Insight into the long-term impact of birth weight on intestinal development, microbial settlement, and the metabolism of weaned piglets.

Infant mortality of low birth body weight (LBBW) piglets can reach 10% and is mainly due to gut and immune system immaturity which can lead to a higher risk in the long term. This study aimed to assess the impact of birth body weight (BBW) on piglet metabolism, gut status, and microbial profile from weaning to 21 d postweaning. At birth, 32 piglets were selected for their BBW and inserted into the normal BBW (NBBW:1.38 ±â€…0.09 g) or the LBBW (0.92 ±â€…0.07 g) group. The piglets were weighed weekly from weaning (d0) to d21. At d9 and d21, 8 piglets/group were slaughtered to obtain the distal jejunum for morphology, immunohistochemistry, and gene expression analysis, colon content for microbiota and short-chain fatty acid (SCFA) analysis, and intestinal content for pH measurement. Blood was collected for metabolomic, haptoglobin (Hp), and reactive oxygen metabolite (ROM) analysis. The LBBW group had a lower body weight (BW) throughout the study (P < 0.01), a lower average daily gain from d9-d21 (P = 0.002), and lower feed intake (P = 0.02). The LBBW piglets had lower Hp at d9 (P = 0.03), higher ROMs at d21 (P = 0.06), and a net alteration of the amino acid (AA) metabolism at d9 and d21. A higher expression of NFKB2 was observed in the LBBW piglets at d9 (P = 0.003) and d21 (P < 0.001). MYD88 expression was enhanced in NBBW piglets at d9 (P < 0.001). The LBBW piglets had a lower villus height, absorptive mucosal surface (P = 0.01), and villus height:crypt depth ratio (P = 0.02), and a greater number of T-lymphocytes in both the epithelium and the crypts (P < 0.001) at d21. At d21, the LBBW piglets had higher lactic acid, acetate, butyrate, and valerate, and also higher SCFA in the colon (P < 0.05). The LBBW piglets had a higher Shannon index (P = 0.01) at d9 and a higher abundance of SCFA-fermenting bacteria. In conclusion, the present study confirmed that LBBW could impact the gut mucosal structure, immunity, and inflammatory and oxidative status, leading to an altered AA metabolism, and delaying the recovery from weaning.

The drawback of the high prolificacy selection in the swine industry in the past decades is an increase in the number of piglets born with a low birth body weight (LBBW). This study aimed to assess performance, metabolism, gut status, and microbial profile in piglets born with low (0.92 ±â€…0.07 g) and normal birth body weight (1.38 ±â€…0.09 g). Piglets were weighed weekly from weaning (25 d) until 3 weeks postweaning (end of the trial). At d9 and d21, 8 piglets/group were slaughtered to obtain blood for metabolomic, haptoglobin, reactive oxygen metabolite analyses, colon content for microbiota and short-chain fatty acid, intestinal content for pH measurement, distal jejunum for morphology, immunohistochemistry, and gene expression. The LBBW resulted in lower body weight through the study (P < 0.001), lower average daily gain from d9 to d21 (P = 0.002), and lower feed intake (P = 0.02). The LBBW piglets had a lower villus height, absorptive mucosal surface (P = 0.01), and villus height:crypt depth ratio (P = 0.02), and a greater number of T-lymphocytes in both the epithelium and the crypts (P < 0.001) at d21. In conclusion, the present study confirmed that LBBW could impact the gut mucosal structure, immunity, and inflammatory and oxidative status, leading to an altered AA metabolism, and delaying the recovery from weaning.

Effect of high dose vitamin D3 on the HIV-1 reservoir: A pilot randomised controlled trial.

Introduction: Antiretroviral therapy for people living with HIV-1 must be taken lifelong due to the persistence of latent virus in long-lived and proliferating CD4+ T cells. Vitamin D3 is a steroidal gene transcription regulator which exerts diverse effects on immune and epithelial cells including reductions in CD4+ T cell proliferation and improvement in gut barrier integrity. We hypothesised that a high dose of vitamin D3 would reduce the size of the HIV-1 reservoir by reducing CD4+ T cell proliferation. Methods: We performed a randomised placebo-controlled trial evaluating the effect of 24 weeks of vitamin D3 (10,000 international units per day) on the HIV-1 reservoir and immunologic parameters in 30 adults on antiretroviral therapy; participants were followed for 12 weeks post-treatment. The primary endpoint was the effect on total HIV-1 DNA at week 24. Parameters were assessed using mixed-effects models. Results: We found no effect of vitamin D3 on the change in total HIV-1 DNA from week 0 to week 24 relative to placebo. There were also no changes in integrated HIV-1 DNA, 2-long-terminal repeat (2-LTR) circles or cell-associated HIV-1 RNA. Vitamin D3 induced a significant increase in the proportion of central memory CD4+ and CD8+ T cells, a reduction in the proportion of senescent CD8+ T cells and a reduction in the natural killer cell frequency at all time points including week 36, 12 weeks after the study drug cessation. At week 36, there was a significant reduction in total HIV-1 DNA relative to placebo and persistently elevated 25-hydroxyvitamin D levels. No significant safety issues were identified. Conclusions: Vitamin D3 administration had a significant impact on the T cell differentiation but overall effects on the HIV-1 reservoir were limited and a reduction in HIV-1 DNA was only seen following cessation of the study drug. Additional studies are required to determine whether the dose and duration of vitamin D3 can be optimised to promote a continued depletion of the HIV-1 reservoir over time. Trial registration: ClinicalTrials.gov NCT03426592.

