PD-1-mediated inhibition of T cell activation: Mechanisms and strategies for cancer combination immunotherapy.

The programmed cell death 1 (PD-1) immune checkpoint of co-inhibitory signaling plays crucial roles in controlling the magnitude and duration of T cell activation to limit tissue damage and maintain self-tolerance. Cancer cells hijack the co-inhibitory pathway and escape immune surveillance by overexpressing the PD-1 ligand PD-L1. Immune checkpoint inhibitors, such as PD-1 blocking antibody have been approved for tumor immunotherapy. However, not all patients can benefit from PD-1 monotherapy. Combination immunotherapy based on PD-1 axis blockade substantially improves clinical anti-tumor efficacy. In this review, we briefly summarize the current progress on the mechanisms of PD-1-mediated inhibition of T cell activation and strategies for cancer combination immunotherapy.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Aromatic and magnetic properties in a series of heavy rare earth-doped Ge6 cluster anions.

A series of pentagonal bipyramidal anionic germanium clusters doped with heavy rare earth elements, REGe 6 - (RE = Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu), have been identified at the PBE0/def2-TZVP level using density functional theory (DFT). Our findings reveal that the centrally doped pentagonal ring structure demonstrates enhanced stability and heightened aromaticity due to its uniform bonding characteristics and a larger charge transfer region. Through natural population analysis and spin density diagrams, we observed a monotonic decrease in the magnetic moment from Gd to Yb. This is attributed to the decreasing number of unpaired electrons in the 4f orbitals of the heavy rare earth atoms. Interestingly, the system doped with Er atoms showed lower stability and anti-aromaticity, likely due to the involvement of the 4f orbitals in bonding. Conversely, the systems doped with Gd and Tb atoms stood out for their high magnetism and stability, making them potential building blocks for rare earth-doped semiconductor materials.

Exploring the stability and aromaticity of rare earth doped tin cluster MSn16- (M = Sc, Y, La).

Rare earth elements have high chemical reactivity, and doping them into semiconductor clusters can induce novel physicochemical properties. The study of the physicochemical mechanisms of interactions between rare earth and tin atoms will enhance our understanding of rare earth functional materials from a microscopic perspective. Hence, the structure, electronic characteristics, stability, and aromaticity of endohedral cages MSn16- (M = Sc, Y, La) have been investigated using a combination of the hybrid PBE0 functional, stochastic kicking, and artificial bee colony global search technology. By comparing the simulated results with experimental photoelectron spectra, it is determined that the most stable structure of these clusters is the Frank-Kasper polyhedron. The doping of atoms has a minimal influence on density of states of the pure tin system, except for causing a widening of the energy gap. Various methods such as ab initio molecular dynamics simulations, the spherical jellium model, adaptive natural density partitioning, localized orbital locator, and electron density difference are employed to analyze the stability of these clusters. The aromaticity of the clusters is examined using iso-chemical shielding surfaces and the gauge-including magnetically induced currents. This study demonstrates that the stability and aromaticity of a tin cage can be systematically adjusted through doping.

PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity.

Combination therapy with PD-1 blockade and IL-2 substantially improves anti-tumor efficacy comparing to monotherapy. The underlying mechanisms responsible for the synergistic effects of the combination therapy remain enigmatic. Here we show that PD-1 ligation results in BATF-dependent transcriptional induction of the membrane-associated E3 ubiquitin ligase MARCH5, which mediates K27-linked polyubiquitination and lysosomal degradation of the common cytokine receptor γ chain (γc). PD-1 ligation also activates SHP2, which dephosphorylates γcY357, leading to impairment of γc family cytokine-triggered signaling. Conversely, PD-1 blockade restores γc level and activity, thereby sensitizing CD8+ T cells to IL-2. We also identified Pitavastatin Calcium as an inhibitor of MARCH5, which combined with PD-1 blockade and IL-2 significantly improves the efficacy of anti-tumor immunotherapy in mice. Our findings uncover the mechanisms by which PD-1 signaling antagonizes γc family cytokine-triggered immune activation and demonstrate that the underlying mechanisms can be exploited for increased efficacy of combination immunotherapy of cancer.

Dissecting the efficacy of the use of acupuncture and Chinese herbal medicine for the treatment of premature ovarian insufficiency (POI): A systematic review and metaanalysis.

