五虎汤对咳嗽变异性哮喘大鼠血清IL-33和TSLP水平的影响及机制研究

摘要 目的：探讨与研究五虎汤对咳嗽变异性哮喘大鼠血清白介素（Interleukin,IL）-33和胸腺基质淋巴细胞生成素（Thymic stromal lymphopoietin,TSLP）水平的影响及机制。方法：咳嗽变异性哮喘大鼠（n=48）随机平分为三组-模型组、孟鲁司特钠组与五虎汤组,模型组给予蒸馏水10 mL/(kg?d)灌胃,孟鲁司特钠组给予孟鲁司特钠10 mg/(kg?d)灌胃,五虎汤组给予五虎汤药液50 g/(kg?d)灌胃,1次/d,持续4周。结果：孟鲁司特钠组与五虎汤组治疗第2周、第4周的1min内桡鼻、打喷嚏次数少于模型组（P<0.05）,五虎汤少于孟鲁司特钠组(P<0.05)。孟鲁司特钠组与五虎汤组治疗第4周、第8周的白细胞计数及嗜酸性粒细胞百分率少于模型组,五虎汤少于孟鲁司特钠组（P<0.05）。孟鲁司特钠组与五虎汤组治疗第4周、第8周的血清IL-33、TSLP含量少于模型组（P<0.05）,五虎汤少于孟鲁司特钠组（P<0.05）。孟鲁司特钠组与五虎汤组治疗第4周、第8周的肺组织紧密连接蛋白-1（Zonula occludens-1,ZO-1）、钙粘附蛋白E（E-cadherin）蛋白相对表达水平高于模型组（P<0.05）,五虎汤组高于孟鲁司特钠组（P<0.05）。结论：五虎汤在咳嗽变异性哮喘大鼠的应用可能通过上调ZO-1、E-cadherin蛋白的表达,抑制血清IL-33和TSLP水平的表达,能促进缓解症状,降低白细胞计数及嗜酸性粒细胞百分率。     

重症肺炎患者血清IL-18、IL-23、IL-33与肠道菌群和临床转归的关系分析

摘要 目的：研究重症肺炎（SP）患者血清白细胞介素-18（IL-18）、白细胞介素-23（IL-23）、白细胞介素-33（IL-33）与肠道菌群和临床转归的关系。方法：选取2019年12月～2022年6月济南市人民医院收治的90例SP患者,记作研究组。另取同期收治的90例普通肺炎患者作为对照组。对比两组血清IL-18、IL-23、IL-33与肠道菌群含量,并以Pearson相关性分析两者的关系。此外,将研究组患者按照治疗后临床转归情况的差异分为好转组60例及恶化组30例,多因素Logistic回归分析SP患者临床转归的影响因素。结果：研究组血清IL-18、IL-23、IL-33水平高于对照组（均P＜0.05）。研究组大肠埃希菌、肠球菌含量均高于对照组,而拟杆菌、双歧杆菌及乳酸杆菌含量均低于对照组（均P＜0.05）。经Pearson相关性分析可得：血清IL-18、IL-23、IL-33与大肠埃希菌、肠球菌含量呈正相关关系,而与拟杆菌、双歧杆菌及乳酸杆菌含量呈负相关关系（均P＜0.05）。好转组年龄及血清IL-18、IL-23、IL-33水平均低于恶化组,且机械通气与长期卧床人数占比均低于恶化组（均P＜0.05）。经多因素Logistic回归分析发现：年龄大、机械通气、长期卧床以及血清IL-18、IL-23、IL-33水平高均是SP患者治疗后恶化的危险因素（均P＜0.05）。结论：SP患者血清IL-18、IL-23、IL-33与肠道菌群密切相关,且随着上述三项血清指标水平的升高,患者临床转归越差。     

上皮源性细胞因子IL-33、IL-25和TSLP在哮喘发病机制中的作用

哮喘(asthma)是一种以气道高反应性、慢性气道炎症、气道重塑和可逆性的气流受阻为特征的常见慢性呼吸系统疾病。近年来,研究发现气道上皮细胞在霉菌、尘螨、花粉、病毒感染、空气污染物等各种损伤因素的作用下,可释放细胞因子白细胞介素-33(interleukin-33,IL-33)、白细胞介素-25(interleukin-25,IL-25)和胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP),这些细胞因子不仅可作用于2型辅助性T细胞(type 2 helper T cells,Th2 cells),同时也可作用于固有淋巴样2型细胞(group 2 innate lymphoid cells,ILC2s),通过释放Th2型细胞因子,参与哮喘的发生与发展。尽管这3种细胞因子在哮喘的发生与发展中均起到重要作用,但其在哮喘病理、生理学效应及作用方式上并非完全相同。现就这3种上皮源性细胞因子IL-33、IL-25和TSLP在哮喘发病机制中的作用作一概述。     

