Contrasting Roles of Mannan-Specific Monoclonal Immunoglobulin M Antibodies in the Activation of Classical and Alternative Pathways by Candida albicans

Polyclonal antimannan immunoglobulin G (IgG) activates the classical complement pathway and accelerates initiation of the alternative pathway by Canidida albicans. This dual role was assessed for two antimannan IgM monoclonal antibodies (MAbs). MAb B6.1 is specific for an epitope on the acid-labile portion of C. albicans phosphomannan; MAb B6 is specific for an epitope on the acid-stable region. Both MAbs were potent activators of the classical pathway but poor facilitators of alternative pathway initiation.Candida albicans activates the human complement system via both the classical and the alternative pathways, leading to deposition of opsonic complement fragments on the yeast cell surface (8, 10, 18). In previous studies, we described a critical role for naturally occurring antimannan immunoglobulin G (IgG) in complement activation by C. albicans. Those studies used a kinetic assay for C3 deposition on the yeast and immunofluorescence evaluation of the sites of C3 binding (10, 17, 18). Deposition of C3 onto C. albicans cells incubated in normal human serum (NHS) occurs rapidly via the classical pathway and can be detected within the first 2 min of incubation. If the classical pathway is blocked by chelation of Ca²⁺ with EGTA, C3 deposition occurs via the alternative pathway, but C3 deposition is delayed and a 6-min incubation is required before bound C3 is readily detectable on the yeast surface. Removal of naturally occurring antimannan IgG from the serum by mannan absorption profoundly delays accumulation of C3 on the yeast cell surface, with 12 min or more of incubation being required before appreciable amounts of bound C3 are detected. However, this 12-min delay can be overcome by supplementation of the mannan-absorbed serum with affinity-purified human antimannan IgG in the absence of EGTA to mediate classical pathway initiation or shortened to 6 min in the presence of EGTA to allow antibody-facilitated activation of the alternative pathway. These observations demonstrate a dual role for antimannan IgG in serum from healthy adults in complement activation by C. albicans. Antimannan IgG mediates activation of the classical pathway and facilitates initiation of the alternative pathway (17, 18).In studies described above, we used polyclonal antimannan IgG purified from pooled human plasma. Since C. albicans cells express a number of immunodominant mannan components recognized by rabbits (15, 16), the human polyclonal antimannan IgG likely contains a range of specificities for distinct mannan determinants. It has been shown that rabbit antibodies that are reactive with three different cell wall determinants of group A streptococci display differential abilities to activate the classical or alternative pathway (2). Although the antibodies specific for three different cell wall epitopes all activated the classical pathway, only antibody specific for the N-acetyl-d-glucosamine epitope activated the alternative pathway (2). In a separate study, capsular as well as noncapsular antibodies were found to direct classical-pathway-mediated killing of Haemophilus influenzae type b, whereas only the capsular antibodies promoted killing by the alternative pathway (12). These studies provide evidence that epitope specificity may influence the ability of an antibody to activate the alternative pathway and prompted us to examine whether antibodies that recognize different mannan determinants are able to mediate activation of the classical and alternative pathways by C. albicans.Two IgM monoclonal antibodies (MAbs) that recognize distinct mannan determinants were compared for their abilities to activate the classical or alternative pathway. MAb B6.1 is specific for an acid-labile component of the Candida phosphomannan complex, and MAb B6 is specific for an acid-stable component (5). The MAbs were produced commercially (Montana ImmunoTech, Inc., Bozeman, Mont.).C. albicans CA-1 was grown as yeast forms to stationary phase in glucose (2%)-yeast extract (0.3%)-peptone (1%) broth for 24 h at 37°C as described elsewhere (4, 6, 10). The mannan of CA-1 yeast was purified as described previously (7, 18) and coupled to CNBr-Sepahrose 4B (Pharmacia Biotech, Uppsala, Sweden) (18).Pooled NHS was prepared from peripheral blood from at least 10 healthy adult donors and stored at −80°C. C3 was isolated from frozen human plasma (9, 13) and stored at −80°C until used. C3 was labeled with ¹²⁵I as described previously (3) by use of IODO-GEN reagent (Pierce, Rockford, Ill.). NHS was absorbed with mannan-Sepharose 4B to remove antimannan antibodies (18).Kinetics of C3 binding were assayed by the method of Kozel et al. (10). To determine whether MAb B6 or B6.1 activates the classical pathway, 2 × 10⁶ yeast cells were incubated at 37°C in 1 ml of a complement binding medium that contained (i) 40% NHS, mannan-absorbed serum, or mannan-absorbed serum supplemented with MAb B6 or B6.1, (ii) sodium Veronal (5 mM)-buffered saline (142 mM, pH 7.3) containing 0.1% gelatin, 1.5 mM CaCl₂, and 1 mM MgCl₂, and (iii) ¹²⁵I-labeled C3. To study whether MAb B6 or B6.1 plays a role in alternative pathway initiation, yeast cells were incubated in the manner described above except that the binding medium was not supplemented with Ca²⁺ and contained 5 mM EGTA and 5 mM MgCl₂. At various time intervals from 2 to 16 min, 50-μl samples were withdrawn in duplicate and added to 200 μl of phosphate-buffered saline–0.1% sodium dodecyl sulfate–20 mM EDTA in Millipore MABX-N12 filter plates fitted with BV 1.2-μm-pore-size filter membranes (Millipore, Bedford, Mass.). The cells were washed with phosphate-buffered saline–0.1% sodium dodecyl sulfate, and filter-bound radioactivity was determined with a gamma counter. Nonspecific binding was estimated from cells incubated in NHS containing EDTA and was subtracted from the total counts.Mannan absorption of serum profoundly delayed C3 accumulation on yeast from 2 min to approximately 10 min (Fig. ​(Fig.11 and ​and2).2). However, addition of either MAb B6 or MAb B6.1 at 50 μg per ml of reaction mixture to the absorbed serum generated rapid activation kinetics characteristic of C3 deposition via the classical pathway (Fig. ​(Fig.1)1) (10, 17, 18). This observation was not unexpected, as polyvalent IgM is known to be a potent activator of the classical pathway. Open in a separate windowFIG. 