Gastrodin synergistically increases migration of interleukin-13 receptor α2 chimeric antigen receptor T cell to the brain against glioblastoma multiforme: A preclinical study.

Therapy with chimeric antigen receptor T (CAR-T) cells involves using reformative T lymphocytes that have three domains, antigen recognition, transmembrane, and costimulating to achieve the therapeutic purpose. CAR-T therapy on malignant hematologic has been successful; however, its effectiveness in patients with solid tumors is still limited. Few studies exist confirming the efficacy of natural products on the function of CAR-T cells. The purpose of this study is to assess the effect of gastrodin (GAS) on CAR-T cells that target interleukin-13 receptor α2 antigen (IL-13Rα2 CAR-T) in the brain against glioblastoma multiforme. Migration of IL-13Rα2 CAR-T was evaluated using the Transwell assay. The effects of GAS on IL-13Rα2 CAR-T cells were assessed both in vitro and situ glioblastoma models. The cytoskeleton was stained with Fluorescein 5-isothiocyanate (FITC)-phalloidin. Cytokines expression in cells was determined by flow cytometry and ELISA assay. Western blotting was used to detect the S1P1 expression, and quantitative PCR assay was used to determine the IL-13Rα2 gene level. GAS increased the migratory and destructive capacity of IL-13Rα2 CAR-T cells with no effect on cytokine release. By increasing the expression of S1P1, GAS encouraged the entry of CAR-T cells into the brain and bone marrow. Transcriptomic analysis revealed that genes related to skeletal migration such as add2 and gng8 showed increased expression in GAS-treated CAR-T cells. We found that GAS synergistically improves the mobility of IL-13Rα2 CAR-T, enhancing their ability to recognize the tumor antigen of glioblastoma, which could be advantageous for the application of CAR-T for the treatment of solid tumors.

Adjuvant dendritic cell-based immunotherapy after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in patients with malignant peritoneal mesothelioma: a phase II clinical trial.

BACKGROUND: Malignant peritoneal mesothelioma (MPM) is an aggressive malignancy with a poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival outcomes, but recurrence rates remain high. Dendritic cell-based immunotherapy (DCBI) showed promising results in patients with pleural mesothelioma. The primary aim of this trial was to determine feasibility of adjuvant DCBI after CRS-HIPEC. METHODS: This open-label, single-center, phase II clinical trial, performed in the Erasmus MC Cancer Institute Rotterdam, the Netherlands, included patients with epithelioid MPM. 4-6 weeks before CRS-HIPEC leukapheresis was performed. 8-10 weeks after surgery, DCBI was administered three times biweekly. Feasibility was defined as administration of at least three adjuvant vaccinations in 75% of patients. Comprehensive immune cell profiling was performed on peripheral blood samples prior to and during treatment. RESULTS: All patients who received CRS-HIPEC (n=16) were successfully treated with adjuvant DCBI. No severe toxicity related to DCBI was observed. Median progression-free survival (PFS) was 12 months (IQR 5-23) and median overall survival was not reached. DCBI was associated with increased proliferation of circulating natural killer cells and CD4+ T-helper (Th) cells. Co-stimulatory molecules, including ICOS, HLA-DR, and CD28 were upregulated predominantly on memory or proliferating Th-cells and minimally on CD8+ cytotoxic T-lymphocytes (CTLs) after treatment. However, an increase in CD8+ terminally differentiated effector memory (Temra) cells positively correlated with PFS, whereas co-expression of ICOS and Ki67 on CTLs trended towards a positive correlation. CONCLUSIONS: Adjuvant DCBI after CRS-HIPEC in patients with MPM was feasible and safe, and showed promising survival outcomes. DCBI had an immune modulatory effect on lymphoid cells and induced memory T-cell activation. Moreover, an increase of CD8+ Temra cells was more pronounced in patients with longer PFS. These data provide rationale for future combination treatment strategies. TRIAL REGISTRATION NUMBER: NTR7060; Dutch Trial Register (NTR).

Immunoregulatory effects of Huaier (Trametes robiniophila Murr) and relevant clinical applications.

