Alteration in gut microbiota is associated with immune imbalance in Graves' disease.

Background: Graves' disease (GD), characterized by immune aberration, is associated with gut dysbiosis. Despite the growing interest, substantial evidence detailing the precise impact of gut microbiota on GD's autoimmune processes remains exceedingly rare. Objective: This study was designed to investigate the influence of gut microbiota on immune dysregulation in GD. Methods: It encompassed 52 GD patients and 45 healthy controls (HCs), employing flow cytometry and enzyme-linked immunosorbent assay to examine lymphocyte and cytokine profiles, alongside lipopolysaccharide (LPS) levels. Gut microbiota profiles and metabolic features were assessed using 16S rRNA gene sequencing and targeted metabolomics. Results: Our observations revealed a disturbed B-cell distribution and elevated LPS and pro-inflammatory cytokines in GD patients compared to HCs. Significant differences in gut microbiota composition and a marked deficit in short-chain fatty acid (SCFA)-producing bacteria, including ASV263(Bacteroides), ASV1451(Dialister), and ASV503(Coprococcus), were observed in GD patients. These specific bacteria and SCFAs showed correlations with thyroid autoantibodies, B-cell subsets, and cytokine levels. In vitro studies further showed that LPS notably caused B-cell subsets imbalance, reducing conventional memory B cells while increasing naïve B cells. Additionally, acetate combined with propionate and butyrate showcased immunoregulatory functions, diminishing cytokine production in LPS-stimulated cells. Conclusion: Overall, our results highlight the role of gut dysbiosis in contributing to immune dysregulation in GD by affecting lymphocyte status and cytokine production.

Aberrant expression of inhibitory receptors on B cells in patients with Graves' disease.

The pathophysiological mechanism underlying Graves' disease (GD) remains incompletely understood. Inhibitory receptors on B cells are critical for humoral immunity, which plays a key role in GD pathogenesis. This study aimed to investigate B cell subsets distribution and inhibitory receptor expression on these subsets in GD patients. Peripheral blood was drawn from 41 healthy controls and 46 GD patients (21 patients with moderate GD, 25 patients with severe GD). B cell subset distribution and CD22, CD32b and CD72 expression on B cells were analyzed by flow cytometry. Serum cytokines were examined by enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, the naïve B cell percentage was increased, while the preswitched memory and conventional memory B cell percentages were decreased. The inhibitory receptors expression, especially CD32b, on B cell subsets was significantly decreased in patients with GD. In addition, the inhibitory receptors expression on B cell subsets from severe GD patients exhibited a decreasing trend compared with those from moderate GD patients. These results suggest that abnormal B cell subset distribution occurs in GD. Impaired inhibitory receptors, in particular CD32b, play a crucial role in GD pathogenesis and might be a therapeutic target to rebuild self-immune tolerance in GD.

Effect of different anaesthesia methods on perioperative cellular immune function and long-term outcome in patients undergoing radical resection of esophageal cancer: a prospective cohort study.

To analyze the effects of different anaesthetic methods on perioperative cellular immunity and long-term outcome in patients who undergo esophageal cancer surgery. PARTICIPANTS: A total of 120 patients with esophageal cancer admitted to Zhengzhou University People's Hospital from January 2016 to January 2017 were recruited and randomly divided into a GA group (general anaesthesia, n = 40), a PG group (paravertebral nerve block with general anaesthesia, n = 40) and an EG group (epidural anaesthesia with general anaesthesia, n = 40). METHODS: Self-rating anxiety scale and visual analogue scale scores were adopted to compare postoperative anxiety and the degree of pain of patients in the three groups. In addition, the adverse reactions of patients in the three groups were compared. The levels of interleukin-6 (IL-6), IL-4, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and the survival of T-cell subsets (CD3+, CD4+, CD8+, CD4+/CD8+) before operation, at the end of operation, and on postoperative day (POD) 1 and POD 2 were measured by either ELISA or flow cytometry. RESULTS: In the PG and EG group, the VAS scores were lower, and fewer opioids and vasoactive agents were used than in the GA group. In both the EG and PG groups, higher CD3+ and CD4+ cell survival and lower levels of Cor, IL-4, and IL-6 were identified at the end of or after the surgery than in the GA group. Moreover, the postoperative survival curves of the PG and EG groups were better than that of the GA group. CONCLUSIONS: The combination of paravertebral nerve block or epidural anaesthesia and general anaesthesia may improve perioperative immune function and long-term outcome in patients who undergo esophageal cancer surgery.

