ACT001 exerts anti-inflammatory and antioxidative activity to alleviate sepsis-induced acute lung injury through STAT1/CIITA/MHC- II pathway / 中国药理学通报

Aim This study aimed to assess the therapeutic potential of ACT001, a micheliolide derivative, in the treatment of acute lung injury ( ALI) induced by sepsis and investigate its pharmacological mechanisms. Methods At the animal level, an ALI model was established in mice through intraperitoneal injection of li-popolysaccharide (LPS). Subsequently, ACT001 was administered to the ALI-afflicted mice. The therapeutic effects of ACT001 were assessed by evaluating factors such as individual survival rate, lung inflammation, and pulmonary edema. At the cellular level, RAW264. 7 cells were stimulated with LPS to explore the pharmacological mechanism of ACT001. The study examined inflammatory response and oxidative stress levels, and proteomics analysis was conducted to investigate the underlying molecular mechanisms. Results At the animal level, ACT001 can improve the survival of mice with ALI, reduce lung inflammation, and reduce the levels of inflammatory cytokines in serum. At the cellular level, ACT001 promotes the polarization of RAW264. 7 cells toward an anti-inflammatory pheno-type by inhibiting MHC-II related pathways, inhibiting the production of NO and related inflammatory cytokines while increasing SOD content and scavenging ROS. Conclusions ACT001 exhibited the potential to alleviate ALI via its anti-inflammatory and antioxidative activity, mainly by inhibiting the STAT1/ CIITA/ MHC-II pathway. ACT001 holds promise as a novel therapeutic candidate for the treatment of ALI induced by sepsis.

The roles of sphingosine-1-phosphate and its receptor in Mycobacterium tuberculosis infection and novel antibiotics discovery / 药学学报

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) infection remains a major public health problem of global concern, largely due to antibiotics resistance, persistence and immune evasion. Sphingolipid bioactive molecules are involved in several important pathophysiological processes. Sphingosine-1-phosphate is a key product of sphingolipid metabolism, and can play a role in two manners: autocrine and/or paracrine. Sphingosine-1-phosphate regulates T cells and a variety of antigen-presenting cells during M. tuberculosis infection, promotes antigen processing and expression in monocytes, is involved in the maturation of phagolysosome, regulates Ca2+ homeostasis, participates in the autophagy of macrophages, inhibits the survival and proliferation of M. tuberculosis within host cells, and effectively reduces the necrosis of the mouse lungs infected by M. tuberculosis. Injection of 20 nmol per mouse sphingosine-1-phosphate inhibited up to 47% of mycobacterial growth in the lung and spleen of mice infected by M. tuberculosis. In this paper, sphingosine-1-phosphate, its receptors and regulatory network were reviewed, and the specific mechanism of sphingosine-1-phosphate inhibiting the survival of M. tuberculosis-infected host cells was elaborated. This will provide novel insights into the new targets for tuberculosis prevention and treatment.

Effects of vIL-10 on MHC-I antigen processing“the operon”in nasopharyngeal carcinoma cell lines / 西安交通大学学报(医学版)

ABSTRACT:Objective To explore the effects of virus interleukin‐10 (vIL‐10 ) on different expressions of MHC‐I antigen processing “the operon” .Methods We collected nasopharyngeal carcinoma cells (CNE‐1 and CNE‐2) treated by vIL‐10 at different time points ,and detected the changes of MHC‐I antigen processing “the operons” (TAP‐1 ,TAP‐2 ,LMP‐2 ,LMP‐7 and HLA‐I) by RT‐PCR and Western blot .Results ① mRNA level :There was no difference in the expression of TAP‐1 in CNE‐1 and CNE‐2 cells at various time points .The expressions of TAP‐2 and LMP‐2 in CNE‐1 and CNE‐2 did not change at 1 ,4 ,6 ,12 h ,but downregulated and even disappeared at 24 h .The expression of LMP‐7 in CNE‐1 decreased 4 h after vIL‐10 was added ,and that in CNE‐2 decreased at 6 h .The expression of HLA‐I in CNE‐1 and CNE‐2 showed significant decrease at 24 h .② Protein expression :The expression of TAP‐1 in CNE‐1 and CNE‐2 showed significant decrease at 24 h .The expression of TAP‐2 in CNE‐1 and CNE‐2 was gradually downregulated at different time points .The expressions of LMP‐2 and LMP‐7 in CNE‐2 were gradually downregulated at different periods ,while that in CNE‐1 was only decreased at 12 h .The expression of HLA‐I in CNE‐1 and CNE‐2 was gradually downregulated ,but there was no significant difference at each period in CNE‐1 ,while the expression of HLA‐I in CNE‐2 at 24 h was significantly downregulated .Conclusion vIL‐10 can inhibit the expression of MHC‐I antigen processing “the operon” in NPC in the time‐dependent manner .