Huaier (Trametes robiniophila Murr) is a medicinal fungus of traditional Chinese medicine with more than 1000 years of history of clinical application. Its remarkable anticancer activities has led to its application in treating diverse malignancies. In recent years, the immunomodulatory effects of Huaier have been uncovered and proved to be beneficial in a plethora of immune-related diseases including cancer, nephropathy, asthma, etc. In this review, we comprehensively summarized the active components of Huaier, its regulatory activities on multifaceted aspects of the immune system, its application in various clinical settings as well as toxicologic evidence. Based on currently available literature, Huaier possesses broad-spectrum regulatory activities on various components of the innate and adaptive immune system, including macrophages, dendritic cells, natural killer cells, T and B lymphocytes, etc. Versatile immunologic reactions are under the regulation of Huaier from expression of damage-associated molecular patterns, immune cell activation and maturation to cell proliferation, differentiation, antibody production, expression of cytokines and chemokines and terminal intracellular signal transduction. Moreover, some modulatory activities of Huaier might be context-dependent, typically promoting the restoration toward normal physiological status. With excellent efficacy and minimal side effects, we foresee more extensive application of Huaier for treating immune-related disorders.

Surface TREM2 on circulating M-MDSCs as a novel prognostic factor for adults with treatment-naïve diffuse large B-cell lymphoma.

INTRODUCTION: Circulating monocytic myeloid-derived suppressive cells (M-MDSCs) are implicated as a poor prognostic factor and cause CAR T-cell failure in diffuse large B-cell lymphoma (DLBCL). Triggering receptors expressed on myeloid cells 2 (TREM2) are a transmembrane glycoprotein that polarize macrophages to anti-inflammation phenotype but have never been explored on M-MDSCs. This study aims to elucidate the expression and clinical impact of surface TREM2 on circulating M-MDSCs derived from DLBCL adults. METHODS: This prospective, observational study enrolled 100 adults with newly diagnosed and treatment-naïve DLBCL from May 2019 to October 2021. Human circulating M-MDSCs were obtained from freshly isolated peripheral blood, and each patient's surface-TREM2 level on M-MDSCs was normalized via a healthy control at the same performance of flow-cytometry analysis. Murine MDSCs derived from bone marrow (BM-MDSCs) were adopted to assess the link between Trem2 and cytotoxic T lymphocytes. RESULTS: More circulating M-MDSCs at diagnosis of DLBCL predicted worse progression-free (PFS) and overall survival (OS). Patients with higher IPI scores, bone marrow involvement, or lower absolute counts of CD4+ or CD8+ T cells in PB had significantly higher normalized TREM2 levels on M-MDSCs. Additionally, normalized TREM2 levels on M-MDSCs could be grouped into low (< 2%), medium (2-44%), or high (> 44%) levels, and a high normalized TREM2 level on M-MDSCs was proven as an independent prognostic factor for both PFS and OS via multivariate Cox regression analysis and associated with worst PFS and OS. Interestingly, normalized levels of surface TREM2 on M-MDSCs were negatively associated with absolute counts of PB CD8+ T cells and positively correlated with levels of intracellular arginase 1 (ARG1) within M-MDSCs. Wild-type BM-MDSCs had significantly higher mRNA levels of Arg1 and showed more prominent ability to suppress the proliferation of co-cultured CD8+ T cells than BM-MDSCs from Trem2 knockout mice, and the suppressive ability could be impaired by adding Arg1 inhibitors (CB1158) or supplementing L-arginine. CONCLUSION: In treatment-naïve DLBCL adults, a high surface-TREM2 level on circulating M-MDSCs is a poor prognostic factor for both PFS and OS and warrants further investigation for its potential as a novel target in immunotherapy.

Randomized, Double-Blind, Placebo-Controlled Trial of Vitamin D Supplementation in the Build-up Phase of House Dust Mite-Specific Immunotherapy.

PURPOSE: Vitamin D (VitD) is an immunomodulatory molecule capable of alleviating allergic symptoms. However, the effectiveness of allergen-specific immunotherapy (AIT) is not commonly evidenced in the early build-up phase. The aim of the study was to determine the potential of VitD supplementation in this treatment phase. METHODS: Thirty-four house dust mite (HDM)-allergic adult patients treated with subcutaneous AIT were randomized to receive VitD2 60,000 IU/week or placebo for 10 weeks and followed up for 10 weeks. The primary endpoints were the symptom-medication score (SMS) and the treatment response rate. The secondary endpoints were eosinophil count and levels of plasma IL-10, Der p 2-specific IgG4, and dysfunctional regulatory T (CRTH2+ Treg) cells. RESULTS: Of 34 patients, 15 in each group completed the study. Patients with VitD deficiency receiving a VitD supplement showed significantly lower mean change SMS than the placebo group in weeks 10 (mean difference -54.54%, P = 0.007) and 20 (mean difference -42.69%, P = 0.04). The percentage of treatment responders reached 78% and 50% in the VitD and placebo groups, respectively, and the effect remained in week 20 (89% and 60%). No significant difference was observed for the tested immunological read-outs, with the exception of the frequency of CRTH2+ Treg cells, which was remarkably reduced in the VitD-treated patients. Moreover, improvement in SMS was correlated to the number of CRTH2+ Treg cells. Our in vitro experiment indicated that VitD downregulated activation markers, whereas it improved the function of CRTH2+ Treg cells. CONCLUSIONS: VitD supplementation in the build-up phase of AIT could relieve symptoms and decrease Treg cell dysfunction, especially in patients with VitD deficiency.

