of new approaches for the treatment of MS.

Effects of Molecular Iodine/Chemotherapy in the Immune Component of Breast Cancer Tumoral Microenvironment.

Molecular iodine (I2) induces apoptotic, antiangiogenic, and antiproliferative effects in breast cancer cells. Little is known about its effects on the tumor immune microenvironment. We studied the effect of oral (5 mg/day) I2 supplementation alone (I2) or together with conventional chemotherapy (Cht+I2) on the immune component of breast cancer tumors from a previously published pilot study conducted in Mexico. RNA-seq, I2 and Cht+I2 samples showed significant increases in the expression of Th1 and Th17 pathways. Tumor immune composition determined by deconvolution analysis revealed significant increases in M0 macrophages and B lymphocytes in both I2 groups. Real-time RT-PCR showed that I2 tumors overexpress T-BET (p = 0.019) and interferon-gamma (IFNγ; p = 0.020) and silence tumor growth factor-beta (TGFß; p = 0.049), whereas in Cht+I2 tumors, GATA3 is silenced (p = 0.014). Preliminary methylation analysis shows that I2 activates IFNγ gene promoter (by increasing its unmethylated form) and silences TGFß in Cht+I2. In conclusion, our data showed that I2 supplements induce the activation of the immune response and that when combined with Cht, the Th1 pathways are stimulated. The molecular mechanisms involved in these responses are being analyzed, but preliminary data suggest that methylation/demethylation mechanisms could also participate.

Gomisin M2 alleviates psoriasis­like skin inflammation by inhibiting inflammatory signaling pathways.

Psoriasis, a chronic inflammatory skin disease, is characterized by the excessive proliferation and impaired differentiation of epidermal keratinocytes and is accompanied by the increased infiltration of inflammatory cells. The condition requires long­term treatment and has no definitive cure. Hence, supplements and therapeutic agents have been intensely investigated. Gomisin M2 (GM2), a lignan extracted from Schisandra chinensis (Turcz). Baill. (Schisandraceae; S. chinensis), has demonstrated diverse pharmacological properties, including anticancer, anti­inflammatory and antiallergic effects. Based on these findings, the present study examined the effects of GM2 on an imiquimod (IMQ)­induced psoriasis mouse model and on keratinocytes stimulated by tumor necrosis factor (TNF)­α and interferon­Î³. IMQ was topically applied to the back skin of mice for 7 consecutive days, and the mice were orally administered CD. These results showed that the oral administration of GM2 suppressed the symptoms of psoriasis, as evidenced by reductions in skin thickness, psoriasis area severity index scores for psoriasis lesions, transepidermal water loss and myeloperoxidase (MPO)­associated cell infiltration. Furthermore, GM2 reduced the pathologically increased levels of immunoglobulin G2a, MPO and TNF­α in the serum and T helper (Th)1 and Th17 cell populations in the spleen. GM2 decreased the gene expression of inflammatory­related cytokines and chemokines and inhibited the expression of signal transducer and activator of transcription 1 and nuclear factor­κB in the activated keratinocytes. These results suggested that GM2 from S. chinensis is a potential therapeutic candidate to alleviate psoriasis­like skin inflammation.

Schisandrin B promotes TH1 cell differentiation by targeting STAT1.

Schisandrin B (Sch B) is the major active ingredient of the traditional Chinese medicine Schisandra chinensis and has antitumor activity, anti-inflammatory activity. CD4+ Th subsets orchestrate immune responses to plenty of pathogen infections and participate in the pathogenesis of many immune-related diseases. However, little is known about the relationship between Sch B and T cell differentiation. Here, we showed that Sch B might participate in T cell receptor signaling pathway by using the TCMIO database. Importantly, Sch B promoted TH1 cell differentiation. Furthermore, Sch B did not affect TH2 cell and Treg differentiation. Mechanismly, Sch B increased the level of IFN-γ of CD4+ T cells by upregulating the phosphorylation of STAT1 protein. Then, STAT1 promoted T-bet expression in CD4+ T cells. In conclusion, Sch B modulates the differentiation of naïve CD4+ T cells into TH1 subset by STAT1/T-bet signaling, which may have the potential for the treatment of T cell-mediated-immune diseases.