1Effect of MAb B6 or B6.1 on the kinetics of C3 deposition on C. albicans cells via the classical pathway. Yeast cells were incubated in a C3 binding medium containing (i) 40% NHS (•), (ii) 40% mannan-absorbed NHS (○), (iii) 40% mannan-absorbed NHS supplemented with MAb B6 (▴), or (iv) 40% mannan-absorbed NHS supplemented with MAb B6.1 (▿) at 50 μg per ml of reaction mixture. C3 deposition patterns from three independent assays were similar; results from one representative assay are shown.Open in a separate windowFIG. 2Effect of MAb B6 or B6.1 on the kinetics of C3 deposition on C. albicans cells via the alternative pathway. Yeast cells were incubated in a C3 binding medium containing (i) 40% NHS (•), (ii) 40% NHS–EGTA (■), (iii) 40% mannan-absorbed NHS containing EGTA (○), (vi) 40% mannan-absorbed NHS containing EGTA supplemented with MAb B6 (▴), or (iv) 40% mannan-absorbed NHS supplemented with MAb B6.1 (▿) at 50 μg per ml of reaction mixture. C3 deposition patterns from four independent assays were similar; results from one representative assay are shown.The effects of MAbs B6 and B6.1 on activation of the alternative pathway were assessed by addition of the antibodies to mannan-absorbed serum in the presence of EGTA. The results (Fig. ​(Fig.2)2) showed that neither MAb B6 nor MAb B6.1 at 50 μg per ml of reaction mixture altered the alternative pathway activity of the mannan-absorbed serum. To determine whether the inability of MAb B6 or B6.1 to facilitate initiation of the alternative pathway was influenced by antibody concentration, the experiment represented in Fig. ​Fig.22 was repeated with mannan-absorbed serum that was supplemented with 10 to 160 μg of MAb B6 or B6.1 per ml. These antibody concentrations were chosen because in our previous studies we found that affinity-purified human antimannan IgG activates both the classical and alternative pathways (17). However, at 10, 40, or 160 μg per ml of reaction mixture, both antibodies failed to enhance alternative pathway activity of mannan-absorbed serum but promoted classical pathway activity (data not shown).The observation that both MAbs were unable to enhance alternative pathway activity was unexpected. Our previous studies showed that addition of polyclonal antimannan IgG to mannan-absorbed NHS containing EGTA produced C3 binding kinetics that were indistinguishable from the kinetics observed with nonabsorbed NHS containing EGTA (17). We further demonstrated IgG-dependent initiation of the alternative pathway by C. albicans using the six purified alternative pathway proteins (17).There are at least three possible explanations for the failure of MAbs B6 and B6.1 to facilitate activation of the alternative pathway. First, it is possible that antimannan antibodies of the IgM class are unable to enhance C3 deposition via the alternative pathway. However, there is evidence that polyclonal IgM is able to enhance alternative pathway-mediated lysis of rabbit erythrocytes by NHS (11, 14). Second, the ability of an antibody to facilitate deposition of C3 via the alternative pathway could be epitope specific; MAbs B6 and B6.1 could have the wrong epitope specificity. As noted above, Eisenberg and Schwab (2) found that polyclonal antibodies specific for one antigen found on group A streptococcal cell walls were able to facilitate initiation of the alternative pathway, whereas antibodies specific for two other antigens were not. If antibody-facilitated activation of the alternative pathway is dependent on epitope specificity, such a finding might influence strategies for induction of protective immunity to Candida. Optimal immunization may require an immunogen that induces antibodies with epitope specificities needed to facilitate activation of the alternative pathway. Finally, we cannot exclude the possibility that human antimannan antibodies are able to facilitate activation of the alternative pathway, whereas mouse antibodies lack this capability.In studies involving a murine model of disseminated candidiasis, MAb B6.1 was shown to be protective, whereas MAb B6 was not (4). However, the protection mechanisms remain to be elucidated. In an in vitro assay, MAb B6.1 but not MAb B6 was found to enhance candidacidal activity of polymorphonuclear leukocytes in the presence of fresh mouse serum, suggesting the involvement of mouse complement in the killing (1). Although assessing the role of complement in MAb B6.1-mediated protection was beyond the scope of this study, our observation that the two antibodies mediate similar kinetics of C3 deposition for C. albicans does not preclude the possibility that the composition and/or accessibility of opsonic complement fragments bound to the yeast cells might differ following complement activation by these two antibodies. Alternatively, the concerted action of several protective functions, including activation of the complement system, may be required for MAb B6.1-mediated protection.     

MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.     

动脉导管溶栓治疗急性肢体动脉栓塞14例分析

目的 探讨动脉导管溶栓治疗急性肢体动脉栓塞的方法和疗效。方法回顾性分析14例肢体动脉栓塞的诊断方法和介入手术治疗方案。结果治愈11例，治愈率78．6％(11／14)；2例好转。无患肢缺血坏死或截肢，无术后因肾功能衰竭死亡。结论动脉导管溶栓是治疗急性肢体动脉栓塞有效的方法，值得进一步开展和探索。     

乙型肝炎病毒前S1抗原检测及其临床意义

为探讨检测乙型肝炎病毒前S1抗原(Pre-S1Ag)的临床意义,本文对338例各型乙型肝炎患者进行Pre-S1Ag检测,同时检测HBV标志物和HBV-DNA,对其阳性率及相互关系进行分析比较.结果表明:338例患者,Pre-S1Ag阳性检测率为63.02%,HBeAg阳性检测率为48.52%,HBV-DNA阳性检测率为68.05%.Pre-S1Ag阳性与HBV-DNA阳性的符合率为78.56%;Pre-S1Ag与HBeAg阳性的符合率为81.17%;Pre-S1Ag与HBeAg、HBV-DNA具有显著相关性(P＜0.01),提示Pre-S1Ag能够较好地反映病毒复制状况,有可能作为体内病毒复制存在的实验室指标.     