Yi-Qi-Jian-Pi formula ameliorates immune function in acute-on-chronic liver failure by upregulating autophagy and mitochondrial biogenesis in CD8+ T lymphocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: A key event in the pathogenesis of acute-on-chronic liver failure (ACLF) is the imbalance in the systemic immune response; immunosuppression in patients with ACLF contributes to poor prognosis. The Yi-Qi-Jian-Pi formula (YQJPF) may improve T lymphocyte immune function in patients with ACLF. AIM OF THE STUDY: To investigate the immune mechanism of YQJPF in vivo and in vitro. MATERIALS AND METHODS: An ACLF rat model was established by injection of CCl4, lipopolysaccharide, and D-galactosamine. We examined the effect of different doses of YQJPF (6.43, 12.87, 25.74 g/kg) on liver injury and immune function in the ACLF rat model. Magnetic-activated cell sorting was used to sort the CD8+ T lymphocytes in the spleen for lymphocyte function detection. In primary CD8+ T lymphocytes and Jurkat cell lines, the expression of mitochondrial function and biogenesis and autophagy related markers were detected using molecular biological methods and flow cytometry analysis. RESULTS: YQJPF improved the peripheral blood lymphocyte count and proportion of CD8+ T lymphocytes in ACLF rats, increased pro-inflammatory factors (IL-2, IFN-λ, and TNF-α), and reduced anti-inflammatory factors (IL-10 and TGF ß1). YQJPF also improved metabolism and mitochondrial homeostasis in CD8+ T lymphocytes, alleviated lymphocyte immune dysfunction by promoting autophagy, upregulated mitochondrial biogenesis by promoting PGC-1α, NRF-1, and TFAM expression, and regulated the relationship between autophagy and mitochondrial biogenesis via PGC-1α. CONCLUSIONS: Our results suggest that YQJPF could improve immune function in a rat model of ACLF, possibly via affecting the homeostasis of lymphatic mitochondria in CD8+ T lymphocytes. YQJPF may enhance lymphocyte mitochondrial biosynthesis and promote lymphocyte autophagy. PGC-1α is a possible upstream regulatory target of YQJPF.

Impairment of Endogenous Synthesis of Omega-3 DHA Exacerbates T-Cell Inflammatory Responses.

Omega-3 (ω-3) polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are involved in numerous biological processes and have a range of health benefits. DHA is obtained through the action of elongases (ELOVLs) and desaturases, among which Elovl2 is the key enzyme involved in its synthesis, and can be further metabolized into several mediators that regulate the resolution of inflammation. Our group has recently reported that ELOVL2 deficient mice (Elovl2-/-) not only display reduced DHA levels in several tissues, but they also have higher pro-inflammatory responses in the brain, including the activation of innate immune cells such as macrophages. However, whether impaired synthesis of DHA affects cells of adaptive immunity, i.e., T lymphocytes, is unexplored. Here we show that Elovl2-/- mice have significantly higher lymphocytes in peripheral blood and that both CD8+ and CD4+ T cell subsets produce greater amounts of pro-inflammatory cytokines in both blood and spleen compared to wild type mice, with a higher percentage of cytotoxic CD8+ T cells (CTLs) as well as IFN-γ-producing Th1 and IL-17-producing Th17 CD4+ cells. Furthermore, we also found that DHA deficiency impacts the cross-talk between dendritic cells (DC) and T cells, inasmuch as mature DCs of Elovl2-/- mice bear higher expression of activation markers (CD80, CD86 and MHC-II) and enhance the polarization of Th1 and Th17 cells. Reintroducing DHA back into the diets of Elovl2-/- mice reversed the exacerbated immune responses observed in T cells. Hence, impairment of endogenous synthesis of DHA exacerbates T cell inflammatory responses, accounting for an important role of DHA in regulating adaptive immunity and in potentially counteracting T-cell-mediated chronic inflammation or autoimmunity.

