Complement components C1q,C1r/C1s,and C1inh in rheumatoid arthritis

Objective. To analyze the synovial site and the cell types expressing C1q, C1r/C1s, and C1–esterase inhibitor (C1INH) and to characterize newly synthesized C1q in patients with rheumatoid arthritis (RA). Methods. Tissue and primary cell cultures of synovium from RA patients were analyzed for C1q, C1r/C1s, and C1INH by Northern blotting, in situ hybridization, and pulse-chase experiments for C1q. Results. The de novo synthesis of C1q, C1r/C1s, and C1INH in synovium and primary cell cultures was proven by Northern blot and by antigenic and functional analysis. In in situ hybridization experiments, the synovial lining cell layer was identified as the site of C1q, C1r, and C1INH expression. In contrast, immunohistologic analysis showed that C1q, C1s, and C1INH proteins were present in a thin film covering the synovial lining cells. In situ hybridization performed on primary cell cultures provided evidence that only macrophages were able to express C1q, whereas fibroblasts and stellate cells synthesized C1r. Conclusion. The synovium is important for the synthesis and secretion of C1q and C1r/C1s, as well as the control protein C1INH, which supports the idea of a locally occurring inflammatory process in RA patients.     

Serum levels of C1q,C1r and C1s in normal and pathologic sera

C1q, C1r and C1s protein concentrations were simultaneously assayed in sera of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, autoimmune hemolytic anemia, and viral hemorrhagic fever as well as in healthy adult control subjects. Correlation coefficients between the three subunits of C1 were calculated and the sequential evolution of C1q, C1r, and C1s levels was studied in patients with SLE. In the majority of cases there was a good correlation between C1q, C1r, and C1s; subunit levels increased or decreased in a coordinated fashion. A marked discrepancy between C1q, C1r, and C1s serum concentration was observed in 3 instances. Low C1q coexisted with extremely high C1r and C1s values. It appears that C1r and C1s are closely linked while C1q may vary in a more independent way.     

Evaluation of C3c,C4 and C1-esterase inhibitor (C1-INH) during unstable angina

BACKGROUND: The complement system plays an important role in the physiopathology of acute myocardial infarction (AMI) taking part in myocardial damage and reperfusion injury. The aim of this study is to investigate the plasmatic levels of some complement components (C3c, C4 and C1-INH) during unstable angina (C1-INH) and their different concentrations in relation to the different myocardial areas affected by ischemia. METHODS: The plasmatic levels of C1-INH, C3c and c4 in 30 patients affected by unstable angina, and those of 22 clinically healthy subjects (control group) were evaluated (Nefelometer Behering). The patients were divided into four groups according to the different myocardial area affected by ischemia (anterior, antero-lateral, lateral or inferior ischemia), RESULTS: No statistically significant differences were found in plasmatic levels of C3c, C4 and C1-INH between the group of patients and the control group. There is a statistically significant difference between the C1-INH levels of the patients with inferior ischemia and the plasmatic concentrations of the whole patients' group (p<0,01), the control group (p<0,01) and the group of patients with lateral ischemia (p<0,02). CONCLUSIONS: There seems to be a different activation of the complement system during unstable angina, in relation to the different myocardial area affected by ischemia.     

AKR1C1 and AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer

Endometrial cancer is the most common malignancy of the female genital tract. Its incidence correlates with prolonged estrogen stimulation unopposed by progesterone or synthetic progestins. Estrogen and progestin action is regulated at the pre-receptor level, by interconversion of active hormones (estradiol (E2), progesterone (P)) with their inactive counterparts (estrone (E1), 20alpha-hydroxyprogesterone (20alpha-OHP)) in target tissues. Expression of enzymes that control the ratio of E2 and P may thus play role in the disease process. We first confirmed that AKR1C1 (human 20alpha-hydroxysteroid dehydrogenase) in a cellular context inactivates P by forming 20alpha-OHP but does not catalyze the reverse reaction. We next examined the expression of AKR1C1 and AKR1C3 (type 5 17beta-hydroxysteroid dehydrogenase) in 16 paired specimens of endometrial cancer and adjacent normal endometrium. Quantification by isoform specific real-time PCR revealed higher expression of AKR1C1 in nine specimens and higher expression of AKR1C3 in four specimens of endometrial cancer. Importantly, upregulation of both enzymes in the same specimen was observed. Since AKR1C1 inactivates P its elevated expression in diseased endometrium may contribute to diminished protection by P, while elevated expression of AKR1C3 which forms E2 in vivo, may contribute to the enhanced estrogen action. It is suggested that the expression of AKR1C1 and AKR1C3 in endometrial cancer will govern the ratio of P:E2.     

Hepatitis C and HIV-1 coinfection

Hepatitis C virus (HCV) has emerged as the cause of the second major epidemic of viral infection after human immunodeficiency virus (HIV) within the past two decades, and coinfection of HIV and HCV represents a growing problem for the future. This article reviews the current evidence on the epidemiology and clinical implications of an interaction between HIV-1 and HCV infection, and the current status of the management of patients with combined infection.     

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca²⁺ ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.     

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca²⁺-coordinating residues. The data explain the Ca²⁺-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.     

