尿毒症患者多形核白细胞糖皮质激素受体与趋化移动的改变？…

应用放射配体结合分析，检测尿毒症患者外周血多形白细胞（ＰＭＬ）的糖皮质激素受体（ＧＲ），尿毒症患者ＰＭＬ ＧＲ的最大结合容量（Ｂａｍｘ）明显低于对照组，而其解离常数（Ｋｄ）比对照组的显著同。同时检测ＧＲ的效应指标：皮质醇（Ｆ）对ＰＭＬ的趋化移动（ＣｈｔＭ）的抑制率（ＦＩ），Ｆ对ＣｈｔＭ的ＦＩ明显低于对照组，与降低的ＧＲ呈正相关（ｒ＝０．７８５，Ｐ〈０．０１）。尿毒症患者ＰＭＬ的ＧＲ降低的同时，伴效     

小鼠杏仁内侧核中白细胞介素1β的表达调控依赖于雌激素受体a

研究雌激素受体(ER)敲除小鼠脑内，ERa和ERβ在介导内侧杏仁核中自细胞介素1β(IL-1β)表达的作用。IL-1β表达有显著的性别差异，并且在ER敲除小鼠含量减少。细菌脂多糖(LPS)或卵巢切除能够促进野生型和ERβ敲除小鼠(BERKO)IL-1β表达，但对ERa敲除小鼠(ERKO)无作用。相似的是，外源性雌激素能抑制野生型和BERKO小鼠IL-1β表达，后者时间稍有延搁，但对ERKO IL-1β表达没有影响。结果表明，ERa是内侧杏仁核IL-1β表达调节的重要机制，提示ERs可作为选择性靶基因治疗和预防神经功能失常。     

大鼠运动核内5-羟色胺1A、2A、5A受体的定位分布

为了阐明５ 羟色胺在中枢神经系统内与运动神经元结合的精确部位,本研究用免疫细胞化学技术分别观察了大鼠躯体运动核和内脏运动核内５ 羟色胺１Ａ、２Ａ、５Ａ 受体的定位分布。在躯体运动核内观察到：（１）５ 羟色胺１Ａ 受体样阳性神经元和纤维主要分布于动眼神经核、滑车神经核、三叉神经运动核、面神经核、舌下神经核和脊髓前角;（２）５ 羟色胺２Ａ 受体样阳性神经元主要见于动眼神经核、三叉神经运动核、面神经核、舌下神经核和脊髓前角,但阳性纤维和终末却密集地分布于三叉神经运动核、面神经核、舌下神经核和脊髓前角等处,除此之外动眼神经核、滑车神经核、展神经核和疑核内也能见到中等密度的阳性纤维和终末,纤维和终末的分布范围和染色浓度、密度都较神经元为明显;（３）少量淡染的５ 羟色胺５Ａ 受体样阳性神经元和稀疏的阳性纤维及终末主要见于三叉神经运动核、面神经核、舌下神经核和脊髓前角。在内脏运动核内观察的结果是：（１）动眼神经副交感核（Ｅ Ｗ 核）、上涎核、迷走神经背核、骶髓副交感核和胸髓侧角内仅有少量５ 羟色胺１Ａ 受体样阳性神经元、纤维和终末分布;（２）５ 羟色胺２Ａ 受体样阳性神经元和较密集的阳性纤维和终末见于Ｅ Ｗ 核、迷走神经背核、骶?     

Roles of the ribosomal protein S19 dimer and the C5a receptor in pathophysiological functions of phagocytic leukocytes

Monocytes and neutrophils, the major phagocytic leukocytes, migrate to inflammatory sites by sensing chemoattractants such as anaphylatoxin C5a with membrane receptors such as C5a receptor. Upon stimulation, the leukocytes increase cytoplasmic Ca(2+) concentration and generate radical oxygen species. These leukocytes have different functions in inflammation. Neutrophils migrate more rapidly and induce vascular plasma leakage upon infiltration. Monocytes infiltrate tissue more slowly but have superior capacities of phagocytosis and antigen presentation. There must be mechanisms to separately recruit the leukocyte species at an inflammatory site. Ribosomal protein S19 (RP S19) is a component of ribosome. During apoptosis, RP S19 is dimerized and obtains a ligand capacity to C5a receptor. The RP S19 dimer attracts monocytes to phagocytically clear the apoptotic cells that released the dimer molecules. The phagocytic monocytes/macrophages then translocate to regional lymph nodes and present apoptotic cell-derived antigens. Oppositely, the RP S19 dimer inhibits C5a-induced neutrophil migration and promotes apoptosis of neutrophils via the C5a receptor. The RP S19 dimer seems to prevent excessive tissue destruction induced by neutrophils. Skp is a molecular chaperon of Gram-negative bacteria. Skp also attracts monocytes and neutrophils as a ligand of C5a receptor. However, it promotes neither cytoplasmic Ca(2+) enhancement nor radical oxygen generation.     

Generation of chimeric C5a/formyl peptide receptors: towards the identification of the human C5a receptor binding site

We employed the polymerase chain reaction to produce a series of chimeric C5a/formyl peptide receptors. Chinese hamster ovary cells transfected with these constructs were tested for their ability to bind C5a. Substitution of three of the extracellular domains of the C5a receptor with the corresponding domains of the formyl peptide receptor abolished C5a binding, whilst replacement of the first extracellular loop of the C5a receptor with that of the formyl peptide receptor had little effect on the affinity of the receptor for C5a. We therefore conclude that this first outer loop of the C5a receptor does not participate in ligand binding, whilst involvement of the other extracellular domains of the receptor cannot be ruled out.     