介绍一种制备特异性TF和特异性iRNA的方法

以乙肝疫苗、人喉癌细胞膜抗原为抗原，猪脾细胞为效应细胞，经体外免疫后收集应答细胞，制备ＰＳＨＢＶ－ＴＦ ＰＳＡＣ－ｉＲＮＡ。通过抗原特异性细胞免疫功能试验证实，ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ都能转移特异性细胞免疫功能。采用体外免疫法制备ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ是可行的，并且具有诸多优点。     

Multi-virulence-locus sequence typing clarifies epidemiology of recent listeriosis outbreaks in the United States

Multi-virulence-locus sequence typing (MVLST) was used to analyze isolates from two major listeriosis outbreaks in the United States in 1998 and 2002 that were due to consumption of contaminated hot dogs and turkey deli meat, respectively. MVLST demonstrated high epidemiological relevance and indicated that the two outbreaks were the result of one epidemic.     

氧化苦参碱对LAK细胞活性的影响

ＬＡＫ细胞具有很强的广谱杀瘤作用，而氧化苦参碱具有较强的免疫抑制作用。本文研究了氧化苦参碱对ＬＡＫ细胞活力的影响，结果表明：氧化苦参碱可抑制ＩＬ－２对小鼠脾细胞的促增殖作用，并且对ＩＬ－２活化ＬＡＫ细胞杀伤Ｐ８１５的能力也有抑制作用。当ＩＬ－２（５００ｕ／ｍｌ）与２００μｇ／ｍｌ的氧化苦参碱共同孵育４ｄ后，可使ＬＡＫ细胞杀瘤能力（在效靶比为１００：１时）的８２．５％被抑制。同时氧化苦参碱本身对Ｐ８     

Endocarditis with ruptured cerebral aneurysm caused by Cardiobacterium valvarum sp. nov

A fastidious gram-negative bacterium was isolated from the blood of a 37-year-old man who had insidious endocarditis with a sudden rupture of a cerebral aneurysm. Characterization of the organism through phylogenetic and phenotypic analyses revealed a novel species of Cardiobacterium, for which the name Cardiobacterium valvarum sp. nov. is proposed. C. valvarum will supplement the current sole species Cardiobacterium hominis, a known cause of endocarditis. Surgeries and antibiotic treatment cured the patient's infection and associated complications. During cardiac surgery, a congenital bicuspid aortic valve was found to be the predisposing factor for his endocarditis.     

Inhibitory effect of ketamine on phosphorylation of the extracellular signal-regulated kinase1/2 following brain ischemia and reperfusion in rats with hyperglycemia

To determine if the inhibitory effects of ketamine on the extracellular signal-regulated kinase (ERK) 1/2 are involved in reduction of the hyperglycemia-exaggerated cerebral ischemic lesion, rats with normoglycemia, hyperglycemia, or hyperglycemia supplemented with ketamine were subjected to 15 min of forebrain ischemia, and then, reperfusion for 0.5, 1, and 3h. Phosphorylation of ERK1/2 in the brain tissues was assessed by immunohistochemistry and Western blot analysis. In rats with normoglycemia, we demonstrated a moderate increase of the ERK1/2 phosphorylation in the cingulum cortex and hippocampus CA3 following an ischemic intervention. It quickly dropped to control levels after reperfusion for 0.5h. In rats with hyperglycemia, however, the increase of the ERK1/2 phosphorylation in these areas was significantly higher in all animals reperfused. The neuronal death, detected by the TdT-mediated-dUTP nick end labeling assays, was found in the cingulum cortex (5.23+/-2.34, per high power feild) and hippocampus CA3 areas (6.29+/-3.68, per 1mm(2)) in hyperglycemic group after reperfusion for 3h. With ketamine treatment, the ERK1/2 phosphorylation in cingulum cortex and hippocampus CA1 and CA3 areas was found to be the same as that in normoglycemia rats. Our results suggest that hyperglycemia may increase the ischemic insult through modulation of the signal transduction pathways involving ERK1/2. The inhibitory effects of ketamine on the hyperglycemia-activated ERK1/2 phosphorylation are probably through inhibition of the N-methyl d-aspartate-mediated calcium influx, which subsequently reduce the hyperglycemia-exaggerated cerebral damage.