A Perspective on Withania somnifera Modulating Antitumor Immunity in Targeting Prostate Cancer.

Medicinal plants serve as a lead source of bioactive compounds and have been an integral part of day-to-day life in treating various disease conditions since ancient times. Withaferin A (WFA), a bioactive ingredient of Withania somnifera, has been used for health and medicinal purposes for its adaptogenic, anti-inflammatory, and anticancer properties long before the published literature came into existence. Nearly 25% of pharmaceutical drugs are derived from medicinal plants, classified as dietary supplements. The bioactive compounds in these supplements may serve as chemotherapeutic substances competent to inhibit or reverse the process of carcinogenesis. The role of WFA is appreciated to polarize tumor-suppressive Th1-type immune response inducing natural killer cell activity and may provide an opportunity to manipulate the tumor microenvironment at an early stage to inhibit tumor progression. This article signifies the cumulative information about the role of WFA in modulating antitumor immunity and its potential in targeting prostate cancer.

Yiqi Jiemin decoction alleviates allergic rhinitis in a guinea pig model by suppressing inflammation, restoring Th1/Th2 balance, and improving cellular metabolism.

We investigated the mechanisms underlying the therapeutic effects of Yiqi Jiemin decoction (YJD), a traditional Chinese medicine (TCM), in the ovalbumin (OVA)-induced allergic rhinitis (AR) model in guinea pigs. YJD significantly decreased infiltration of mast cells and eosinophils into the nasal mucosa of AR model guinea pigs. YJD also increased expression of TGF-ß in the nasal mucosa, restored the balance of Th1/Th2 immune cell responses, and decreased serum levels of various pro-inflammatory mediators, including histamine (HA), neuropeptide Y (NPY), acetylcholine (ACH), norepinephrine and immunoglobulin E (IgE). Metabolic analyses using liquid chromatography coupled with high-resolution mass spectrometry revealed that YJD improved cellular metabolism in AR model guinea pigs and increased serum levels of glycocholic acid while decreasing levels 1-palmitoyl lysophosphatidic acid. RNA-sequencing analysis identified BPIFB2 as a potential diagnostic biomarker and therapeutic target for AR. Functional enrichment analyses showed that YJD significantly inhibited cytokine secretion pathways in AR model guinea pigs. These findings demonstrate that YJD protects against OVA-induced AR in guinea pigs by suppressing inflammation in the nasal mucosa, restoring Th1/Th2 balance, and improving cellular metabolism.

Evaluation of Th1 and Th2 mediated cellular and humoral immunity in patients with COVID-19 following the use of melatonin as an adjunctive treatment.

Coronavirus (SARS-CoV-2) is spreading rapidly in the world and is still taking a heavy toll. Studies show that cytokine storms and imbalances in T-helper (Th)1/Th2 play a significant role in most acute cases of the disease. A number of medications have been suggested to treat or control the disease but have been discontinued due to their side effects. Melatonin, as an intrinsic molecule, possesses pharmacological anti-inflammatory and antioxidant properties that decreases in concentration with age; as a result, older people are more prone to various diseases. In this study, patients who were hospitalized with a diagnosis of coronavirus disease 2019 (COVID-19) were given a melatonin adjuvant (9 mg daily, orally) for fourteen days. In order to measure markers of Th1 and Th2 inflammatory cytokines (such as interleukin (IL)-2, IL-4, and interferon (IFN)-γ) as well as the expression of Th1 and Th2 regulatory genes (signal transducer and activator of transcription (STAT)4, STAT6, GATA binding protein 3 (GATA3), and T-box expressed in T cell (T-bet)), blood samples were taken from patients at the beginning and end of the treatment. Adjuvant therapy with melatonin controlled and reduced inflammatory cytokines in patients with COVID-19. Melatonin also controlled and modulated the dysregulated genes that regulate the humoral and cellular immune systems mediated by Th1 and Th2. In this study, it was shown for the first time that melatonin can be used as a medicinal adjuvant with anti-inflammatory mechanism to reduce and control inflammatory cytokines by regulating the expression of Th1 and Th2 regulatory genes in patients with COVID-19.