Total glucosides of paeony alleviates scleroderma by inhibiting type I interferon responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Type I interferon (IFN) is believed to play a pathogenic role in systemic sclerosis (SSc, also called scleroderma), which is an autoimmune rheumatic disease. Our previous studies have found that Chinese medicine formula Si-Ni-San (SNS, composed of Glycyrrhiza uralensis Fisch., Bupleurum chinense DC., Paeonia lactiflora Pall., and Citrus aurantium L.) had inhibitory effects on type I IFN responses. Among these herbal products, Paeonia lactiflora Pall. has been traditionally used to treat inflammation-related diseases, yet its therapeutic effects against type I IFN-related diseases and potential bioactive ingredients are not characterized. AIM OF THE STUDY: We aim to identify bioactive ingredient with anti-type I IFN activity from herbal products in SNS and further elucidate its therapeutic effect against scleroderma and underlying mechanisms. MATERIALS AND METHODS: We constructed a Gaussia-luciferase (Gluc) reporter assay system to identify ingredients with anti-type I IFN activities from SNS. In RAW264.7 cells, real-time PCR (RT-PCR) and western blotting were used to investigate the induction of type I IFN pathway. Additionally, in a bleomycin (BLM)-induced experimental scleroderma model, the expression of fibrotic genes, type I IFN-related genes, inflammatory cytokines, and cytotoxic granules were measured by RT-PCR, and the histopathological changes were determined by H&E staining, Masson's staining and immunohistochemistry analysis. RESULTS: Our data demonstrated that total glucosides of paeony (TGP) was the bioactive component of SNS that selectively inhibited TLR3-mediated type I IFN responses and blocked type I IFN-induced downstream JAK-STAT signaling pathways. In the BLM-induced scleroderma mouse model, TGP ameliorated skin fibrosis by inhibiting multiple targets in the upstream and downstream of type I IFN signaling. Further research found that TGP hindered polarization of M2 macrophages and their profibrotic effects and reduced cytotoxic T lymphocytes and their cytotoxic granules by suppressing Cxcl9 and Cxcl10 in the skin tissue of scleroderma mice. CONCLUSIONS: Our study not only sheds novel lights into the immunoregulative effects of TGP but also provides convincing evidence to develop TGP-based therapies in the treatment of scleroderma and other autoimmune diseases associated with type I IFN signatures. CLASSIFICATION: Skin.

Polysaccharides from Astragalus membranaceus elicit T cell immunity by activation of human peripheral blood dendritic cells.

Astragalus membranaceus is a widely used herbal medicine in Asia. It has been recognized as possessing various biological properties, however, studies on the activity of the A. membranaceus polysaccharide (AMP), a major component of A. membranaceus, on human peripheral blood dendritic cells (PBDCs) have not been thoroughly investigated. In this study, we found that AMP induced changes in dendritic morphology and the upregulation of activation marker expression and inflammatory cytokine production in human blood monocyte-derived dendritic cells (MDDCs). The AMP promoted the activation of both blood dendritic cell antigen 1+ (BDCA1+) and BDCA3+ PBDCs. AMP-induced secretion of cytokines in the peripheral blood mononuclear cells (PBMCs) was mainly due to PBDCs. Finally, activated BDCA1+ and BDCA3+ PBDCs by AMP elicited proliferation and activation of autologous T cells, respectively. Hence, these data demonstrated that AMPs could activate dendritic and T cells in human blood, and may provide a new direction for the application of AMPs in the regulation of human immunity.

Enhanced efficacy of direct immunochemotherapy for hepatic cancer with image-guided intratumoral radiofrequency hyperthermia.

BACKGROUND: It is still a challenge to prevent tumor recurrence post radiofrequency ablation (RFA) of medium-to-large hepatocellular carcinomas (HCC). Immunochemotherapy, a combination of immunotherapy with chemotherapy, has demonstrated a great potential in augmenting the treatment efficacy for some malignancies. In this study, we validated the feasibility of using radiofrequency hyperthermia (RFH)-enhanced intratumoral immunochemotherapy of LTX-315 with liposomal doxorubicin for rat orthotopic HCC. METHODS: Different groups of luciferase-labeled rat HCC cells and rat orthotopic HCC models were treated by: (1) phosphate buffered saline; (2) RFH; (3) LTX-315; (4) RFH+LTX-315; (5) liposomal doxorubicin; (6) RFH+liposomal doxorubicin; (7) LTX-315+liposomal doxorubicin; and (8) RFH+LTX-315+liposomal doxorubicin. Cell viabilities and apoptosis of different treatment groups were compared. Changes in tumor sizes were quantified by optical and ultrasound imaging, which were confirmed by subsequent histopathology. The potential underlying biological mechanisms of the triple combination treatment (RFH+LTX-315+liposomal doxorubicin) were explored. RESULTS: Flow cytometry and MTS assay showed the highest percentage of apoptotic cells and lowest cell viability in the triple combination treatment group compared with other seven groups (p<0.001). Tumors in this group also presented the most profound decrease in bioluminescence signal intensities and the smallest tumor volumes compared with other seven groups (p<0.001). A significant increase of CD8+ T cells, CD8+/interferon (IFN)-γ+ T cells, CD8+/tumor necrosis factor (TNF)-α+ T cells, and natural killer cells, and a significant decrease of regulatory T cells were observed in the tumors (p<0.001). Meanwhile, a significantly higher level of Th1-type cytokines in both plasma (interleukin (IL)-2, IL-12, IL-18, IFN-γ) and tumors (IL-2, IL-18, IFN-γ, TNF-α), as well as a significantly lower Th2-type cytokines of IL-4 and IL-10 in plasma and tumor were detected. CONCLUSIONS: Intratumoral RFA-associated RFH could enhance the efficacy of immunochemotherapy of LTX-315 with liposomal doxorubicin for HCC, which may provide a new strategy to increase the curative efficacy of thermal ablation for medium-to-large HCC.

Targeting cathepsin B by cycloastragenol enhances antitumor immunity of CD8 T cells via inhibiting MHC-I degradation.

