Metabolomics analysis reveals the metabolite profiles of Rheum tanguticum grown under different altitudinal gradients.

BACKGROUND: Plant growth and quality are often affected by environmental factors, including geographical location, climate, and soil. In this study, we describe the effect of altitudinal differences on the growth and active ingredients in Rheum tanguticum Maxim. ex Balf. (R. tanguticum), a traditional Chinese medicinal herb known for its laxative properties. RESULTS: The results showed that plants grown at lower altitudes had better growth performances than those in higher altitude areas. The yield varied by 2.45-23.68 times with altitude, reaching a maximum of 102.01 t/ha. In addition, total anthraquinone and total sennoside contents decreased with increasing altitude, whereas total tannins increased with increasing altitude. The total anthraquinone content of the indicator compound reached 5.15% at five experimental sites, which exceeded the Chinese Pharmacopoeia standard by 70.87%. The content of the other two categories of active ingredients reached a maximum value of 0.94% (total sennosides) and 2.65% (total tannins). Redundancy analysis revealed that annual rainfall, annual average temperature, annual sunshine hours, and pH significantly affected growth and active ingredients. Moreover, key metabolites, such as flavonoids, amino acids and their derivatives, phenolic acids, lipids, and terpenes, were differentially expressed between samples from low- and high-altitude cultivation areas. These metabolites were enriched in the flavonoid and flavonol biosynthetic pathway and the monoterpene biosynthetic pathway. CONCLUSIONS: These results suggest that high anthraquinone content was observed in the lowest-latitude cultivation area due to low rainfall and alkaline soil pH. Key metabolites were significantly upregulated in high-latitude cultivation areas. These results provide a scientific basis for quality control and the systematic cultivation of R. tanguticum.

Exogenous regulation of macronutrients promotes the accumulation of alkaloid yield in anisodus tanguticus (Maxim.) pascher.

BACKGROUND: Anisodus tanguticus (Maxim.) Pascher (A. tanguticus) is a valuable botanical for extracting tropane alkaloids, which are widely used in the pharmaceutical industry. Implementing appropriate cultivation methods can improve both the quality and yield of A. tanguticus. A two-year field experiment was conducted from 2021 to 2023 using a single-factor randomized complete block design replicated three times. The study examined the effects of different nutrient levels (nitrogen: 0, 75, 150, 225, 300, 375 kg/ha; phosphorus: 0, 600, 750, 900, 1050, 1200 kg/ha; potassium: 0, 75, 112.5, 150, 187.5, 225 kg/ha) on the growth, primary alkaloid contents, and alkaloid yield of A. tanguticus at different growth stages (S-Greening, S-Growing, S-Wilting; T-Greening, T-Growing, and T-Wilting) in both the roots and aboveground portions. RESULTS: Our results demonstrate that nutrient levels significantly affect the growth and alkaloid accumulation in A. tanguticus. High nitrogen levels (375 kg/ha) notably increased both root and aboveground biomass, while phosphorus had a minimal effect, especially on aboveground biomass. For alkaloid content (scopolamine, anisodamine, anisodine, atropine), a moderate nitrogen level (225 kg/ha) was most effective, followed by low potassium (75 kg/ha), with phosphorus showing a limited impact. Increased phosphorus levels led to a decrease in scopolamine content. During the T-Growing period, moderate nitrogen addition (225 kg/ha) yielded the highest alkaloid levels per unit area (205.79 kg/ha). In the T-Wilting period, low potassium (75 kg/ha) and low phosphorus (750 kg/ha) resulted in alkaloid levels of 146.91 kg/ha and 142.18 kg/ha, respectively. This indicates nitrogen has the most substantial effect on alkaloid accumulation, followed by potassium and phosphorus. The Douglas production function analysis suggests focusing on root biomass and the accumulation of scopolamine and atropine in roots to maximize alkaloid yield in A. tanguticus cultivation. CONCLUSIONS: Our findings show that the optimum harvesting period for A. tanguticus is the T-Wilting period, and that the optimal nitrogen addition is 225 kg/ha, the optimal potassium addition is 75 kg/ha, and the optimal phosphorus addition is 600 kg/ha or less.