Kurarinone Attenuates Collagen-Induced Arthritis in Mice by Inhibiting Th1/Th17 Cell Responses and Oxidative Stress.

Kurarinone is a flavanone, extracted from Sophora flavescens Aiton, with multiple biological effects. Here, we determine the therapeutic potential of kurarinone and elucidate the interplay between kurarinone and the autoimmune disease rheumatoid arthritis (RA). Arthritis was recapitulated by induction of bovine collagen II (CII) in DBA/1 mice as a collagen-induced arthritis (CIA) model. After the establishment of the CIA, kurarinone was given orally from day 21 to 42 (100 mg/kg/day) followed by determination of the severity based on a symptom scoring scale and with histopathology. Levels of cytokines, anti-CII antibodies, and the proliferation and lineages of T cells from the draining lymph nodes were measured using ELISA and flow cytometry, respectively. The expressional changes, including STAT1, STAT3, Nrf2, KEAP-1, and heme oxygenase-1 (HO-1) changes in the paw tissues, were evaluated by Western blot assay. Oxidative stress featured with malondiadehyde (MDA) and hydrogen peroxide (H2O2) activities in paw tissues were also evaluated. Results showed that kurarinone treatment reduced arthritis severity of CIA mice, as well as their levels of proinflammatory cytokines, TNF-α, IL-6, IFN-γ, and IL-17A, in the serum and paw tissues. T cell proliferation was also reduced by kurarinone even under the stimulation of CII and anti-CD3 antibody. In addition, kurarinone reduced STAT1 and STAT3 phosphorylation and the proportions of Th1 and Th17 cells in lymph nodes. Moreover, kurarinone suppressed the production of MDA and H2O2. All while promoting enzymatic activities of key antioxidant enzymes, SOD and GSH-Px. In the paw tissues, upregulation of Nrf-2 and HO-1, and downregulation of KEAP-1 were observed. Overall, kurarinone showed an anti-inflammatory effect by inhibiting Th1 and Th17 cell differentiation and an antioxidant effect exerted in part through activating the Nrf-2/KEAP-1 pathway. These beneficial effects in CIA mice contributed to the amelioration of their arthritis, indicating that kurarinone might be an adjunct treatment option for rheumatoid arthritis.

Design and application of nanoparticles as vaccine adjuvants against human corona virus infection.

In recent years, some viruses have caused a grave crisis to global public health, especially the human coronavirus. A truly effective vaccine is therefore urgently needed. Vaccines should generally have two features: delivering antigens and modulating immunity. Adjuvants have an unshakable position in the battle against the virus. In addition to the perennial use of aluminium adjuvant, nanoparticles have become the developing adjuvant candidates due to their unique properties. Here we introduce several typical nanoparticles and their antivirus vaccine adjuvant applications. Finally, for the combating of the coronavirus, we propose several design points, hoping to provide ideas for the development of personalized vaccines and adjuvants and accelerate the clinical application of adjuvants.

Cadmium induces endoplasmic reticulum stress-mediated apoptosis in pig pancreas via the increase of Th1 cells.

Cadmium (Cd), an environmental pollutant, causes several adverse reactions in animals. High dose of Cd has serious cytotoxicities, including the induction of programmed cell necrosis, autophagy and apoptosis, which has aroused wide public concern. The balance of cytokine network is affected by Th1/Th2 balance which is closely related to immune response and the occurrence, development, treatment and outcome of various diseases. Cd can induce severe apoptosis, but the relationship between Cd induced apoptosis and Th1/Th2 balance has not been clarified. In this study, we established a pig Cd poisoning model, exposing to CdCl2 for 40 days (20 mg Cd/kg diet). Firstly, deviation of Th1/Th2 balance was observed by fluorescence staining, and apoptosis was observed by TUNEL staining. Then, real-time fluorescence quantitative analysis and Western blot were used to detect the expression of related proteins. The results show that Cd can interfere with the balance of Th1/Th2 and shift the balance towards Th1. In addition, through the experiments, we found that Cd exposure can increase the expression of glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78), marker proteins of unfolded protein response (UPR). Cd exposure can increase the expression of pancreatic endoplasmic reticulum kinase (PERK), CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF-6), cysteinyl aspartate specific proteinase (Caspase12), indicating the three branches (ATF6, PERK and IRE-1) of endoplasmic reticulum stress (ER-stress) were activated. Moreover, we found that the expression of pro-apoptosis genes in the downstream pathway of ER-stress increased. In summary, our results indicated that Cd exposure upregulated the expression of pro-apoptosis related genes and caused apoptosis via the activation of the ER-stress signaling pathways in pancreas cells. And these negative effects were correlated with the equilibrium drift of Th1/Th2, increase in the expression and secretion of Th1 cytokines.