BACKGROUND: The loss of tumor antigens and depletion of CD8 T cells caused by the PD-1/PD-L1 pathway are important factors for tumor immune escape. In recent years, there has been increasing research on traditional Chinese medicine in tumor treatment. Cycloastragenol (CAG), an effective active molecule in Astragalus membranaceus, has been found to have antiviral, anti-aging, anti-inflammatory, and other functions. However, its antitumor effect and mechanism are not clear. METHODS: The antitumor effect of CAG was investigated in MC38 and CT26 mouse transplanted tumor models. The antitumor effect of CAG was further analyzed via single-cell multiomics sequencing. Target responsive accessibility profiling technology was used to find the target protein of CAG. Subsequently, the antitumor mechanism of CAG was explored using confocal microscopy, coimmunoprecipitation and transfection of mutant plasmids. Finally, the combined antitumor effect of CAG and PD-1 antibodies in mice or organoids were investigated. RESULTS: We found that CAG effectively inhibited tumor growth in vivo. Our single-cell multiomics atlas demonstrated that CAG promoted the presentation of tumor cell-surface antigens and was characterized by the enhanced killing function of CD8+ T cells. Mechanistically, CAG bound to its target protein cathepsin B, which then inhibited the lysosomal degradation of major histocompatibility complex I (MHC-I) and promoted the aggregation of MHC-I to the cell membrane, boosting the presentation of the tumor antigen. Meanwhile, the combination of CAG with PD-1 antibody effectively enhanced the tumor killing ability of CD8+ T cells in xenograft mice and colorectal cancer organoids. CONCLUSION: Our data reported for the first time that cathepsin B downregulation confers antitumor immunity and explicates the antitumor mechanism of natural product CAG.

The Impact of Vitamin D Supplementation on the IFNγ-IP10 Axis in Women with Hashimoto's Thyroiditis Treated with Levothyroxine: A Double-blind Randomized Placebo-controlled Trial.

Hashimoto's thyroiditis (HT) results from chemoattraction of inflammatory cells toward the thyroid gland by inducing the production of interferon-gamma (IFNγ)-induced protein 10 (IP10) by T helper (Th) 1 cells. Vitamin D may suppress the IFNγ-IP10 axis, but this new function of vitamin D has not yet been investigated in HT patients. In an intervention and control group, patients received 50000 IU cholecalciferol or placebo every week for three months, respectively. The CD4+ T cells of 40 patients were isolated, and the mRNA expression levels of vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPAR)-α, and PPAR-γ genes were determined by real-time PCR. ELISA method was used to determine serum levels of vitamin D, tumor necrosis factor-alpha (TNF-α), IFN-γ, and IP10. Vitamin D levels in the intervention group were significantly higher than in the placebo group after supplementation. PPAR-α and PPAR-γ gene expression levels did not differ significantly between the two groups. The serum levels of IP10, IFNγ, and TNF-α decreased significantly in the vitamin D group, as well as in the placebo group.  During this study, vitamin D levels significantly increased in the intervention group and inflammatory factors decreased. Based on the similar results obtained in the placebo group, further studies with larger sample sizes and longer intervention times are recommended.

Effective Treatment of Diabetes Mellitus by Resonance Medicine.

The metabolic disorder known as diabetes mellitus (DM) has several different causes, distinguished by recurring hyperglycemia due to inadequate insulin secretion, insulin action, or both. T-lymphocytes target such cells for destruction, which include beta cells. Transplants of the pancreas, islets of Langerhans, and individual beta cells are all effective treatments for DM. Additionally, treating DM using stem cells is popular currently. The basis of stem cell therapy for DM is the replacement of beta cells, or dead pancreatic cells, with stem cells. After attaching to the tissues of the pancreas, the stem cells differentiate into active cells. An X-ray scanner is used to place a catheter into the pancreatic artery in DM, and the process lasts 90 minutes. The use of stem cells to replace dead pancreatic beta cells forms the cornerstone of stem cell treatment for DM. Transplants of the pancreas, islets of Langerhans, and individual beta cells are all effective treatments for insulin-dependent DM. In contrast to prior studies, where we only used low potencies of nosodes and organopreparations, our research used both high and low potencies of these substances. Choosing the strength of the nosode stomach cancer in the computer-connected device selector so that it will resonate with the nosode that is tested in the patient's device is the doctor's responsibility when using the bioresonance therapy method. The initial nosode, which is in the computer programme of the device for bioresonance therapy, is no longer tested when the stomach cancer nosode is tested in a patient along with the chosen potency of this nosode. The initial nosode in the bioresonance therapy device itself is still being studied in case the chosen nosode's potency is inadequate (the frequency of oscillations of the nosode is lower than the frequency of oscillations of the tumour).

L-5-hydroxytryptophan promotes antitumor immunity by inhibiting PD-L1 inducible expression.