Injectable Hydrogels Based on Hyaluronic Acid and Gelatin Combined with Salvianolic Acid B and Vascular Endothelial Growth Factor for Treatment of Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) leads to structural damage in the brain, and is one of the major causes of disability and death in the world. Herein, we developed a composite injectable hydrogel (HA/Gel) composed of hyaluronic acid (HA) and gelatin (Gel), loaded with vascular endothelial growth factor (VEGF) and salvianolic acid B (SAB) for treatment of TBI. The HA/Gel hydrogels were formed by the coupling of phenol-rich tyramine-modified HA (HA-TA) and tyramine-modified Gel (Gel-TA) catalyzed by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2). SEM results showed that HA/Gel hydrogel had a porous structure. Rheological test results showed that the hydrogel possessed appropriate rheological properties, and UV spectrophotometry results showed that the hydrogel exhibited excellent SAB release performance. The results of LIVE/DEAD staining, CCK-8 and Phalloidin/DAPI fluorescence staining showed that the HA/Gel hydrogel possessed good cell biocompatibility. Moreover, the hydrogels loaded with SAB and VEGF (HA/Gel/SAB/VEGF) could effectively promote the proliferation of bone marrow mesenchymal stem cells (BMSCs). In addition, the results of H&E staining, CD31 and α-SMA immunofluorescence staining showed that the HA/Gel/SAB/VEGF hydrogel possessed good in vivo biocompatibility and pro-angiogenic ability. Furthermore, immunohistochemical results showed that the injection of HA/Gel/SAB/VEGF hydrogel to the injury site could effectively reduce the volume of defective tissues in traumatic brain injured mice. Our results suggest that the injection of HA/Gel hydrogel loaded with SAB and VEGF might provide a new approach for therapeutic brain tissue repair after traumatic brain injury.

Phylogeography and ecological niche modeling implicate multiple microrefugia of Swertia tetraptera during quaternary glaciations.

BACKGROUND: Climate fluctuations during the Pleistocene and mountain uplift are vital driving forces affecting geographic distribution. Here, we ask how an annual plant responded to the Pleistocene glacial cycles. METHODS: In this study, we analyzed the population demographic history of the annual herb Swertia tetraptera Maxim (Gentianaceae) endemic to Qinghai-Tibetan Plateau (QTP). A total of 301 individuals from 35 populations of S. tetraptera were analyzed based on two maternally inherited chloroplast fragments (trnL-trnF and trnS-trnG). Phylogeographic analysis was combined with species distribution modeling to detect the genetic variations in S. tetraptera. RESULTS: The genetic diversity of S. tetraptera was high, likely due to its wide natural range, high proportion of endemic haplotypes and evolutionary history. Fifty-four haplotypes were identified in S. tetraptera. Only a few haplotypes were widespread (Hap_4, Hap_1, Hap_3), which were dispersed throughout the present geographical range of S. tetraptera, while many haplotypes were confined to single populations. The cpDNA dataset showed that phylogeographic structuring was lacking across the distribution range of S. tetraptera. Analyses of molecular variance showed that most genetic variation was found within populations (70.51%). In addition, the relationships of the haplotypes were almost completely unresolved by phylogenetic reconstruction. Both mismatch distribution analysis and neutrality tests showed a recent expansion across the distribution range of S. tetraptera. The MaxEnt analysis showed that S. tetraptera had a narrow distribution range during the Last Glacial Maximum (LGM) and a wide distribution range during the current time, with predictions into the future showing the distribution range of S. tetraptera expanding. CONCLUSION: Our study implies that the current geographic and genetic distribution of S. tetraptera is likely to have been shaped by Quaternary periods. Multiple microrefugia of S. tetraptera existed during Quaternary glaciations. Rapid intraspecific diversification and hybridization and/or introgression may have played a vital role in shaping the current distribution patterns of S. tetraptera. The distribution range of S. tetraptera appeared to have experienced contraction during the LGM; in the future, when the global climate becomes warmer with rising carbon dioxide levels, the distribution of S. tetraptera will expand.

A core root bacteria contribute to plant growth and anisodine accumulation of Anisodus tanguticus.