LncRNA GIHCG Promotes the Development of Esophageal Cancer by Modulating miR-29b-3p/ANO1 Axis.

BACKGROUND: Esophageal cancer is one of the most frequent cancers with a higher mortality worldwide. Although many long non-coding RNAs (LncRNAs) are reported to play important roles in the progression of esophageal cancer, the function of lncRNA GIHCG in esophageal cancer remains unclear. METHODS: The expression of GIHCG in esophageal cancer tissues and cancer cell lines was detected by qRT-PCR. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay, EdU staining assay and colony formation assay. Cell invasion and migration were measured by transwell assay. Cell apoptosis was detected by a flow cytometer. Luciferase reporter assay and RIP assay were used to determine the interaction between GIHCG and miR-29b-3p, and their subsequent regulation of anoctamin 1 (ANO1). The expression of ANO1 in esophageal cancer tissues and cell lines was detected by Western blot. The effect of GIHCG/miR-29b-3p in tumor formation was assessed by the xenograft nude mice model in vivo. RESULTS: GIHCG was significantly upregulated in esophageal cancer tissues and relevant cancer cell lines. Downregulation of GIHCG significantly inhibited the growth, colony formation, invasion, migration and induced apoptosis of esophageal cancer cells in vitro. Bioinformatic analysis and RIP assay determined that GIHCG was a sponge of miR-29b-3p, and ANO1 was a direct target of miR-29b-3p. Moreover, functional experiments showed that GIHCG upregulated ANO1 expression by directly sponging miR-29b-3p. Furthermore, in vivo experiment revealed that knockdown of GIHCG significantly inhibited tumor growth in nude mice. CONCLUSION: Our study revealed that lncRNA GIHCG promoted the progression of esophageal cancer by targeting the miR-29b-3p/ANO1 axis, suggesting that GIHCG might be a novel therapeutic target for esophageal cancer.

Antidepressant-like effect of active fraction of Polyrhachisvicina Roger in a rat depression model.

OBJECTIVE: To investigate the antidepressant-like effect of active fraction of Polyrhachis vicina Roger (AFPR) in a rat depression model, and to elucidate the underlying mechanism. METHODS: AFPR was extracted with ethanol followed by petroleum ether. Its antidepressant-like effect was investigated in mice by tail suspension test (TST), forced swimming test (FST) and open field test (OPT). A repeated dose of reserpine (0.5 mg/kg, daily for 14 d) was used to establish a rat depression model. Fluoxetine was used as positive control agent. The effect of AFPR on reserpine-induced ptosis, hypothermia and akinesia, the levels of monoamines and their metabolites, and the activity of monoamine oxidase (MAO) in hippocampus and prefrontal cortex were determined. RESULTS: Administration of AFPR by gavage at 160 and 320 mg/kg significantly reduced the duration of immobility in the FST and TST, and did not affect locomotor activity in the OPT. In the reserpine-induced depression model, AFPR attenuated anhedonia, demonstrated by reversing hypothermia, akinesia and sucrose consumption. AFPR significantly increased the concentration of monoamines, including dopamine, serotonin, noradrenaline and acetylcholine. CONCLUSION: AFPR normalized the metabolism rates of noradrenaline, serotonin and dopamine, and the activity of MAO, which were altered by chronic reserpine exposure. The findings suggest that modulation of the monoaminergic neurotransmitter system likely underlies the antidepressant-like effect of AFPR.

[Chemical Constituents from Macaranga denticulata Root].