Development of a pharmacodynamic model of murine malaria and antimalarial treatment with dihydroartemisinin

Antimalarial treatment strategies based on in vitro studies are limited by the paucity of pharmacodynamic information for dosage regimen design. We postulated that a murine model could be used for pre-clinical stages of drug development, especially in dose–response studies and evaluation of combination therapies. Swiss mice infected with Plasmodium berghei parasites (2–5% starting parasitaemia) were given dihydroartemisinin (0–100 mg/kg single dose). Parasite density was regularly determined from thin blood films. A parasite population growth model comprising parasite multiplication, decline in erythrocyte count with increasing parasitaemia and parasite clearance after drug administration was developed. This model described the rise in parasitaemia following inoculation, the nadir following dihydroartemisinin administration, and the subsequent resurgence of parasitaemia (analogous to ‘recrudescence’). At doses of 10, 30 and 100 mg/kg dihydroartemisinin, there was a graded response with 2.5 ± 1, 5 ± 1 and 12 ± 4-fold decreases in parasitaemia, respectively. The nadir parasitaemia (at 21–27 h) was also dose-dependent. This study demonstrates that a murine malaria pharmacodynamic model is a valuable tool for understanding how single drugs and their dosing schedules alter the time course and level of infection.     

Development of experimental cerebral malaria is independent of IL-23 and IL-17

Cerebral malaria (CM) is the most severe complication of Plasmodium infection. Although inappropriate immune responses to Plasmodium falciparum are reported as the major causes of CM, the precise mechanisms for development remain unclear. IL-23 and IL-17 have critical roles in the onset of autoimmunity and inflammatory diseases triggered by microbial infections. Thus, we investigated the influence of IL-23 and IL-17 on experimental CM (ECM) using Plasmodium berghei ANKA infection of C57BL/6 mice. Both IL-23 deficient mice and wild-type (WT) mice developed ECM. IL-17 deficient mice also developed ECM, while IL-17 producing cells other than CD4⁺ T cells (Th17) were increased in WT mice that developed ECM. In conclusion, this study showed that IL-23 and IL-17 are not involved in ECM development.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.     

The C5 convertase is not required for activation of the terminal complement pathway in murine experimental cerebral malaria

Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5(-/-) mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4(-/-) and factor B(-/-) mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3(-/-) mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3(-/-) mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway.     

The IL-1 receptor accessory protein (AcP) is required for IL-33 signaling and soluble AcP enhances the ability of soluble ST2 to inhibit IL-33

Interleukin (IL)-33 (or IL-1F11) was recently identified as a ligand for the orphan IL-1 receptor family member T1/ST2 (ST2). IL-33 belongs to the IL-1 cytokine family and, upon binding to ST2, induces intracellular signals similar to those utilized by IL-1. The effects of other IL-1 family cytokines are mediated by their binding to a specific receptor and the recruitment of a co-receptor required for elicitation of signaling. The aim of this study was to characterize the co-receptor involved in IL-33 signaling. Immunoprecipitation confirmed that IL-33 specifically binds ST2 and revealed that cellular IL-1 receptor accessory protein (AcP) associates with ST2 in a ligand-dependent manner. Receptor binding measurements demonstrated that the affinity of mouse (m)IL-33 for ST2 is increased by 4-fold in presence of AcP. IL-33 dose-dependently stimulated IL-6 secretion from wild-type (WT) mast cells, while no effect of IL-33 was observed with mast cells derived from AcP-deficient mice. Finally, soluble (s)ST2-Fc and sAcP-Fc acted synergistically to inhibit IL-33 activity. These observations identify AcP as a shared co-receptor within the IL-1 family that is essential for IL-33 signaling and suggest a novel role for sAcP in modulating the activity of IL-33.     

Modulation of IL-33/ST2 signaling as a potential new therapeutic target for cardiovascular diseases

IL-33 belongs to the IL-1 family of cytokines, which function as inducers of Th2 cytokine production by binding with ST2L and IL-1RAcP. This, in turn, activates various signaling pathways, including the mitogen-activated protein kinase (MAPK), the inhibitor of Kappa-B kinase (IKK) pathway, and the phospholipase D-sphingosine kinase pathway. IL-33 has demonstrated protective effects against various cardiovascular diseases (CVDs) by inducing Th2 cytokines and promoting alternative activating M2 polarization. However, the soluble decoy form of ST2 (sST2) mitigates the biological effects of IL-33, exacerbating CVDs. Furthermore, IL-33 also plays a significant role in the development of asthma, arthritis, atopic dermatitis, and anaphylaxis through the activation of Th2 cells and mast cells. In this review, we aim to demonstrate the protective role of IL-33 against CVDs from 2005 to the present and explore the potential of serum soluble ST2 (sST2) as a diagnostic biomarker for CVDs. Therefore, IL-33 holds promise as a potential therapeutic target for the treatment of CVDs.     