BACKGROUND: Although it is well recognized that core root microorganisms contribute to plant health and productivity, little is known about their role to the accumulation of secondary metabolites. The roots of Anisodus tanguticus, a traditional herbal medication utilized by Tibetan medicine, are rich in tropane alkaloids. We collected wild A. tanguticus populations throughout a 1500 km transect on the Qinghai-Tibetan Plateau. RESULTS: Our results showed that despite sampling at a distance of 1500 km, the root of A. tanguticus selectively recruits core root bacteria. We obtained 102 root bacterial core OTUs, and although their number only accounted for 2.99% of the total, their relative abundance accounted for 73% of the total. Spearman correlation and random forest analyses revealed that the composition of core root microbiomes was related to anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. Among them, the main role is played by Rhizobacter, Variovorax, Polaromonas, and Mycobacterium genus that are significantly enriched in roots. Functional prediction by FAPROTAX showed that nitrogen-cycling microorganisms and pathogenic bacteria are strongly associated with anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. CONCLUSIONS: Our findings show that the root selectively recruits core root bacteria and revealed that the core microbiomes and microbial functions potentially contributed to the anisodine contents, aboveground biomass and nitrogen contents of the plant. This work may increase our understanding of the interactions between microorganisms and plants and improve our ability to manage root microbiota to promote sustainable production of herbal medicines.

Soluble Protein Content, Bioactive Compounds and the Antioxidant Activity in Seeds of Ten Rheum tanguticum Lines from Qinghai-Tibet Plateau.

Rheum tanguticum (Rh. tanguticum) is a Chinese medicinal plant traditionally used in the treatment of constipation. As a byproduct, the seeds of this plant are rich in nutrients and phytochemicals. This study aimed to determine and assess seed germination ability, seed physical characteristics, soluble protein content, chemical constituents and antioxidant capacity from different breeding lines, to promote the development and utilization of seed resources. Significant differences were observed for the soluble protein content and antioxidant assays among the ten lines. The contents of aloe-emodin, rhein and catechins accumulated in seeds were extremely low and significantly different from those in roots. In contrast, emodin and chrysophanol were abundant in seeds, and significant differences were observed between seeds and roots. It was found that associations between gallic acid and catechins were not significant for either soluble protein or antioxidant capacity. There was a significantly positive correlation between the contents of four anthraquinones (aloe-emodin, rhein, emodin and chrysophanol) and soluble protein. Seeds have potent antioxidative capacity and relatively high levels of soluble protein content. The rich chemical composition of seeds can be widely used in the medical industry for further development.

Salvianolic-Acid-B-Loaded HA Self-Healing Hydrogel Promotes Diabetic Wound Healing through Promotion of Anti-Inflammation and Angiogenesis.

Inflammatory dysfunction and angiogenesis inhibition are two main factors leading to the delayed healing of diabetic wounds. Hydrogels with anti-inflammatory and angiogenesis-promoting effects have been considered as promising wound care materials. Herein, a salvianolic acid B (SAB)-loaded hyaluronic acid (HA) self-healing hydrogel (HA/SAB) with anti-inflammatory and pro-angiogenesis capacities for diabetic wound healing is reported. The HA hydrogel was prepared via the covalent cross-linking of aldehyde groups in oxidized HA (OHA) and hydrazide groups in adipic dihydrazide (ADH)-modified HA (HA-ADH) with the formation of reversible acylhydrazone bonds. The obtained HA hydrogel exhibited multiple favorable properties such as porous structures, excellent self-healing properties, a sustainable release capacity of SAB, as well as excellent cytocompatibility. In addition, the effects of the SAB-loaded HA self-healing hydrogel were investigated via a full-thickness skin defect model using diabetic rats. The HA/SAB hydrogel showed enhanced skin regeneration effects with accelerated wound closure, shorter remaining dermal space length, thicker granulation tissue formation, and more collagen deposition. Furthermore, reduced inflammatory response and enhanced vascularization were found with HA/SAB2.5 hydrogel-treated wounds, indicating that the hydrogel promotes diabetic wound healing through the promotion of anti-inflammation and angiogenesis. Our results suggest that the fabricated SAB-loaded HA self-healing hydrogel is promising as a wound dressing for the treatment of diabetic wounds.

The Signaling Pathways Induced by Exosomes in Promoting Diabetic Wound Healing: A Mini-Review.