OBJECTIVE: To study the chemical constituents from Macaranga denticulata Root. METHODS: The chemical constituents were isolated and purified by silica-gel column chromatography and recrystallization, and their structures were identified by physicochemical properties and spectral data. RESULTS: Nine compounds were isolated and identified as: gheddic acid (1), aleuritolic acid-3-acetate (2), ß-sitosterol (3), stigmast-4-en-6ß-ol-3 -one (4), 2α-hydroxyaleuritolic acid 3-p-hydroxybenzoate (5), scopoletin (6), daucosterol (7), 2, 6-dimethoxy-1,4-benzoquinone (8) and maslinic acid(9). CONCLUSION: Compounds 1-9 are obtained from this plant for the first time.

[Chemical constituents of Desmodium caudatum].

OBJECTIVE: To study the chemical constituents of Desmodium caudatum. METHODS: Silica column chromatography, Sephadex LH-20 column chromatography and recrystallization were used to separate and purify the chemical composition of Desmodium caudatum. Their chemical structures were identified by infrared spectrum (IR), mass spectrum (MS), nuclear magnetic resonance (NMR) and other physicochemical methods. RESULTS: Twelve compounds were isolated and identified as lacceroic acid(1), gheddic acid(2), stigmasterol(3), betulin(4), citrusinol(5), yukovanol(6), kaempferol(7), protocatechuic acid(8), sophocarpine(9), matrine(10), N, Ndimethyltryptamine(11) and 5-hydroxy-N,N-dimethyltryptamine(12). CONCLUSION: Compounds 1, 2, 4 and 8-12 are isolated from this plant for the first time.

Tumor microenvironment macrophage inhibitory factor directs the accumulation of interleukin-17-producing tumor-infiltrating lymphocytes and predicts favorable survival in nasopharyngeal carcinoma patients.

The accumulation of an intratumoral CD4(+) interleukin-17-producing subset (Th17) of tumor-infiltrating lymphocytes (TILs) is a general characteristic in many cancers. The relationship between the percentage of Th17 cells and clinical prognosis differs among cancers. The mechanism responsible for the increasing percentage of such cells in NPC is still unknown, as is their biological function. Here, our data showed an increase of Th17 cells in tumor tissues relative to their numbers in normal nasopharynx tissues or in the matched peripheral blood of NPC patients. Th17 cells in tumor tissue produced more IFNγ than did those in the peripheral blood of matched NPC patients and healthy controls. We observed high levels of CD154, G-CSF, CXCL1, IL-6, IL-8, and macrophage inhibitory factor (MIF) out of 36 cytokines examined in tumor tissue cultures. MIF promoted the generation and recruitment of Th17 cells mediated by NPC tumor cells in vitro; this promoting effect was mainly dependent on the mammalian target of rapamycin pathway and was mediated by the MIF-CXCR4 axis. Finally, the expression level of MIF in tumor cells and in TILs was positively correlated in NPC tumor tissues, and the frequency of MIF-positive TILs was positively correlated with NPC patient clinical outcomes. Taken together, our findings illustrate that tumor-derived MIF can affect patient prognosis, which might be related to the increase of Th17 cells in the NPC tumor microenvironment.

Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy.

Establishing Epstein-Barr virus(EBV)-specific cytolytic T lymphocytes(EBV-CTLs) from peripheral blood mononuclear cells(PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC). In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3), recombinant human interleukin(IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

Distribution, characterization, and induction of CD8+ regulatory T cells and IL-17-producing CD8+ T cells in nasopharyngeal carcinoma.