A case of episodic angioedema associated with blood eosinophilia: upregulated C5a receptor expression on eosinophils

A 47-year-old woman was admitted to hospital complaining of swelling and pain of the extremities, accompanied by high fever and generalized erythema. Laboratory examination showed marked blood eosinophilia with elevation of IgM, IgE, and C-reactive protein. All autoantibodies examined were negative. The heart and lungs showed no untoward findings. Biopsies of the skin and muscle revealed cellular infiltration of eosinophils around small blood vessels. Ouantitation of C5a receptor (C5aR) expression by flow cytometry using anti-C5aR antibody showed upregulated expression of C5aR on blood eosinophils but downregulated expression on neutrophils. The abnormal C5aR expression on eosinophils and neutrophils became normal after spontaneous resolution of symptoms and blood eosinophilia. The possibility that C5aR expression on granulocytes is related to the pathogenesis of this syndrome may be considered.     

Guinea pig S19 ribosomal protein as precursor of C5a receptor-directed monocyte-selective leukocyte chemotactic factor

Objective: To reveal the C5a receptor-mediated monocyte-selective chemoattraction of the homo-dimer of guinea pig S19 ribosomal protein (RP S19), and to study the topological relationship between the RP S19 and C5a receptor genes.Methods: cDNA cloning and nucleotide sequencing, leukocyte chemotaxis measurement, and fluorescent in situ hybridization (FISH) were performed in the guinea pig.Results: The amino acid sequence of the guinea pig RP S19 deduced from the cDNA nucleotide sequence was identical to the human protein. The dimer of a recombinant RP S19 attracted guinea pig monocytes but suppressed neutrophil chemotactic movement. Both effects were C5a receptor-mediated. In the FISH analysis, the signals denoting the guinea pig RP S19 gene and C5a receptor gene completely overlapped each other.Conclusions: The guinea pig RP S19 dimer possessed a dual ligand effect, agonistic to the monocyte C5a receptor and antagonistic to the neutrophil receptor. The RP S19 and C5a receptor genes co-localized on the same chromosome.Received 24 April 2004; returned for revision 14 June 2004; accepted by M. Katori 21 June 2004     

Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice

Complement activation has a deep pathogenic influence in immunoglobulin (Ig)A nephropathy (IgAN). C3a and C5a, small cleavage fragments generated by complement activation, are key mediators of inflammation. The fragments exert broad proinflammatory effects by binding to specific receptors (C3aR and C5aR, respectively). However, no studies thus far have investigated the effects of C3a, C5a and their receptors on IgAN. We observed that C3aR and C5aR antagonists repressed IgA‐induced cell proliferation and interleukin (IL)‐6 and monocyte chemotactic protein 1 (MCP‐1) production in cultured human mesangial cells (HMCs). Furthermore, an IgAN mouse model induced by Sendai virus infection was employed to investigate the effects of C3aR and C5aR on IgAN in vivo for the first time. Wild‐type (WT) and several knock‐out mouse strains (C3aR^–/– or C5aR^–/–) were immunized intranasally with increasing doses of inactivated virus for 14 weeks and were subjected to two intravenous viral challenges during the time‐period indicated. In the Sendai virus‐induced IgAN model, C3aR/C5aR‐deficient mice had significantly reduced proteinuria, lower renal IgA and C3 deposition, less histological damage and reduced mesangial proliferation compared with WT mice. Both C3aR deficiency and C5aR deficiency, especially C3aR deficiency, inhibited renal tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, IL‐1β, IL‐6 and MCP‐1 expression significantly. However, C3aR/C5aR‐deficient and WT mice with IgAN did not differ with respect to their blood urea nitrogen (BUN) and serum creatinine levels. Our findings provide further support for the idea that C3aR and C5aR are crucially important in IgAN, and suggest that pharmaceutically targeting C3aR/C5aR may hold promise for the treatment of IgAN.     

Purification of human C5a des arg by immunoadsorbent and molecular sieve chromatography

Human C5a des arg was isolated from complement-activated serum by immunoadsorption followed by Sephadex G-75 chromatography. C5a des arg obtained by this 2-step procedure was shown to be immunologically identical to C5a des arg purified by a conventional multi-step method, homogeneous on SDS-polyacrylamide gels, and biologically active. Although this technique yields approximately the same amount of C5a des arg/liter of activated serum as that obtained by conventional methods, its simplicity and relative rapidity make it a practical alternative.     

The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, Gα16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.     

中和C5a过敏毒素的分子设计研究

目的 从蛋白质结构与功能的关系出发,探讨Ｃ５ａＲ与其配体Ｃ５ａ的结合位。方法 按分子设计理论,采用亲水性方案寻打Ｃ５ａ胞外区高亲水性区域,Ｆｍｏｃ方案人工合成Ｃ５ａ第９～３０位氨基酸基序（Ｐ２２肽）,经高压液相色谱纯化,毛细管电泳鉴定。结果合成多肽（Ｐ２２）的纯度为９５．１９％,每次缩合的平均效率为９９．７８％;能与ａｎｔｉ－Ｃ５ａＲ ＭｃＡｂ（Ｓ５／Ⅰ,Ｓｅｒｏｔｉｃ公司）有效地结合,酶联ＯＤ４