Separation and purification of benzylester glucosides and derivatives from tubers of Gymnadenia conopsea (L.) R. Br. by linear gradient counter-current chromatography combined with elution-extrusion mode.

Tubers of Gymnadenia conopsea (L.) R. Br. (Orchidaceae), a traditional medicine and food homologous plant, has a broad application and development prospect in the food and drug industries. Benzylester glucosides, the main effective active components in this plant, are difficult to separate due to their similar structures and high polarity. In this study, linear gradient counter-current chromatography was used to separate benzylester glucosides and derivatives, combined with elution-extrusion mode. The main separation parameters were optimized, including the ratio of mobile phase and sample loading. Finally, seven compounds were successfully separated, including 4-hydroxybenzyl alcohol (1), 4-hydroxybenzaldehyde (2), dactylorhin B (3), loroglossin (4), dactylorhin A (5), 4-(ethoxymethyl) phenol (6), and militarine (7). The structures were analyzed by mass spectrometry and nuclear magnetic resonance spectrometry. According to our findings, the established method was an efficient approach to separate benzylester glucosides and derivatives from tubers of G. conopsea. The established strategy could be applied to purify other similar high-polarity compounds from complex natural products.

Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity.

UNLABELLED: An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity. IMPORTANCE: A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that their fine specificities require a structural fitness of the antibody combining site involving heavy and light chain variable domains shaped by somatic hypermutation and affinity maturation of B cells in the germinal center. Perhaps more importantly, our results demonstrate that the majority of MPER-specific antibodies are not inherently polyspecific and/or autoreactive, suggesting that polyreactivity of MPER-specific antibodies is separable from their antigen specificity.

Dioscin ameliorates silica-aggravated systemic lupus erythematosus via suppressing apoptosis and improving LC3-associated phagocytosis in MRL/lpr mice.

Inhalation of silica not only directly leads to silicosis locally, but also results in various types of autoimmune diseases systemically, most commonly systemic lupus erythematosus (SLE). Little is known about the etiopathogenesis of silica-aggravated SLE to date, however, abnormal apoptosis and impaired apoptotic clearance have been reported to be closely related to the occurrence of SLE. LC3-associated phagocytosis (LAP) is a non-canonical form of autophagy, which plays a crucial role in mediating the clearance of apoptotic cells. Here we showed that the excessive accumulation of apoptotic debris in MRL/lpr mice exposed to silica might be due to the increased cell apoptosis and defective LAP caused by silica, thus accelerating the occurrence and progression of silica-aggravated SLE. Dioscin is an active ingredient in the family of Dioscoreaceae and is reported to possess multiple pharmacological activities, including anti-inflammatory, anti-apoptotic and autophagy-promoting properties. However, its role in SLE aggravated by silica exposure has not been investigated. In our study, we confirmed that dioscin decreased the accumulation of apoptotic debris by suppressing the excessive cell apoptosis and improving the LAP of immune cells in lung and spleen, leading to subsequent dramatically ameliorated lupus-like symptoms in silica-exposed MRL/lpr mice.

Targeting STING-mediated pro-inflammatory and pro-fibrotic effects of alveolar macrophages and fibroblasts blunts silicosis caused by silica particles.

Silica is utilized extensively in industrial and commercial applications as a chemical raw material, increasing its exposure and hazardous potential to populations, with silicosis serving as an important representative. Silicosis is characterized by persistent lung inflammation and fibrosis, for which the underlying pathogenesis of silicosis is unclear. Studies have shown that the stimulating interferon gene (STING) participates in various inflammatory and fibrotic lesions. Therefore, we speculated that STING might also play a key role in silicosis. Here we found that silica particles drove the double-stranded DNA (dsDNA) release to activate the STING signal pathway, contributing to alveolar macrophages (AMs) polarization by secreting diverse cytokines. Then, multiple cytokines could generate a micro-environment to exacerbate inflammation and promote the activation of lung fibroblasts, hastening fibrosis. Intriguingly, STING was also crucial for the fibrotic effects induced by lung fibroblasts. Loss of STING could effectively inhibit silica particles-induced pro-inflammatory and pro-fibrotic effects by regulating macrophages polarization and lung fibroblasts activation to alleviate silicosis. Collectively, our results have revealed a novel pathogenesis of silica particles-caused silicosis mediated by the STING signal pathway, indicating that STING may be regarded as a promising therapeutic target in the treatment of silicosis.

The cGAS-STING Pathway: A Promising Immunotherapy Target.

With the continuous development of immunotherapy, researchers have paid more attention to the specific immune regulatory mechanisms of various immune responses in different diseases. As a novel and vital innate immune signal pathway, the cGAS-STING signal pathway activated by nucleic acid substances, interplays with other immune responses, by which it participates in regulating cancer, autoimmune and inflammatory diseases, microbial and parasitic infectious diseases, and other diseases. With the exception of its role in innate immunity, the growing list of researches demonstrated expanding roles of the cGAS-STING signal pathway in bridging the innate immunity (macrophage polarization) with the adaptive immunity (T lymphocytes differentiation). Macrophages and T lymphocytes are the most representative cells of innate immunity and adaptive immunity, respectively. Their polarization or differentiation are involved in the pathogenesis and progression of various diseases. Here we mainly summarized recent advanced discoveries of how the cGAS-STING signal pathway regulated macrophages polarization and T lymphocytes differentiation in various diseases and vaccine applications, providing a promising direction for the development and clinical application of immunotherapeutic strategies for related diseases.