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.

Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.

"Wang-Bi tablet, a patented Chinese medicine, maintains the balance of Th1/Th2 in mice with collagen-induced arthritis".

OBJECTIVE: To investigate the pharmacological mechanism of Wang-Bi tablets (WBTs), a Chinese patented medicine, in rheumatoid arthritis (RA) using mice with collagen-induced arthritis (CIA). METHODS: A mouse model of CIA was induced using bovine type Ⅱ collagen. WBT treatment was administered and efficacy was evaluated. The levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-4 (IL-4) were examined using an enzyme-linked immunosorbent assay, and the proportions of Th1 and Th2 were detected using flow cytometry. T-bet and GATA-binding protein 3 (GATA3) expression were demonstrated using Western blot analysis. RESULTS: Paw swelling and the arthritis index decreased significantly following WBT treatment. Histopathological analysis revealed markedly alleviated damage to synovium tissue in the WBT and methotrexate treatment groups. WBT regulated the production of IFN-γ, IL-2, and IL-4 and modulated Th1 and Th2 cell populations, which might have been induced by the attenuation of Th1 and Th2 cell differentiation through a decrease in the expression of T-bet and an increase in the expression of GATA3 in the synovial tissue in CIA mice. CONCLUSION: These results indicate that WBT may produce a therapeutic effect on CIA through maintaining the balance of Th1/Th2 cells, which could result in a decrease in the autoinflammatory disorder observed in RA.

Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).

Notch signaling pathway mediates the immunomodulatory mechanism of Yangfei Huoxue decoction alleviating bleomycin-induced pulmonary fibrosis in rats.

OBJECTIVE: To investigate the immunomodulatory mechanism by which Yangfei Huoxue decoction (YHD) alleviates bleomycin (BLM)-induced pulmonary fibrosis (PF) in rats. METHODS: Rats were randomly divided into two time-point groups (day 14 and 28), and each time-point group comprised the following six subgroups: control, BLM, dexamethasone (DXM), YHD high dose (YHD-H), YHD middle dose (YHD-M), and YHD low dose (YHD-L). Haematoxylin and eosin and Masson staining, flow cytometry, enzyme-linked immunosorbent assay, Western blotting and UPLC-QT of analyses were examined. RESULTS: The results showed that YHD reduced the degree of alveolar inflammation and fibrosis; downregulated the expression of CD28, CD80, CD86, Delta-like 1, Notch2, and Notch3; and upregulated the proportions of Th1/Th2 and Tc1/Tc2. The seven components of YHD were detected. CONCLUSION: The current study indicates that YHD mainly functions by regulating the immune system and that the molecular mechanism may be related to the regulation of the Notch signaling pathway.