Premature ovarian insufficiency is a multi-factor gynecological disease that has become a major global health problem. In recent years, several trials have explored the treatment of premature ovarian insufficiency using Chinese herbal medicine and acupuncture, but the efficacy and safety of this combination remains controversial. This systematic review and meta-analysis aimed to comprehensively evaluate the efficacy and safety of combining Chinese herbal medicine with acupuncture to treat premature ovarian insufficiency. From eight different databases, we retrieved randomized controlled trials wherein Chinese herbal medicine and acupuncture had been compared with western medicine in the treatment of premature ovarian insufficiency. The bias risk assessment stipulated by the Cochrane Collaboration's tool was utilized to evaluate the quality of the chosen randomized controlled trials. This meta-analysis was executed with the help of Review Manager 5.3 and Stata 10.0. The quality of evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation framework. A total of 10 randomized controlled trials involving 594 premature ovarian insufficiency patients were included in the analysis. Compared with western medicine, co-treatment with acupuncture and Chinese herbal medicine exhibited a significantly higher total effective rate (relative risk: 1.21; 95% confidence interval: 1.12-1.31; P < 0.01, I2 = 0%), but lower levels of luteinizing hormone (standardized mean difference: -0.57; 95% confidence interval: -1.06, -0.08; P < 0.05, I2 = 80%), follicle-stimulating hormone, and Kupperman index score. Moreover, the combined intervention increased estradiol level in the serum. Overall, the data demonstrate that acupuncture plus Chinese herbal medicine is an efficacious and safe treatment option for POI patients. These findings must be verified by conducting large-scale, multicenter, high-quality, and long-term randomized controlled trials.

The novel small molecule BH3 mimetic nobiletin synergizes with vorinostat to induce apoptosis and autophagy in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly lethal subtype of lung cancer with few therapeutic options; therefore, the identification of new targets and drugs with potent combination therapy is desirable. We previously screened BH3 mimetics from a natural product library, and in this study, we validated nobiletin as a BH3 mimetic. Specifically, we observed its combination potential and mechanism with vorinostat in SCLC in vitro and in vivo. The results showed that combination treatment with nobiletin and vorinostat reduced the proliferation of SCLC H82 cells and increased the levels of apoptotic proteins such as cleaved caspase-9 and cleaved PARP. The combination treatment increased LC3-II expression and induced autophagic cell death. In addition, this treatment significantly inhibited H82 cell xenograft SCLC tumor growth in nude mice. The combination treatment with nobiletin and vorinostat efficiently increased autophagy by inhibiting the PI3K-AKT-mTOR pathway and promoting dissociation of the BCL-2 and Beclin 1 complex, increasing the level of isolated Beclin 1 to stimulate autophagy. Molecular docking and surface plasmon resonance analysis showed that nobiletin stably bound to the BCL-2, BCL-XL and MCL-1 proteins with high affinity in a concentration-dependent manner. These results suggest that nobiletin is a BH3-only protein mimetic. Furthermore, the combination of nobiletin with vorinostat increased histone H3K9 and H3K27 acetylation levels in SCLC mouse tumor tissue and enhanced the expression of the BH3-only proteins BIM and BID. We conclude that nobiletin is a novel natural BH3 mimetic that can cooperate with vorinostat to induce apoptosis and autophagy in SCLC.

Making Sense of the Growth Behavior of Ultra-High Magnetic Gd2-Doped Silicon Clusters.

The growth behavior, stability, electronic and magnetic properties of the Gd2Sin- (n = 3-12) clusters are reported, which are investigated using density functional theory calculations combined with the Saunders 'Kick' and the Artificial Bee Colony algorithm. The lowest-lying structures of Gd2Sin- (n = 3-12) are all exohedral structures with two Gd atoms face-capping the Sin frameworks. Results show that the pentagonal bipyramid (PB) shape is the basic framework for the nascent growth process of the present clusters, and forming the PB structure begins with n = 5. The Gd2Si5- is the potential magic cluster due to significantly higher average binding energies and second order difference energies, which can also be further verified by localized orbital locator and adaptive natural density partitioning methods. Moreover, the localized f-electron can be observed by natural atomic orbital analysis, implying that these electrons are not affected by the pure silicon atoms and scarcely participate in bonding. Hence, the implantation of these elements into a silicon substrate could present a potential alternative strategy for designing and synthesizing rare earth magnetic silicon-based materials.

The Triglyceride Glucose (TyG) Index as a Sensible Marker for Identifying Insulin Resistance and Predicting Diabetic Kidney Disease.