A cell-free Salmonella typhimurium extract modulates interleukin-2 (IL-2) receptor expression but not IL-2-stimulated responses of murine splenic lymphocytes

Abstract In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2). In this study, we found that a cell-free S. typhimurium extract modulated IL-2 receptor (IL-2R) expression on phytohemagglutinin (PHA)-stimulated murine spleen cells and this was a mechanism of T-cell non-responsiveness to IL-2, but did not affect IL-2 binding to IL-2R and the consequent responses. Western blotting using anti-phosphotyrosine antibodies showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in PHA-activated murine splenic T-cells, which express a high-affinity IL-2R (α- and β-chains), was not affected by treatment with the S. typhimurium cell-free extract. Furthermore, PHA-activated spleen T-cells responded to recombinant IL-2 and this was not inhibited by the extract. Surprisingly, IL-2R expression was augmented by treatment with the extract, although this was independent of IL-2 production. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract was associated with augmentation of IL-2R expression, rather than down-regulation of the IL-2 response. This may be a mechanism responsible for the Salmonella extract-evoked suppression of mitogen-induced T-cell proliferation.     

The effect of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> on TSLP,IL-33 and IL-25 expression in respiratory epithelium

Background

Airway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D₃ in asthma severity and treatment response.

Objective

Our study evaluated the influence of an active form of vitamin D₃ on the expression of selected mediators of allergic inflammation in the respiratory epithelium.

Material and Methods

Primary nasal and bronchial epithelial cells were exposed to1,25D₃ for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants usingELISAandmRNAlevels in cells by real time PCR.

Results

1,25D₃ increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D₃ mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D₃ decreased IL-33 expression. Simultaneously 1,25D₃ augmented IL-25 production in the same model of stimulation.

Conclusion

Our study revealed the dual nature of vitamin D₃ manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.

     

Interleukin-33/ST2 signaling promotes production of interleukin-6 and interleukin-8 in systemic inflammation in cigarette smoke-induced chronic obstructive pulmonary disease mice

Interleukin-33 is a newly described member of the interleukin-1 family. Recent research suggests that IL-33 is increased in lungs and plays a critical role in chronic airway inflammation in cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mice. To determine the role of IL-33 in systemic inflammation, we induced COPD mice models by passive cigarette smoking and identified the IL-33 expression in bronchial endothelial cells and peripheral blood mononuclear cells (PBMCs) of them. After isolation, PBMCs were cultured and stimulated in vitro. We measured expressions of interleukin-6 and interleukin-8 in PBMCs in different groups. The expression of IL-33 in bronchial endothelial cells and PBMCs of COPD mice were highly expressed. Stimulated by cigarette smoke extract (CSE), the expression of IL-6 and IL-8 were induced and enhanced by IL-33. PBMCs of COPD mice produced more IL-6 and IL-8 stimulated by CSE and IL-33. Expression of IL-6 and IL-8 were decreased when stimulated by IL-33 together with soluble ST2. The mRNA production of ST2 in IL-33 stimulated PBMCs was increased. Being pretreated with several kinds of MAPK inhibitors, the secretions of IL-6 and IL-8 in PBMCs did not decrease except for the p38 MAPK inhibitor. We found that IL-33 could induce and enhance the expression of IL-6 and IL-8 in PBMCs of COPD mice via p38 MAPK pathway, and it is a promoter of the IL-6 and IL-8 production in systemic inflammation in COPD mice.     