BACKGROUND: The repression or downregulation of programmed death-ligand 1 (PD-L1) can release its inhibition of T cells and activate antitumor immune responses. Although PD-1 and PD-L1 antibodies are promising treatments for diverse tumor types, their inherent disadvantages and immune-related adverse events remain significant issues. The development of small molecule inhibitors targeting the interaction surface of PD-1 and PD-L1 has been reviving, yet many challenges remain. To address these issues, we aimed to find small molecules with durable efficacy and favorable biosafety that alter PD-L1 surface expression and can be developed into a promising alternative and complementary therapy for existing anti-PD-1/PD-L1 therapies. METHODS: Cell-based screen of 200 metabolic molecules using a high-throughput flow cytometry assay of PD-L1 surface expression was conducted, and L-5-hydroxytryptophan (L-5-HTP) was found to suppress PD-L1 expression induced by interferon gamma (IFN-γ). Inhibition of PD-L1 induction and antitumor effect of L-5-HTP were evaluated in two syngeneic mouse tumor models. Flow cytometry was performed to investigate the change in the tumor microenvironment caused by L-5-HTP treatment. RESULTS: We discovered that L-5-HTP suppressed IFN-γ-induced PD-L1 expression in tumor cells transcriptionally, and this effect was directly due to itself. Mechanistically, L-5-HTP inhibited IFN-γ-induced expression of RTK ligands and thus suppressed phosphorylation-mediated activation of RTK receptors and the downstream MEK/ERK/c-JUN signaling cascade, leading to decreased PD-L1 induction. In syngeneic mouse tumor models, treatment with 100 mg/kg L-5-HTP (intraperitoneal) inhibited PD-L1 expression and exhibited antitumor effect. L-5-HTP upregulated the ratio of granzyme B+ CD8+ activated cytotoxic T cells. An intact immune system and PD-L1 expression was critical for L-5-HTP to exert its antitumor effects. Furthermore, L-5-HTP acted synergistically with PD-1 antibody to improve anticancer effect. CONCLUSION: Our study illustrated L-5-HTP's inhibitory effect on PD-L1 induction stimulated by IFN-γ in tumor cells and also provided insight into repurposing L-5-HTP for use in tumor immunotherapy.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 expression and its relationship with T cell activation in human immunodeficiency virus/acquired immune deficiency syndrome patients with lung-spleen deficiency syndrome pattern.

OBJECTIVE: To investigate the relationship between antiviral restriction factor Sterile Alpha Motif and Histidine-Aspartic acid domain-containing protein 1 (SAMHD1) expression and T cell activation, furthermore, identifying objective indexes of lung-spleen deficiency symptom pattern. METHODS: We assessed the profile of T lymphocyte subsets, characteristics of SAMHD1 and human leukocyte antigen DR (HLA-DR) expression in lung-spleen deficiency patients. At the same time, people living with human immunodeficiency virus / acquired immune deficiency syndrome (HIV/AIDS) (PLWHA) without obvious clinical symptoms and healthy donors in this area were used as controls. RESULTS: Immunohematologic indexes lower CD4 count, lower CD4/CD8 ratio and higher SAMHD1 level were found in lung-spleen deficiency patients. Furthermore, we demonstrated a positive relationship between SAMHD1 and HLA-DR level as well as with interferon factor in lung-spleen deficiency syndrome and patients without obvious clinical signs and symptoms groups. CONCLUSIONS: These data indicated the positive relationship between SAMHD1 and T cell activation which further elucidated the role of SAMHD1 in cellular immune response. Furthermore, combination of T lymphocyte subsets counts and SAMHD1 level may be used as clinical and biological reference basis for the differentiation and diagnosis of HIV / AIDS traditional Chinese medicine syndromes.

Vitamin D3 reverses the transcriptional profile of offspring CD4+ T lymphocytes exposed to intrauterine inflammation.

Chorioamnionitis profoundly influences multiple fetal organs as well as the immune system. Maternal vitamin D (VitD) supplementation may modulate the immune function of offspring. Here, we sought to uncover the immunomodulatory potential of intrauterine inflammation and VitD in offspring CD4+ T cells. Pregnant C57BL/6 mice were treated with intrauterine lipopolysaccharide (LPS) injections, with or without VitD. Splenic CD4+ T cells were negatively selected using anti-biotin microbeads at 28 days after birth. Differentially expressed genes (DEGs) in the offspring CD4+ T cells were identified via RNA sequencing. In total, 181 DEGs induced by LPS exposure were identified in offspring CD4+ T cells. Furthermore, 2461 DEGs were detected after VitD supplementation in addition to LPS exposure. VitD supplementation showed an unexpected ability to counteract the LPS-induced transcriptional responses. VitD supplementation downregulated lymphocyte differentiation (GO: 0030098) and lymphocyte activation (GO: 0046649), and upregulated the responses to viruses (GO: 0009615) and bacteria (GO:0009617) in offspring CD4+ T cells with intrauterine LPS exposure. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that several pathways, including the T cell receptor signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, Th17 cell differentiation, and autophagy, were downregulated by intrauterine VitD intervention following LPS exposure. Subsequently, we confirmed the counteracting effect of VitD against LPS on the expression of several genes (Insr, Foxo1, and Peli1) using qRT-PCR and western blot analyses. We also demonstrated that intrauterine VitD supplementation interferes with offspring Th17 cell differentiation induced by intrauterine LPS exposure. Our study revealed that VitD reverses the transcriptional and Th17 differential profiles of offspring CD4+ T lymphocytes induced by intrauterine LPS, and indicated the contribution of maternal VitD supplementation to immune protection in offspring affected by intrauterine inflammation.