BACKGROUND Patients with insulin-resistant diabetes have the highest risk of kidney disease. The triglyceride glucose (TyG) index is considered a reliable and simple marker of insulin resistance. We studied the relationship between the TyG index, diabetic kidney disease (DKD), and related metabolic disorders in patients with type 2 diabetes. MATERIAL AND METHODS This retrospective study included a consecutive case series from January 2021 to October 2022 in the Department of Endocrinology at Hebei Yiling Hospital. In total, 673 patients with type 2 diabetes met the inclusion criteria. The TyG index was calculated by napierian logarithmic (ln) (fasting triglyceride×fasting glucose /2). Patient demographic and clinical indicators were obtained from medical records, and statistical analysis was conducted using SPSS version 23. RESULTS The TyG index was significantly related to metabolic indicators (low-density lipoprotein, high-density lipoprotein, alanine aminotransferase, plasma albumin, serum uric acid, triglyceride, and fasting glucose) and urine albumin (P<0.01) but not with serum creatinine and estimated glomerular filtration rate. In multiple regression analysis, an increase in the TyG index was revealed to be an independent risk factor for DKD (OR: 1.699, P<0.001). CONCLUSIONS The TyG index was independently related to DKD and related metabolic disorders; therefore, the TyG index can be used as an early sensitive target for clinical guidance in the treatment of DKD with insulin resistance.

Lomatogonium rotatum extract alleviates diabetes mellitus induced by a high-fat, high-sugar diet and streptozotocin in rats.

BACKGROUND: Lomatogonium rotatum (LR) is traditionally used in Mongolian folk medicine as a hypoglycemic agent, but its evidence-based pharmacological effects and me-chanisms of action have not been fully elucidated. AIM: To emphasize the hypoglycemic action mechanism of LR in a type 2 diabetic rat model and examine potential biomarkers to obtain mechanistic understanding regarding serum metabolite modifications. METHODS: A high-fat, high-sugar diet and streptozotocin injection-induced type 2 diabetic rat model was established. The chemical composition of the LR was identified by high performance liquid chromatography. LR extract administrated as oral gavage at 0.5 g/kg, 2.5 g/kg, and 5 g/kg for 4 wk. Anti-diabetic effects of LR extract were evaluated based on histopathological examination as well as the measurement of blood glucose, insulin, glucagon-like peptide 1 (GLP-1), and lipid levels. Serum metabolites were analyzed using an untargeted metabolomics approach. RESULTS: According to a chemical analysis, swertiamarin, sweroside, hesperetin, coumarin, 1.7-dihydroxy-3,8-dimethoxyl xanthone, and 1-hydroxy-2,3,5 trimethoxanone are the principal active ingredients in LR. An anti-diabetic experiment revealed that the LR treatment significantly increased plasma insulin and GLP-1 levels while effectively lowering blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and oral glucose tolerance test compared to the model group. Furthermore, untargeted metabolomic analysis of serum samples detected 236 metabolites, among which 86 were differentially expressed between the model and the LR group. It was also found that LR considerably altered the levels of metabolites such as vitamin B6, mevalonate-5P, D-proline, L-lysine, and taurine, which are involved in the regulation of the vitamin B6 metabolic pathway, selenium amino acid metabolic pathway, pyrimidine metabolic pathway, and arginine and proline metabolic pathways. CONCLUSION: These findings indicated that LR may have a hypoglycemic impact and that its role may be related to changes in the serum metabolites and to facilitate the release of insulin and GLP-1, which lower blood glucose and lipid profiles.

Investigation of the material basis of Xiexin Tang to alleviate type 2 diabetes mellitus based on spectrum-effect analysis by UPLC-Q-TOF/MS.

Xiexin Tang (XXT) is a classic prescription for treating diabetes in clinical practices for thousands of years in China, which has been also proved by a large number of modern pharmacological studies. However, due to its complex composition, the bioactive ingredients of XXT is still unclear. In present researches, spectrum-effect relationship analysis is widely used to explore the material basis of traditional medical herbs, so this method was adopted in this study. Firstly, the extract of XXT was separated and enriched into 5 fractions by macroporous adsorption resin. Then, UPLC-Q-TOF/MS method was used for qualitative identification of components in each eluting part, and efficacy of each fraction was assessed by the T2DM rat model. Based on grey relational analysis and pearson bivariate correlation analysis, it was found that the components such as berberine, gallic acid, catechin, epicatechin, acteoside, berberastine and 1-O-galloyl-ß-D-glucose might be the main effective basis of XXT to improve T2DM.