Impaired healing of diabetic wounds harms patients' quality of life and even leads to disability and death, which is an urgent issue to be solved clinically. Despite the great progress that has been achieved, it remains a worldwide challenge to develop effective therapeutic treatments for diabetic wounds. Recently, exosomes have attracted special attention because they can be involved in immune response, antigen presentation, cell migration, cell differentiation, tumor invasion and other processes. Meanwhile, exosomes have been proven to hold great potential in the treatment of diabetic wounds. Mechanistic studies of exosomes based on signaling pathways could not only help to uncover the mechanisms by which exosomes promote diabetic wound healing but could also provide a theoretical basis for the clinical application of exosomes. Herein, our mini-review aims to summarize the progress of research on the use of various exosomes derived from different cell types to promote diabetic wound healing, with a focus on the classical signaling pathways, including PI3K/Akt, Wnt, NF-κB, MAPK, Notch, Nrf2, HIF-1α/VEGF and TGF-ß/Smad. The results show that exosomes could regulate these signaling pathways to down-regulate inflammation, reduce oxidative stress, increase angiogenesis, promote fibroblast proliferation, induce re-epithelization and inhibit scar formation, making exosomes attractive candidates for the treatment of diabetic wounds.

Immune suppressive checkpoint interactions in the tumour microenvironment of primary liver cancers.

Liver cancer is one of the most prevalent cancers, and the third most common cause of cancer-related mortality worldwide. The therapeutic options for the main types of primary liver cancer-hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA)-are very limited. HCC and CCA are immunogenic cancers, but effective immune-mediated tumour control is prevented by their immunosuppressive tumour microenvironment. Despite the critical involvement of key co-inhibitory immune checkpoint interactions in immunosuppression in liver cancer, only a minority of patients with HCC respond to monotherapy using approved checkpoint inhibitor antibodies. To develop effective (combinatorial) therapeutic immune checkpoint strategies for liver cancer, in-depth knowledge of the different mechanisms that contribute to intratumoral immunosuppression is needed. Here, we review the co-inhibitory pathways that are known to suppress intratumoral T cells in HCC and CCA. We provide a detailed description of insights from preclinical studies in cellular crosstalk within the tumour microenvironment that results in interactions between co-inhibitory receptors on different T-cell subsets and their ligands on other cell types, including tumour cells. We suggest alternative immune checkpoints as promising targets, and draw attention to the possibility of combined targeting of co-inhibitory and co-stimulatory pathways to abrogate immunosuppression.

Modelling immune cytotoxicity for cholangiocarcinoma with tumour-derived organoids and effector T cells.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

Major Chemical Compounds and Mineral Elements of Astragalus membranaceus Cultivated on the Qinghai-Tibet Plateau with Different Planting Densities.

Astragalus membranaceus is a well-known herb that is widely used in the food and pharmaceutical industries. However, its commercial development has been limited due to wild resource shortages. This study was conducted in 2018 and 2019 to assess the effect of planting density on the major chemical compounds and mineral elements and biomass yield of A. membranaceus. The biomass yield (7,700.956 kg) reached the maximum at M2 planting density in 2018. In 2019, astragaloside IV reached its maximum concentration (0.117 %) at M2 group, which was significantly different from the concentrations obtained at the other groups. Calycosin-7-O-ß-D-glucoside (0.062 %) reach its maximum concentration in 2019 at M5, but not significant with M2. The concentration of major chemical compounds among the five groups in 2018 and 2019 all conformed to the Chinese Pharmacopoeia standards. In 2018, the mineral elements (Al, Ba, Fe, Li and Mn) content was higher at M2 than other groups. However, a general decrease in the mineral elements content was observed at M2 group in 2019. Enrichment analysis demonstrated that the enrichment capacity was highest for phosphorus. In conclusion, according to the TOPSIS results, M2 planting density was recommended as the optimal application. For optimal economic benefits, A. membranaceus should be harvested when it is 2 years old.

Non-target metabolomics revealed the differences between Rh. tanguticum plants growing under canopy and open habitats.