BACKGROUND: CD8+ effector cells often have an antitumor function in patients with cancer. However, CD8+Foxp3+ regulatory T cells (Tcregs) and interleukin (IL)-17-producing CD8+ T cells (Tc17 cells) also derive from the CD8+ T cell lineage. Their role in the antitumor response remains largely unknown. In the present study, we aimed to investigate the distribution, characterization, and generation of CD8+ Tcregs and Tc17 cells in NPC patients. METHODS: Peripheral blood and tumor biopsy tissues from 21 newly diagnosed patients with nasopharyngeal carcinoma (NPC) were collected, along with peripheral blood from 21 healthy donors. The biological characteristics of Tcregs and Tc17 cells from blood and tumor tissues were examined by intracellular staining, tetramer staining and fluorescence-activated cell sorting (FACS) analysis. The suppressive function of Tcregs was investigated using a proliferation assay that involved co-culture of sorted CD8+CD25+ T cells with naïve CD4+ T cells in vitro. RESULTS: We observed an increased prevalence of Tcregs and Tc17 cells among tumor-infiltrating lymphocytes (TILs) and different distribution among peripheral blood mononuclear cells (PBMCs) in NPC patients. Cytokine profiles showed that the Tcregs expressed a high level of IL-10 and low level of transforming growth factor ß, whereas Tc17 cells expressed a high level of tumor necrosis factor α. Interestingly, both subsets expressed a high level of interferon γ in TILs, and the Tcregs suppressed naïve CD4+ T cell proliferation by a cell contact-dependent mechanism in vitro. Moreover, we demonstrated the existence of Epstein-Barr virus latent membrane protein (LMP) 1 and LMP2 antigen-specific Tcregs in NPC. CONCLUSIONS: Our data provide new insights into the composition and function of CD8+ T-cell subsets in NPC, which may have an important influence on NPC immunotherapy.

Circulating and tumor-infiltrating Foxp3⁺ regulatory T cell subset in Chinese patients with extranodal NK/T cell lymphoma.

Foxp3⁺ regulatory T lymphocytes (Tregs) usually act as an immune suppressor and correlate with poorer survival in malignancies. This study aims to investigate the distribution and characterization of Foxp3(+) subset in peripheral blood mononuclear cells (PBMCs) and tumor tissues from extranodal NK/T cell lymphoma (ENKTL). Our study showed the percentage of Foxp3⁺ subset from PBMC was significantly higher than that of healthy individuals (P<0.001). The Foxp3⁺ subset from PBMCs expressed CD45RO, CTLA4, GITR, CCR7, and had an IL-10(high)IFNγ⁺TGFß⁺IL-2(low)IL-17(low) cytokine secreting phenotype. Interestingly, the existence of EBV antigen-specific CD8⁺Foxp3⁺ Tregs was discovered in ENKTL. Furthermore, the high density of Foxp3⁺ TILs was associated with improved progression-free survival (PFS) in ENKTL patients (P<0.05). Collectively, our study implicates that EBV antigens could induce antigen-specific CD8⁺Foxp3⁺ Tregs in ENKTL, and Foxp3⁺ TILs is an independent factor for PFS in ENKTL.

Immunophenotyping at the time of diagnosis distinguishes two groups of nasopharyngeal carcinoma patients: implications for adoptive immunotherapy.

BACKGROUND: Adoptive immunotherapy with EBV-specific CTLs (EBV-CTL) has been used to treat EBV-associated nasopharyngeal carcinoma (NPC) but only a fraction of the patients shows noticeable clinical response. PATIENTS AND METHODS: Sixty-seven newly diagnosed NPC patients from 2005 to 2007 and 21 healthy donors were collected. Immunological parameters and immune function of PBMCs and EBV-CTL were analyzed by flow cytometer analysis (FACS) and 5¹Cr releasing experiment; Molecular characteristics on NPC tumor cells were investigated by immunochemical staining and statistic analysis. RESULTS: NPC patients can be classified into two groups based on the percentage of CD3+ T cells in peripheral blood before accepted any treatment, (>52.6%, mean-2SE from healthy controls, NPC Group 1; <52.6%, NPC Group 2). The patients in Group 2 showed a significant decrease of CD3+CD8+ T-cells, CD3+CD4+ T-cells and CD3+CD45RO+ memory T cells, and increase of CD3⁻CD16+ NK cells compared to Group 1 patients and healthy controls (P<0.001). EBV-specific T cell responses, were weaker in this group of patients and their tumor cells expressed lower levels of the EBV encoded latent membrane protein (LMP)-1 and HLA class II protein compared with the patients of NPC Group 1 (P<0.05). CONCLUSION: These findings demonstrate that NPC patients could be distinguished on the basis of their immune status which will affect the efficacy of EBV-CTL immunotherapy.