Ultraestructura del compartimiento clase II en macrófagos humanos en rosetas macrófago-linfocitarias autólogas / Ultrastructure of the class II compartment in human macrophages in autologous macrophage-lymphocyte rosettes

Introducción: El procesamiento y presentación de antígenos está involucrado en el fenómeno de múltiples sinapsis inmunológicas de la Roseta Macrófago-Linfocitaria humana (RML) entre macrófagos derivados de monocitos y linfocitos T CD4+ de cultivos autólogos leucocitarios totales extraídos de la sangre; aquí los antígenos autólogos de los neutrófilos apoptóticos son presentados por la vía endocítica o vía Clase II. El Compartimiento Clase II (CCMII) ha sido caracterizado en células B y células dendríticas en modelos murinos. Objetivo: estudiar la evolución ultraestructural de la organización espacial CCMII en macrófagos en el fenómeno de RML humana. Métodos: Se utilizaron muestras de sangre humana sana, anticoagulada con heparina (n=10) donadas por Banco de Sangre, UNC, en anonimato. Cultivos leucocitarios autólogos en medio TC199 (SIGMA, St. Louis, MO). Se tomaron muestras de cultivo celular a: 1, 2, 3, 20, 48 y 96 horas. Se aplicó la técnica de RML. Las citopreparaciones se sometieron a técnicas de procesamiento para su estudio ultraestructural con el MET: Zeiss LEO-906E. Resultados: Observamos cuerpos multivesiculares, multilaminares y tubulares en la organización espacial del CCMII a lo largo del tiempo de cultivo. Estructuras tubulares aparecieron a las 48 horas de cultivo. Se concluye que organización espacial del CCMII toma diversos aspectos en coincidencia con la ocurrencia de transformación macrofágica en cultivo y su rol como célula presentadora de antígenos (CPA) en RMLs. Dado el origen autólogo de los antígenos presentados postulamos que el perfil de los macrófagos en RMLs podría corresponder al alternativo o M2

Introduction: Processing and presentation of antigens is involved in the phenomenon of multiple immunological synapses of human Macrophage-Lymphocyte Rosette (MLR) between monocyte-derived macrophages and CD4 + T cells of total leukocyte cultures autologous extracted from blood; here autologous apoptotic neutrophil antigens are presented by the endocytic pathway or via Class II. Class II Compartment (MIIC) has been characterized by B cells and dendritic cells in murine models. Objective: To study the ultrastructural evolution in the spatial organization MIIC in macrophages in the phenomenon of human MLR. Methods: healthy human blood samples were used, anticoagulated with heparin (n = 10) donated by Blood Bank, UNC, anonymous. Autologous leukocyte cultures in TC199 medium (SIGMA, St. Louis, MO). Cell culture samples were taken at 1, 2, 3, 20, 48 and 96 h. MLR technique was applied. The citopreparations underwent processing techniques for ultrastructural study with MET: Zeiss LEO-906E. Results: We observed multivesicular, multilamellar, and tubular bodies in the spatial organization of MIIC throughout the culture time. Tubular structures appeared at 48 h of culture. We conclude that spatial organization of MIIC takes various aspects coinciding with the occurrence of macrophage transformation in culture and its role as antigen presenting cell (APC) in MLRs. Given the autologous antigens presented we postulate that the profile of macrophages in MLRs could correspond to alternative or M2 (AU) .

Evidence for Direct Inhibition of MHC-Restricted Antigen Processing by Dexamethasone

Dexamethasone (Dex) was shown to inhibit the differentiation, maturation, and antigen-presenting function of dendritic cells (DC) when added during DC generation or maturation stages. Here, we examined the direct effects of Dex on MHC-restricted antigen processing. Macrophages were incubated with microencapsulated ovalbumin (OVA) in the presence of different concentrations of Dex for 2 h, and the efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Dex inhibited both class I- and class II-restricted presentation of OVA to T cells; this inhibitory effect on antigen presentation was much more potent in immature macrophages than in mature macrophages. The presentation of the exogenously added OVA peptide SIINFEKL was not blocked by Dex. In addition, short-term treatment of macrophages with Dex had no discernible effects on the phagocytic activity, total expression levels of MHC molecules or co-stimulatory molecules. These results demonstrate that Dex inhibits intracellular processing events of phagocytosed antigens in macrophages.