Deficiencies of C1 inhibitor

Hereditary and acquired deficiencies of the C1 inhibitor result in a single prominent symptom, namely angioedema. Angioedema may involve the skin, the gastrointestinal tract or the upper airway. Genetically determined defects in C1INH cause hereditary angioedema. The defect may be acquired as the result of an auto-antibody to C1INH or be due to the generation of anti-idiotypic antibody to monoclonal immunoglobulins as occurs in various B cell lymphoproliferative diseases. Androgens provide prophylaxis against attacks of angioedema. There is no widely approved treatment for acute attacks of angioedema although several promising drugs are now in the final stages of clinical trials.     

Posterior temporary fixation of C1–C2 screw-rod system for unstable C1 burst fracture

Whether an unstable C1 burst fracture should be treated surgically or conservatively is controversial. The purpose of this study is to evaluate the effectiveness and motion-preserving function of temporary fixation of C1–C2 screw-rod system for the reduction and fixation of unstable C1 burst fracture.We retrospectively reviewed 10 patients who were treated with posterior C1–C2 temporary fixation without fusion. We assessed age at surgery, gender, pre- and postoperative visual analog scale (VAS), Neck Disability Index (NDI), atlanto-dens interval (ADI), lateral mass distance (LMD), and rotation function of C1–C2 complex.Six males and 4 females were included in our study. The average follow-up duration was 14.1 ± 1.37 months. The left-to-right ROMs of C1–C2 rotation was 9.6° ± 1.42°. The preoperative cervical VAS was 8.30 ± 0.48; the postoperative cervical VAS of C1–C2 fusion was 2.90 ± 0.57. The preoperative VAS for removal was 2.0 ± 0.00, and the postoperative VAS for removal was 2.3 ± 0.48. The preoperative cervical NDI was 81.40% ± 2.07%, the postoperative cervical NDI of C1–C2 fusion was 18.10% ± 1.52%. The preoperative NDI for removal was 15.9% ± 1.20%. The postoperative NDI for removal was 14.5% ± 1.08%. The preoperative ADI was 4.43 ± 0.34 mm, and postoperative ADI was 1.94 ± 0.72 mm. The preoperative LMD was 6.36 ± 0.58 mm, and postoperative LMD was 1.64 ± 0.31 mm.Posterior temporary C1–C2 fixation can achieve a good fusion and satisfied reduction of C1 fracture, relieve the pain, improve the cervical function outcome, but may reduce the rotational range of motion of C1–C2. Posterior C1–C2 temporary fixation without fusion was not suitable for C1 burst fracture. We recommend permanent C1–C2 fixation and fusion for C1 burst fracture if surgery is necessary.     

Human genes for complement components C1r and C1s in a close tail-to-tail arrangement.

Complementary DNA clones for human C1s were isolated from cDNA libraries that were prepared with poly(A)+ RNAs of human liver and HepG2 cells. A clone with the largest cDNA insert of 2664 base pairs (bp) was analyzed for its complete nucleotide sequence. It contained 202 bp of a 5' untranslated region, 45 bp of coding for a signal peptide (15 amino acid residues), 2019 bp for complement component C1s zymogen (673 amino acid residues), 378 bp for a 3' untranslated region, a stop codon, and 17 bp of a poly(A) tail. The amino acid sequence of C1s was 40.5% identical to that of C1r, with excellent matches of tentative disulfide bond locations conserving the overall domain structure of C1r. DNA blotting and sequencing analyses of genomic DNA and of an isolated genomic DNA clone clearly showed that the human genes for C1r and C1s are closely located in a "tail-to-tail" arrangement at a distance of about 9.5 kilobases. Furthermore, RNA blot analyses showed that both C1r and C1s genes are primarily expressed in liver, whereas most other tissues expressed both C1r and C1s genes at much lower levels (less than 10% of that in liver). Multiple molecular sizes of specific mRNAs were observed in the RNA blot analyses for both C1r and C1s, indicating that alternative RNA processing(s), likely an alternative polyadenylylation, might take place for both genes.     

Lymphoproliferative disease and acquired C1 inhibitor deficiency

Angioedema due to acquired deficiency of the C1-inhibitor is a bridging condition between autoimmunity and lymphoproliferation. We report 32 patients with acquired C1 inhibitor deficiency: 23 have anti C1-inhibitor autoantibodies; 13 have monoclonal gammopathies of unknown significance and 9 have non-Hodgkin's lymphoma. Our series suggest that different forms of B cell disorders coexist and/or evolve into each other in acquired angioedema.     

丙型肝炎病毒C和E1区的DNA改组

目的:利用DNA改组技术进行不同亚型的HCVC和E1区的人工进化.方法:首先利用PCR扩增两段具有较高序列同源性的1kb左右的基因片段,两者相比较同源性大于83%.然后将其等量混合,在Mg2 存在的条件下,用DNase Ⅰ切割成50bp左右的小片段.这些小片段在不外加引物的条件下,利用PCR反应进行重聚,再将重聚物经过一轮正常的PCR扩增.结果:获得了与原来片段大小相当的基因片段.结论:这一技术为进一步筛选高活性的HCV C和E1区打下基础,有利于从一组序列同源性程度较高的基因库构建随机嵌合基因,并为改组其他基因家族提供了借鉴.