[Effects of different extracts of Dendrobium officinale on rats with chronic pharyngitis induced by pepper water combined with ammonia].

Dendrobium officinale can serve as Chinese medicinal material effective in nourishing yin, clearing heat, and producing fluid, and is used to treat throat diseases, but its active substances and mechanism are not clear. To clarify the active fraction and underlying mechanism of D. officinale against chronic pharyngitis(CP), the present study induced a CP model in rats by pepper water combined with low-concentration ammonia, and crude polysaccharides of D. officinale(DOP), non-polysaccharides of D. officinale(DON), and total extract of D. officinale(DOT)(0.33 g·kg~(-1), calculated according to the crude drug) were administered by gavage for six weeks. The changes in oral secretions and pharyngeal conditions of rats with CP were observed and rated. The hematological indicators were determined by an automatic hematology analyzer. The serum levels of pro-inflammatory factors, such as tumor necrosis factor-alpha(TNF-α), interleukin 1ß(IL-1ß), and interleukin 6(IL-6), and T-lymphocyte cytokines, including interferon γ(IFN-γ), interleukin 4(IL-4), interleukin 17(IL-17), and transforming growth factor ß1(TGF-ß1) were detected by the enzyme-linked immunosorbent assay(ELISA). The proportions of CD3~+, CD4~+, and CD8~+cells in peripheral blood T lymphocyte subsets were determined by the flow cytometry. The histomorphological changes of the pharynx were observed by hematoxylin-eosin(HE) staining. The protein expression of nuclear factor-κB P65(NF-κB P65), cyclooxygenase-2(COX-2), F4/80, and monocyte chemoattractant protein-1(MCP-1) in the pharynx were detected by immunohistochemistry and Western blot. The results showed that DOP and DON could significantly relieve pharyngeal lesions, reduce white blood cells(WBC) and lymphocytes(LYMP), decrease the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibit the protein expression of NF-κB P65, COX-2, F4/80, and MCP-1 in the pharynx. DOP was superior in reducing oral secretions and serum IL-17 level and inferior in increasing CD4~+/CD8~+ratio to DON. It is suggested that both polysaccharides and non-polysaccharides of D. officinale have anti-PC effects and the anti-inflammatory mechanism may be related to the regulation of T lymphocyte distribution and inhibition of the inflammatory signaling pathways mediated by NF-κB P65. The anti-inflammatory effect of DOP may be related to the regulation of Th17/Treg balance, while that of DON may be related to the regulation of the Th/Tc ratio.

Efficacy of Wumei Baijiang prescription on regulatory T cells / helper T cells Immune balance in mice with ulcerative coliti.

OBJECTIVE: To observe the therapeutic effect of Wumei Baijiang prescription empirical prescription of Lu Zhizheng, on experimental ulcerative colitis (UC) mice, and to investigate the mechanism of the prescription in UC from the perspective of the immune balance of regulatory T cells (Treg) and helper T cells (Th17). METHODS: Sixty C57BL/6 mice were randomly divided into 6 groups: normal group, model group, Chinese medicine group (high, medium and low dose group of Wumei Baijiang prescription) and control group (mesalazine sustained-release granules). Except for the normal group, the other groups used 2.5% dextran sulfate sodium to induce UC mice model. At the end of the model, the Chinese medicine group was given high, medium and low dose administration of Wumei Baijiang prescription, the control group was given slow-release granules of mesalazine, and the model group was given equal volume saline for 10 d. The changes of food intake, body weight, disease activity index (DAI) score, length of large intestine and histopathology were observed. The number of Treg, Th17, CD4+, CD8+ cells in spleen was detected by flow cytometry. The expression of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and C-reactive protein (CRP) in serum was detected by enzyme-linked immunosorbent assay. RESULTS: The middle and high-dose groups of Wumei Baijiang prescription were superior to the model group in terms of increasing food intake and body weight of colitis mice, restoring colon morphology, improving pathological damage, and reducing DAI (P < 0.05). There was no statistical difference with the mesalazine group (P > 0.05). Compared with the model group, the spleen Treg and CD4+ of the mice in the high and middle dose groups of Wumei Baijiang prescription were higher, while Th17 and CD8+ were lower (P < 0.05), and there was no statistical difference compared with the mesalazine group (P > 0.05). In addition, compared with the model group, the serum levels of TNF- and CRP in mice with high and middle doses of Wumei Baijiang prescription and mesalazine group were lower (P < 0.05), and IL-10 content was higher ( P < 0.05). CONCLUSIONS: Wumei Baijiang prescription can improve the general conditions of colitis mice, such as diarrhea, hematochezia, weight loss, and mucosal damage. The mechanism may be related to the regulation of Treg/Th17 immune balance.