BACKGROUND: Rheum tanguticum (Rh. tanguticum) is an important traditional Chinese medicine plant, "Dahuang", which contains productive metabolites and occupies wide habitats on the Qinghai-Tibet plateau. Plants occupying wide habitats usually vary in phenotypes such as in morphology and metabolism, thereby developing into different ecotypes. Under canopy and open habitats are a pair of dissimilar habitats which possess Rh. tanguticum plants. However, few studies have focused on the effect of habitats on Rh. tanguticum growth, particularly combining morphological and metabolic changes. This study focused on Rh. tanguticum plants growing in under canopy and open habitats where morphology and metabolism changes were quantified using non-target metabolism methods. RESULTS: The obtained results indicated that the two dissimilar habitats led to Rh. tanguticum developing into two distinct ecotypes where the morphology and metabolism were simultaneously changed. Under canopy habitats bred morphologically smaller Rh. tanguticum plants which had a higher level of metabolites (22 out of 31) which included five flavonoids, four isoflavonoids, and three anthracenes. On the other hand, the open habitats produced morphologically larger Rh. tanguticum plants having a higher level of metabolites (9 out of 31) including four flavonoids. 6 of the 31 metabolites were predicted to have effect targets, include 4 represent for under canopy habitats and 2 for open habitats. Totally, 208 targets were connected, among which 42 were communal targets for both under canopy and open habitats represent compounds, and 100 and 66 were unique targets for under canopy superior compounds and open habitats superior compounds, respectively. In addition, aloe-emodin, emodin, chrysophanol, physcion, sennoside A and sennoside B were all more accumulated in under canopy habitats, and among which aloe-emodin, emodin, chrysophanol and physcion were significantly higher in under canopy habitats. CONCLUSIONS: This study determined that Rh. tanguticum growing in under canopy and in open habitats developed into two distinct ecotypes with morphological and metabolic differences. Results of network pharmacology study has indicated that "Dahuang" coming from different habitats, such as under canopy and open habitats, are different in effect targets and thus may have different medicinal use. According to target metabolomics, under canopy habitats may grow better "Dahuang".

Above- and below-ground resource acquisition strategies determine plant species responses to nitrogen enrichment.

BACKGROUND AND AIMS: Knowledge of plant resource acquisition strategies is crucial for understanding the mechanisms mediating the responses of ecosystems to external nitrogen (N) input. However, few studies have considered the joint effects of above-ground (light) and below-ground (nutrient) resource acquisition strategies in regulating plant species responses to N enrichment. Here, we quantified the effects of light and non-N nutrient acquisition capacities on species relative abundance in the case of extra N input. METHODS: Based on an N-manipulation experiment in a Tibetan alpine steppe, we determined the responses of species relative abundances and light and nutrient acquisition capacities to N enrichment for two species with different resource acquisition strategies (the taller Stipa purpurea, which is colonized by arbuscular mycorrhizal fungi, and the shorter Carex stenophylloides, which has cluster roots). Structural equation models were developed to explore the relative effects of light and nutrient acquisition on species relative abundance along the N addition gradient. KEY RESULTS: We found that the relative abundance of taller S. purpurea increased with the improved light acquisition along the N addition gradient. In contrast, the shorter C. stenophylloides, with cluster roots, excelled in acquiring phosphorus (P) so as to elevate its leaf P concentration under N enrichment by producing large amounts of carboxylate exudates that mobilized moderately labile and recalcitrant soil P forms. The increased leaf P concentration of C. stenophylloides enhanced its light use efficiency and promoted its relative abundance even in the shade of taller competitors. CONCLUSIONS: Our findings highlight that the combined effects of above-ground (light) and below-ground (nutrient) resources rather than light alone (the prevailing perspective) determine the responses of grassland community structure to N enrichment.

Microbial abundance and community in constructed wetlands planted with Phragmites australis and Typha orientalis in winter.

NOVELTY STATEMENT: The microbial abundance and communities were characterized in CWs with different plant species during winter. Better removal efficiency with high microbial abundance and diversified microbial community were found in CWs planted with Phragmites australis. This study confirmed that in winter, withered plants in CWs can effectively remove NH4+-N and COD by affecting microbial abundance and community structure.

Enrichment of the tumour immune microenvironment in patients with desmoplastic colorectal liver metastasis.