牙周-正畸联合治疗对侵袭性牙周炎患者牙周功能和龈沟液TSLP、IL-33的影响及其预后的影响因素研究

摘要 目的：观察牙周-正畸联合治疗对侵袭性牙周炎（AgP）患者牙周功能和龈沟液胸腺基质淋巴细胞生成素（TSLP）、白介素-33（IL-33）的影响,并分析其预后的影响因素。方法：选择2017年1月至2021年6月期间我院收治的AgP患者119例,根据治疗方式的不同分为对照组（牙周基础治疗）和联合组（牙周-正畸联合治疗）,例数分别为58例和61例。对比两组患者的牙周功能[牙周袋深度（PD）、牙龈指数（GI）、菌斑指数（PLI）、临床附着丧失（AL）]和龈沟液TSLP、IL-33水平,根据PD判断联合组患者的预后情况,采用单因素、多因素Logistic回归分析预后的影响因素。结果：两组治疗后PD、GI、PLI、AL均较治疗前下降,且联合组低于对照组（P<0.05）。两组治疗后龈沟液TSLP、IL-33水平均较治疗前下降,且联合组低于对照组（P<0.05）。61例采用牙周-正畸联合治疗的AgP患者,6个月后复查发现,PD＜4 mm的患者有38例（预后良好组）,PD≥4 mm患者有23例（预后不良组）。单因素分析结果显示,牙周-正畸联合治疗AgP患者的预后与患牙上下/前后分布位置、患牙最深PD值、牙槽骨高度基线值、PLI、根型态异常情况有关（P<0.05）。多因素Logistic回归分析结果显示：患牙分布位置为下颌牙、根型态存在异常情况、患牙分布位置为前牙、患牙最深PD值偏大、PLI偏高、牙槽骨高度基线值偏高均是牙周-正畸联合治疗AgP患者不良预后的危险因素（P<0.05）。结论：与单纯的牙周基础治疗相比,牙周-正畸联合治疗可有效改善AgP患者的临床症状,控制局部炎性反应。同时,患牙上下/前后分布位置、患牙最深PD值、牙槽骨高度基线值、PLI、根型态异常情况均是牙周-正畸联合治疗AgP患者预后的影响因素,需引起临床重视。     

Signals from the IL-9 receptor are critical for the early stages of human intrathymic T cell development

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the alpha-chain of the IL-9 receptor (IL-9Ralpha) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity alpha-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3-CD8-CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Ralpha signaling is critical in early T lymphoid development.     

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

The interaction between ICOS and B7RP-1 is not required for the development of experimental murine allergic conjunctivitis

It is still unclear whether the interaction between inducible costimulator (ICOS) and its ligand, B7 related protein (B7RP)-1, is important for the development of allergic diseases. We investigated whether blocking the ICOS/B7RP-1 interaction affects the development of murine experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice either by active immunization of ragweed (RW) or by transferring RW-primed splenocytes, followed by challenge with RW-containing eye drops. The mice were treated with anti-B7RP-1 antibody (Ab) or normal rat immunoglobulin G (IgG) during either the induction or effector phase. Regardless of the induction method or when the animals were treated, eosinophil infiltration into the conjunctiva was not affected by the anti-B7RP-1 Ab treatment. Splenocyte responses were not largely affected by this treatment. However, serum Ig levels were significantly reduced. These data suggest that blocking the ICOS/B7RP-1 in allergic diseases may not always be therapeutic.     

Characterization of murine and humanized anti-CD33, gelonin immunotoxins reactive against myeloid leukemias

M195 antibodies recognize CD33, an antigen present on acute myeloid leukemia blasts as well as some myeloid progenitor cells, but not on the ultimate hematopoietic progenitor stem cell. Immunotoxins (IT) reactive with human myeloid leukemias were constructed by conjugating gelonin, a single-chain ribosome-inactivating protein, to murine and genetically engineered, humanized M195 antibodies via anN-succinimidyl-3-(2-pyridyldithio)-propionate linkage. No losses of gelonin cytotoxic activity or M195 binding activity were observed after conjugation of up to two toxin molecules per antibody. Toxin conjugates displayed specific, potent toxicity for CD33⁺ cells. The murine and humanized IT were not toxic to CD33^– cells and were 600 and 4500 times more potent, respectively, than free gelonin in inhibiting CD33⁺ HL60 cells. Treatment of HL60 cells with 1 g/ml HuM195-gelonin resulted in more than 1000 times lower colony formation; normal bone marrow mononuclear cell colonyforming units treated with HuM195-IT were reduced by a factor of 10. HL60 leukemia cells could be effectively purged from an excess of normal bone marrow cells. Exposure of target cells to IT for as little as 30 min was as effective as continuous exposure of IT for up to 6 days. However, measures of the efficacy of the immunotoxin were directly related to the length of time of observation after IT exposure and were inversely related to cell concentration. M195-gelonin immunoconjugates are potential candidates for therapeutic use in in vivo or ex vivo bone marrow purging of myeloid leukemias.These studies were supported in part by the Lucille P. Markey Charitable Trust, ACS Grant No. IM551, NIH PO1CA33049, NIH RO1CA55349. Research conducted, in part, by the Clayton Foundation for Research. David A Scheinberg is a Lucille P. Markey Scholar