Schisandrin B Improves the Hypothermic Preservation of Celsior Solution in Human Umbilical Cord Mesenchymal Stem Cells.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) have emerged as promising therapy for immune and inflammatory diseases. However, how to maintain the activity and unique properties during cold storage and transportation is one of the key factors affecting the therapeutic efficiency of hUCMSCs. Schisandrin B (SchB) has many functions in cell protection as a natural medicine. In this study, we investigated the protective effects of SchB on the hypothermic preservation of hUCMSCs. METHODS: hUCMSCs were isolated from Wharton's jelly. Subsequently, hUCMSCs were exposed to cold storage (4 °C) and 24-h re-warming. After that, cells viability, surface markers, immunomodulatory effects, reactive oxygen species (ROS), mitochondrial integrity, apoptosis-related and antioxidant proteins expression level were evaluated. RESULTS: SchB significantly alleviated the cells injury and maintained unique properties such as differentiation potential, level of surface markers and immunomodulatory effects of hUCMSCs. The protective effects of SchB on hUCMSCs after hypothermic storage seemed associated with its inhibition of apoptosis and the anti-oxidative stress effect mediated by nuclear factor erythroid 2-related factor 2 signaling. CONCLUSION: These results demonstrate SchB could be used as an agent for hypothermic preservation of hUCMSCs.

Rural-urban differentials of prevalence and lifestyle determinants of pre-diabetes and diabetes among the elderly in southwest China.

BACKGROUND: Diabetes has become a major public health problem in China. A better understanding of diabetes determinants and urban-rural differences is essential to crafting targeted diabetes prevention measures for the elderly living in both urban and rural areas. This study aimed to compare rural-urban differentials in prevalence and lifestyle determinants of pre-diabetes and diabetes among the elderly in southwest China. METHODS: A cross-sectional health interview and examination survey was conducted among individuals aged ≥ 60 years in both a rural and urban area of China. Anthropometric measurements, including height, weight, and waist circumference, as well as blood pressure and fasting blood glucose measurements were taken. Associated risk factors for pre-diabetes and diabetes were evaluated using multivariate logistic regression analysis. RESULTS: In total, 1,624 urban residents and 1,601 rural residents consented to participate in the study. The urban prevalence of pre-diabetes and diabetes (46.8% and 24.7%, respectively), was higher than the rural prevalence (23.4% and 11.0%, respectively, P<0.01). Urban elderly participants had markedly higher prevalence of obesity, central obesity, and physical inactivity than their rural counterparts (15.3%, 76.0%, and 9.2% vs. 4.6%, 45.6%, and 6.1%, P<0.01). In contrast, rural elderly adults had higher prevalence of smoking than urban ones (23.2% vs. 17.2%, P<0.01). Obese (OR 1.71, 95% CI 1.27-2.30 vs. OR 1.73, 95% CI 1.30-3.28) and centrally obese participants (OR 1.59, 95% CI 1.18-2.15 vs. OR 1.83, 95% CI 1.32-2.54) were more likely to suffer from diabetes in both urban and rural regions. Furthermore, urban current smokers had a higher probability of suffering from diabetes (OR 1.58, 95% CI 1.11-2.25), while hypertension was positively associated with the prevalence of diabetes in the rural area (OR 2.13, 95% CI 1.54-2.95). Obese participants in the rural area were more likely to suffer from pre-diabetes (OR 2.50, 95% CI 1.53-4.08), while physical inactivity was positively associated with prevalence of pre-diabetes in the urban area (OR 1.95, 95% CI 1.37-2.80). CONCLUSION: Pre-diabetes and diabetes are more prevalent among urban older adults than their rural counterparts in southwest China. The identified rural-urban differentials of lifestyle factors have significant impacts on prevalence of pre-diabetes and diabetes. Thus, tailored lifestyle interventions are needed to improve diabetes prevention and management among the elderly in southwest China.