In-vitro (2D and 3D cultures) and in-vivo cytotoxic properties of Zataria multiflora essential oil (ZEO) emulsion in breast and cervical cancer cells along with the investigation of immunomodulatory potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Zataria multiflora is an iranian valuable traditional plants, called Avishan Shirazi in Persian language used to reduce inflammation, spasm, pain, and cancer symptoms. Zataria essential oil (ZEO) is one of the essential oils possessing broad biological activities. AIM OF THE STUDY: The aim was to investigate the anticancer effects of ZEO both in-vitro and in-vivo using mouse mammary carcinoma 4T1 cell line and mouse cervical cancer TC1 cell line. MATERIAL AND METHODS: The in-vitro effects of ZEO on the proliferation of these cell lines were considered in 2D and 3D culture by MTT assay. In the following, to indicate death mode, fluorescence staining, AnnexinV/PI flowcytometry and caspase-3 activity assay of monolayer cells treated with ZEO was done. In order to evaluate the antitumor activities of ZEO, tumor-bearing BALB/c and C57BL/6 mice were intraperitoneally administered with ZEO and the immunomodulatory effects of ZEO were considered through cytokine assay. Additionally, hematobiochemical factors including aspartate aminotransferase and alanine aminotransferase were investigated to confirm the harmless effects of ZEO. RESULTS: The In-vitro results showed that treatment of cells with ZEO leads to significant inhibition of 4T1 and TC1 cell proliferation and apoptosis in monolayer cell culture (2D) and multicellular spheroids (3D). Based on In-vivo results, ZEO was effective in decreasing the tumor weight compared to the control. Furthermore, ZEO was effective in tilting the balance of cytokines in favor of T helper 1 through the increase in the secretion of TNF-α, IFN-γ, IL-2 and decrease in IL-4. During the treatment with ZEO, hematobiochemical factors of mice did not significantly change. CONCLUSION: the present study demonstrated that the ZEO has potent antiproliferative, apoptosis-inducing and immune system stimulant properties in breast and cervical cancer.

Bifidobacterium infantis Relieves Allergic Asthma in Mice by Regulating Th1/Th2.

BACKGROUND Bifidobacteria are among the probiotics used in treating intestinal diseases and are rarely used for allergic asthma treatment. The present study investigated the mechanism of B. infantis in treating allergic asthma in mice. MATERIAL AND METHODS A total of 40 male Balb/c mice were randomized into control, ovalbumin (OVA), montelukast (Mon), and B. infantis (B10) groups, and allergic asthma was induced in the OVA, Mon, and B10 groups. Airway reactivity was measured on day 29 by methacholine at various doses. The numbers of total cells and inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted by blood cell counter and Diff-Quik staining. Hematoxylin-eosin (HE) staining was performed to observe inflammatory cell infiltration in lung tissues. Total IgE and OVA-specific IgE in serum were measured by ELISA. Mucin 5AC expression was detected by Western blot to evaluate airway obstruction. The levels of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-13) cytokines in BALF and tissues were detected by ELISA and qRT-PCR, respectively. RESULTS The mice in the OVA group had airway hyperreactivity, while the symptoms in the B10 group and Mon group were effectively relieved. B10 reduced the number of inflammatory cells in BALF as well as inflammatory cell infiltration in tissues. Moreover, the levels of total serum IgE, OVA-specific IgE, and Mucin 5AC were increased in the OVA group, but were reduced in the Mon group and B10 group. B. infantis increased the levels of Th1 cytokines and decreased those of Th2 cytokines. CONCLUSIONS B. infantis can reduce the infiltration of inflammatory cells induced by OVA-specific antibodies in mice. B. infantis has therapeutic effects on allergic asthma by promoting Th1 and inhibiting Th2 immune responses.

A Solution with Ginseng Saponins and Selenium as Vaccine Diluent to Increase Th1/Th2 Immune Responses in Mice.

Pseudorabies is an important infectious disease of swine, and immunization using attenuated pseudorabies virus (aPrV) vaccine is a routine practice to control this disease in swine herds. This study was to evaluate a saline solution containing ginseng stem-leaf saponins (GSLS) and sodium selenite (Se) as a vaccine adjuvant for its enhancement of immune response to aPrV vaccine. The results showed that aPrV vaccine diluted with saline containing GSLS-Se (aP-GSe) induced significantly higher immune responses than that of the vaccine diluted with saline alone (aP-S). The aP-GSe promoted higher production of gB-specific IgG, IgG1, and IgG2a, neutralizing antibody titers, secretion of Th1-type (IFN-γ, IL-2, IL-12), and Th2-type (IL-4, IL-6, IL-10) cytokines, and upregulated the T-bet/GATA-3 mRNA expression when compared to aP-S. In addition, cytolytic activity of NK cells, lymphocyte proliferation, and CD4+/CD8+ ratio was also significantly increased by aP-GSe. More importantly, aP-GSe conferred a much higher resistance of mice to a field virulent pseudorabies virus (fPrV) challenge. As the present study was conducted in mice, further study is required to evaluate the aP-GSe to improve the vaccination against PrV in swine.