BACKGROUND: Patients with resected colorectal liver metastasis (CRLM) who display only the desmoplastic histopathological growth pattern (dHGP) exhibit superior survival compared to patients with any non-desmoplastic growth (non-dHGP). The aim of this study was to compare the tumour microenvironment between dHGP and non-dHGP. METHODS: The tumour microenvironment was investigated in three cohorts of chemo-naive patients surgically treated for CRLM. In cohort A semi-quantitative immunohistochemistry was performed, in cohort B intratumoural and peritumoural T cells were counted using immunohistochemistry and digital image analysis, and in cohort C the relative proportions of individual T cell subsets were determined by flow cytometry. RESULTS: One hundred and seventeen, 34, and 79 patients were included in cohorts A, B, and C, with dHGP being observed in 27%, 29%, and 15% of patients, respectively. Cohorts A and B independently demonstrated peritumoural and intratumoural enrichment of cytotoxic CD8+ T cells in dHGP, as well as a higher CD8+/CD4+ ratio (cohort A). Flow cytometric analysis of fresh tumour tissues in cohort C confirmed these results; dHGP was associated with higher CD8+ and lower CD4+ T cell subsets, resulting in a higher CD8+/CD4+ ratio. CONCLUSION: The tumour microenvironment of patients with dHGP is characterised by an increased and distinctly cytotoxic immune infiltrate, providing a potential explanation for their superior survival.

Role of the neurovascular unit in the process of cerebral ischemic injury.

Cerebral ischemic injury exhibits both high morbidity and mortality worldwide. Traditional research of the pathogenesis of cerebral ischemic injury has focused on separate analyses of the involved cell types. In recent years, the neurovascular unit (NVU) mechanism of cerebral ischemic injury has been proposed in modern medicine. Hence, more effective strategies for the treatment of cerebral ischemic injury may be provided through comprehensive analysis of brain cells and the extracellular matrix. However, recent studies that have investigated the function of the NVU in cerebral ischemic injury have been insufficient. In addition, the metabolism and energy conversion of the NVU depend on interactions among multiple cell types, which make it difficult to identify the unique contribution of each cell type. Therefore, in the present review, we comprehensively summarize the regulatory effects and recovery mechanisms of four major cell types (i.e., astrocytes, microglia, brain-microvascular endothelial cells, and neurons) in the NVU under cerebral ischemic injury, as well as discuss the interactions among these cell types in the NVU. Furthermore, we discuss the common signaling pathways and signaling factors that mediate cerebral ischemic injury in the NVU, which may help to provide a theoretical basis for the comprehensive elucidation of cerebral ischemic injury.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Nitrogen Fertilizer Levels Affect the Growth and Quality Parameters of Astragalus mongolica.

Owing to overexploitation, wild resources of Astragalus mongolica, a Chinese herbal plant that is widely distributed in the arid and semi-arid areas of Northern China, have gradually become exhausted, and therefore, commercial cultivation is increasingly important to meet the growing demand for astragalus and reduce the pressure on wild populations. Nitrogen level is an important factor that affects the yield and quality of A. mongolica. However, uniform standards for fertilization among production areas have not yet been determined. In this study, the effect of nitrogen fertilizer treatment on the yield and quality of A. mongolica in the Qinghai-Tibet Plateau was explored using a control treatment (no added nitrogen, N0) and five different nutrient levels: 37.5 kg/ha (N1), 75 kg/ha (N2), 112.5 kg/ha (N3), 150 kg/ha (N4), and 187.5 kg/ha (N5). According to grey relational analysis, the optimal nitrogen fertilizer treatment was the N4 level followed by the N5 and N2 levels. Nitrogen fertilizer significantly increased the root biomass, plant height, root length, and root diameter. However, nitrogen fertilization had no significant effect on the content of Astragaloside IV and mullein isoflavone glucoside. The content of ononin and calycosin continually accumulated throughout the growing period. The results showed that the ononin and calycosin content under N4 and N2 is higher than other levels and there is not significantly different between different nitrogen fertilizer levels about them. The content of formononetin decreased gradually with the progression of the growing season. The optimal nitrogen fertilizer treatment for A. mongolica is recommended to be 150 kg/ha and the content of active compounds and yield were observed to reach the maximum in October.

GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+ FoxP3+ regulatory T cells (Treg) showed higher GITR- expression than effector T-cell subsets. The highest expression of GITR was found on CD4+ FoxP3hi CD45RA- activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules.

BACKGROUND & AIMS: Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. METHODS: We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. RESULTS: Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. CONCLUSIONS: Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. LAY SUMMARY: The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.