SIGIRR deficiency contributes to CD4 T cell abnormalities by facilitating the IL1/C/EBPß/TNF-α signaling axis in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a complex autoimmune disease with multiple etiological factors, among which aberrant memory CD4 T cells activation plays a key role in the initiation and perpetuation of the disease. SIGIRR (single immunoglobulin IL-1R-related receptor), a member of the IL-1 receptor (ILR) family, acts as a negative regulator of ILR and Toll-like receptor (TLR) downstream signaling pathways and inflammation. The aim of this study was to investigate the potential roles of SIGIRR on memory CD4 T cells in RA and the underlying cellular and molecular mechanisms. METHODS: Single-cell transcriptomics and bulk RNA sequencing data were integrated to predict SIGIRR gene distribution on different immune cell types of human PBMCs. Flow cytometry was employed to determine the differential expression of SIGIRR on memory CD4 T cells between the healthy and RA cohorts. A Spearman correlation study was used to determine the relationship between the percentage of SIGIRR+ memory CD4 T cells and RA disease activity. An AIA mouse model (antigen-induced arthritis) and CD4 T cells transfer experiments were performed to investigate the effect of SIGIRR deficiency on the development of arthritis in vivo. Overexpression of SIGIRR in memory CD4 T cells derived from human PBMCs or mouse spleens was utilized to confirm the roles of SIGIRR in the intracellular cytokine production of memory CD4 T cells. Immunoblots and RNA interference were employed to understand the molecular mechanism by which SIGIRR regulates TNF-α production in CD4 T cells. RESULTS: SIGIRR was preferentially distributed by human memory CD4 T cells, as revealed by single-cell RNA sequencing. SIGIRR expression was substantially reduced in RA patient-derived memory CD4 T cells, which was inversely associated with RA disease activity and related to enhanced TNF-α production. SIGIRR-deficient mice were more susceptible to antigen-induced arthritis (AIA), which was attributed to unleashed TNF-α production in memory CD4 T cells, confirmed by decreased TNF-α production resulting from ectopic expression of SIGIRR. Mechanistically, SIGIRR regulates the IL-1/C/EBPß/TNF-α signaling axis, as established by experimental evidence and cis-acting factor bioinformatics analysis. CONCLUSION: Taken together, SIGIRR deficiency in memory CD4 T cells in RA raises the possibility that receptor induction can target key abnormalities in T cells and represents a potentially novel strategy for immunomodulatory therapy.

The efficacy of Shenghua Decoction supplementation after early medical abortion: A meta-analysis of randomized controlled trials.

AIMS: Shenghua Decoction (SHD) is a well-known classic herbal formula documented in traditional Chinese medicine (TCM) that has been widely applied during the postpartum period in Chinese communities for several years. We conducted this systematic review and meta-analysis to explore the influence of SHD as an adjuvant treatment for early medical abortion using a combination of mifepristone followed by misoprostol. METHODS: This systematic review and meta-analysis was reported using 2020 PRISMA guidelines. Eight databases were searched from their establishment to February 28, 2022, for randomized controlled trials (RCTs): PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, the Chinese BioMedical database, the Chinese Scientific Journal Database, and the Wanfang database. The Grading of Recommendations Assessment, Development, and Evaluation estimated the quality of evidence. RESULTS: Sixteen RCTs involving 3016 patients were included in the meta-analysis. Overall, compared with no treatment as the control group after early medical abortion, patients treated with SHD were associated with a higher complete abortion rate (RR: 1.14; 95% CI: 1.10 - 1.18; P < 0.01, I2 = 26%, moderate quality), lower incomplete abortion rate (RR: 0.31; 95% CI: 0.24 - 0.41; P < 0.01, I2 = 0%, moderate quality), and lower viable pregnancy rate (RR: 0.26; 95% CI: 0.11 - 0.62; P < 0.01, I2 = 0%, moderate quality). Additionally, SHD supplementation was associated with reduced the induction-abortion time, duration of vaginal bleeding and menstrual recovery time. CONCLUSION: Our findings suggest that SHD supplementation may be beneficial for women seeking a medical abortion before the 7-week gestational period and no adverse events in the experimental group were reported. However, the methodological quality of the included RCTs was unsatisfactory, and therefore it is necessary to further verify the effectiveness of SHD using standardized studies of rigorous design.

Metabolomic Analysis Uncovers Lipid and Amino Acid Metabolism Disturbance During the Development of Ascites in Alcoholic Liver Disease.

Ascites is one of the most common complications of cirrhosis, and there is a dearth of knowledge about ascites-related pathologic metabolism. In this study, 122 alcoholic liver disease (ALD) patients, including 49 cases without ascites, 18 cases with mild-ascites, and 55 cases with large-ascites (1) were established according to the International Ascites Club (2), and untargeted metabolomics coupled with pattern recognition approaches were performed to profile and extract metabolite signatures. A total of 553 metabolites were uniquely discovered in patients with ascites, of which 136 metabolites had been annotated in the human metabolome database. Principal component analysis (PCA) analysis was used to further identify 21 ascites-related fingerprints. The eigenmetabolite calculated by reducing the dimensions of the 21 metabolites could be used to effectively identify those ALD patients with or without ascites. The eigenmetabolite showed a decreasing trend during ascites production and accumulation and was negatively related to the disease progress. These metabolic fingerprints mainly belong to the metabolites in lipid metabolism and the amino acid pathway. The results imply that lipid and amino acid metabolism disturbance may play a critical role in the development of ascites in ALD patients and could be a potent prognosis marker.