[Effect of dachaihu decoction in treating acute mild pancreatitis of Gan-qi stagnant type].

OBJECTIVE: To observe the therapeutic effect of Dachaihu Decoction (DCHD) on acute mild pancreatitis of Gan-qi stagnant syndrome type. METHODS: Thirty-six acute pancreatitis patients were randomized into three groups (Group A, B and C) equally, and they were treated with conventional Western medicine (WM), WM plus placebo and WM plus DCHD, respectively for 7 days. The time for pain relieving, fasting and total hospitalization were observed, and serum amylopsin and C-reactive protein (CRP) levels were measured before treatment and at various time points after treatment. RESULTS: After treatment, amylopsin and CRP level significantly decreased in the 3 groups, comparing with those before treatment (P < 0.05, P < 0.01). In Group C, amylopsin level was obviously higher on dl, d3 and d5, CRP level was higher on dl, d3, d5 and d7, and the remission time of abdominal pain, the fasting time, as well as the hospitalization time were all significantly shorter than those in Group A and B (P < 0.05, P < 0.01). CONCLUSION: The accessory use of DCHD upon conventional Western medical treatment can strikingly shorten the course of disease and enhance the therapeutic effect on mild acute pancreatitis patients.

'Psoriasis 1' reduces psoriasis­like skin inflammation by inhibiting the VDR­mediated nuclear NF­κB and STAT signaling pathways.

'Psoriasis 1', a Chinese herbal medicine (CHM) formulation, is extensively used to treat psoriasis in China. Although this CHM formulation yields good therapeutic effect, the underlying mechanism of how this works remains unknown. The present study aimed to test the hypothesis that the CHM formulation 'psoriasis 1' inhibits vitamin D receptor (VDR)­mediated inflammation in psoriasis. To test this, a model of psoriasis was established by stimulating keratinocytes (HaCaT cells) with tumor necrosis factor (TNF)­α; these cells were subsequently transfected with a lentiviral VDR RNA interference expression vector. The expression levels of 25­hydroxyvitamin D3 (25HVD3), TNF­α, interleukin (IL)­4, IL­1, IL­17C, IL­23 and IL­6 were measured using ELISA, and the expression levels of VDR, inhibitor of nuclear factor (NF)­κB (IKK), NF­κB, signal transducer and activator of transcription (STAT) 3 and STAT4 were measured using reverse transcription­quantitative polymerase chain reaction analysis and western blotting. It was observed that 'psoriasis 1' downregulated the concentrations of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and upregulated the concentration of 25HVD3; furthermore, 'psoriasis 1' downregulated the expression levels of NF­κB, phosphorylated (p)­NF­κB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the expression level of VDR in TNF­α­induced HaCaT cells. These results suggested that 'psoriasis 1' suppressed the inflammatory response and the activation of the NF­κB and STAT signaling pathways. In addition, it was identified that silencing VDR expression decreased the levels of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and increased the level of 25HVD3; silencing VDR expression additionally downregulated the expression levels of NF­ÐºB, p­NF­ÐºB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the level of VDR in TNF­α­induced HaCaT cells. It was concluded that 'psoriasis 1' exerts inflammation­suppressive effects in psoriasis by suppressing the NF­ÐºB and STAT signaling pathways.

[Clinical observation on effect of tongfu xiere recipe in treating surgical abdominal diseases accompanied with systemic inflammatory response syndrome].

OBJECTIVE: To observe the effect of Tongfu Xiere Recipe (TXR) on surgical abdominal diseases accompanied with systemic inflammatory response syndrome (SIRS). METHODS: Forty-six patients in the post-operational stage were treated by basic therapy, including conventional treatment and antibiotics applying, and combined use of TXR, and compared with the 43 patients treated by basic therapy alone as the control group. RESULTS: Compared with the control group, the incidence of complications in the treatment group was significantly lower (P<0.01). The incidence of multiple organ dysfunction syndrome (MODS) and the mortality in the treatment group showed an decreasing tendency. Moreover, levels of plasma endotoxin, TNF-alpha, IL-6 and IL-8 in the treatment group after treatment decreased significantly, while IL-2 level increased (P < 0.01), and showed a significant difference (P < 0.01) as compared with those in the control group. CONCLUSION: The combined therapy can decrease the levels of blood endotoxin and inflammation promoting cytokine and increase the level of cytokine that directly related with immune function to regulate immunity and inhibit the inflammatory reaction, and furthermore, to prevent MODS and decrease the mortality of patients with surgical abdominal diseases accompanied SIRS.

Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41.

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.

Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization.

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.