Cyclooxygenase Inhibitors, Aspirin and Ibuprofen, Inhibit MHC-restricted Antigen Presentation in Dendritic Cells

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve pain, reduce fever and inhibit inflammation. NSAIDs function mainly through inhibition of cyclooxygenase (COX). Growing evidence suggests that NSAIDs also have immunomodulatory effects on T and B cells. Here we examined the effects of NSAIDs on the antigen presenting function of dendritic cells (DCs). METHODS: DCs were cultured in the presence of aspirin or ibuprofen, and then allowed to phagocytose biodegradable microspheres containing ovalbumin (OVA). After washing and fixing, the efficacy of OVA peptide presentation by DCs was evaluated using OVA-specific CD8 and CD4 T cells. RESULTS: Aspirin and ibuprofen at high concentrations inhibited both MHC class I and class II-restricted presentation of OVA in DCs. In addition, the DCs generated in the presence of low concentrations of the drugs exhibit a profoundly suppressed capability to present MHC-restricted antigens. Aspirin and ibuprofen did not inhibit the phagocytic activity of DCs, the expression level of total MHC molecules and co-stimulatory molecules on DCs. Ibuprofen rather increased the expression level of total MHC molecules and co-stimulatory molecules on DCs. CONCLUSION: These results demonstrate that aspirin and ibuprofen inhibit the intracellular processing event of the phagocytosed antigen, and further suggest that prolonged administration of NSAIDs in high doses may impair the capability of DCs to present antigens in asiociation with MHC molecules.

Identification and subcellular localization of transporter associated with antigen processing(TAP)1-EGFP and TAP 2-EGFP fusion proteins in malignant melanoma / 中华皮肤科杂志

Objective To construct an eukaryotic expression vector for TAP genes fused with enhanced green fluorescent protein(EGFP)gene,and to analyze the expression and subcellular localization of the fusion protein in A375 human malignant melanoma cells transfected with the eukaryotic expression vector.Methods A375 cells were cotransfected with the combination of plasmid(P)TAP1-EGFP or pTAP2-EGFP and pDsRed2-endoplasmic reticulum(ER),or with pEGFP-TAP1 and-TAP2,or monotransfected with pTAP1-EGFP or pTAP2-EGFP alone.The monoclonal A375 cells stably expressing TAP were obtained by G418 selection.Then.the distribution and expression of fusion proteins were assessed in A375 cells by using fluorescence microscopy and Western blot,respectively.Flow cytometry was used to measure the expression of HLA class Ⅰ on A375 cells.Results Transfection of A375 cells with pTAP1-EGFP or pTAP1-EGFP and pTAP2-EGFP significantly increased the expression of TAP 1 and TAP 2 in as well as HLA class Ⅰ antigen on A375 cells.The green fluorescence of TAP1-EGFP and TAP2-EGFP overlapped with the red fluorescence of ER marker in cotransfected cells.indicating that TAP was localized subcellularly on the ER.Conclusions The expression vector for TAP-EGFP fusion gene has been constructed cuccessfully and expressed in A375 cells,and the expressed fusion protein is subcelluiarly localized to ER.This study will provide a basis for the research into subsequent immune response following induction of TAP expression.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis / Respuesta de linfocitos T CD4 y CD8 contra la rHSP60 de Klebsiella pneumoniae en células mononucleares de sangre periférica de pacientes con espondilitis anquilosa

Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27 + AS patients and 15 B27 healthy controls was assessed by ³H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Objetivo.Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación. Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H-TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA-B27+y en 15 sujetos sanos HLA-B27-. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H-TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N-acetil-L-leucil-L-leucil-L-nor-leucinal (LLnL), y fijadas con p-formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo. Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno. Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.

PRELIMINARY STUDY ON THE ROLE OF MACROPHAGE IN PROCESSING AND PRESENTING ANTIGENS OF SCHISTOSOMA JAPONICUM / 中国寄生虫学与寄生虫病杂志

The horseradish peroxidase labeled affinity purified mice anti-UEA (Urea soluable egg antigen of Schistosoma japonicum) antibody was used in enzyme-linked immunoe ectrotransfer blot (EITB) te monitor the changes after UEA being processed by M?.The immune respon-sive peptides were delected in the culture supernatant and homogenate of M? pulsed with UEA in vitro (M?+).After processing by M? the high molecular weight UEA was cleaved into low molecular weight peptides,as shown,by the reactive bands.They markedly differed from that native UEA or trypsin-digested UEA; The bands of M? supernatant and homogenate showed similarity with certain quantitative differences.According to the result described above,we considered: 1.UEA could be processed into smaller pieces by M?,the style of processing is cleavage.2.The processed peptides might be released to extracellular environment.