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-γ- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Targeting intestinal flora and its metabolism to explore the laxative effects of rhubarb.

Rhubarb, a traditional herb, has been used in clinical practice for hundreds of years to cure constipation, but its mechanism is still not clear enough. Currently, growing evidence suggests that intestinal flora might be a potential target for the treatment of constipation. Thus, the aim of this study was to clarify the laxative effect of rhubarb via systematically analyzing the metagenome and metabolome of the gut microbiota. In this study, the laxative effects of rhubarb were investigated by loperamide-induced constipation in rats. The gut microbiota was determined by high-throughput sequencing of 16S rRNA gene. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used for fecal metabolomics analysis. The data showed that rhubarb could significantly shorten gastrointestinal transit time, increase fecal water content and defecation frequency, improve gastrointestinal hormone disruption, and protect the colon mucus layer. Analysis of 16S rRNA gene sequencing indicated that rhubarb could improve the disorder of intestinal microbiota in constipated rats. For example, beneficial bacteria such as Ligilactobacillus, Limosilalactobacillus, and Prevotellaceae UCG-001 were remarkably increased, and pathogens such as Escherichia-Shigella were significantly decreased after rhubarb treatment. Additionally, the fecal metabolic profiles of constipated rats were improved by rhubarb. After rhubarb treatment, metabolites such as chenodeoxycholic acid, cholic acid, prostaglandin F2α, and α-linolenic acid were markedly increased in constipation rats; in contrast, the metabolites such as lithocholic acid, calcidiol, and 10-hydroxystearic acid were notably reduced in constipation rats. Moreover, correlation analysis indicated a close relationship between intestinal flora, fecal metabolites, and biochemical indices associated with constipation. In conclusion, the amelioration of rhubarb in constipation might modulate the intestinal microflora and its metabolism. Moreover, the application of fecal metabolomics could provide a new strategy to uncover the mechanism of herbal medicines.Key points• Rhubarb could significantly improve gut microbiota disorder in constipation rats.• Rhubarb could markedly modulate the fecal metabolite profile of constipated rats.

[Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry].

OBJECTIVE: To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood. METHODS: We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed. RESULTS: According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 µg/mL, CD45, CD3, CD14 and CD123 at 4.0 µg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 µg/mL, CD4 at 1.0 µg/mL and DAPI at 0.1 µg/mL. The detection voltages for CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and DAPI were 450 V, 410 V, 400 V, 550 V, 405 V, 500 V, 520 V, 550 V, 550 V, 400 V, 450 V, 400 V, 580 V, and 300 V, respectively. The appropriate fluorescence compensation was determined by single-color staining and fluorescence minus one controls. The 14-color flow cytometry panel was established to analyze the main subsets of leukocytes in human peripheral blood, and peripheral blood samples from 18 healthy adults were examined, obtaining the percentages of each subset of peripheral blood leukocytes and the immunophenotypes of the main subsets. CONCLUSION: We established a 14-color panel for determining leukocyte subsets in human peripheral blood by flow cytometry, which produced stable and reliable results and was easy to operate.

The study of schizogenous formation of secretory ducts in Ferula ferulaeoides (Steud.) Korov.

The secretory ducts of Ferula ferulaeoides (Steud.) Korov. are the main tissue of synthesis, secretion, and accumulation of resin. The formation of secretory ducts is closely related to the harvest and quality of resin, but the lumen formation mode and corresponding mechanism have not been thoroughly studied. This study of F. ferulaeoides investigated the microstructure and ultrastructure of the secretory ducts from a developmental point of view. Stem samples were analyzed by light microscopy, transmission electron microscopy, and fluorescence microscopy. The data results showed (1) the walls of secretory cells were intact during the development of secretory ducts in F. ferulaeoides; (2) the plastids and endoplasmic reticulum of secretory cells participated in the synthesis of resin; (3) pectinase was involved in the degradation of the middle lamella; and (4) no features of programmed cell death during the formation of secretory ducts. The results suggested that the formation of F. ferulaeoides' secretory ducts was schizogenous, and pectinase was involved in its formation. These data may be beneficial to further explore the formation of secretory duct in other species of Ferula L. and the formation mechanism of schizogenous secretory structures.