Enolase antigen, mannan antigen, Cand-Tec antigen, and beta-glucan in patients with candidemia.

We compared the specificities and sensitivities of four tests used for the serodiagnosis of candidemia in 39 patients with candidemia, including 10 patients with superficial Candida colonization, 10 patients with deep mycosis, and 20 healthy subjects. The results obtained by the dot immunoblotting assay for detecting the enolase antigen (48 kDa) were compared with those of assays for detecting mannan antigen, heat-labile antigen (a threshold titer of four times), and beta-glucan (> or = 60 pg/ml). Enolase antigen was detected in 28 (71.8%) patients with candidemia, while 30 (76.9%), 10 (25.6%), and 27 (84.4%) patients were positive for the heat-labile antigen by the Cand-Tec assay, the mannan antigen by the Pastorex Candida assay, and beta-glucan by the limulus test, respectively. Ten patients with superficial Candida colonization, 5 patients with invasive pulmonary aspergillosis, 5 patients with cryptococcosis, and 20 healthy subjects were negative for both enolase antigen and mannan antigen. Two patients with superficial Candida colonization, one patient with invasive pulmonary aspergillosis, and two patients with cryptococcosis were positive by the Cand-Tec assay. The beta-glucan concentration was more than 60 pg/ml in all patients with invasive pulmonary aspergillosis; however, it was less than 10 pg/ml in all patients with cryptococcosis. The specificity of enolase antigen in the serodiagnosis of candidemia was 100%, but the sensitivity was 71.8%. The specificity and sensitivity of Cand-Tec, the assay for mannan antigen, and the assay for beta-glucan were 76.9 and 87.5%, 25.6 and 100%, and 84.4 and 87.5%, respectively. Our results demonstrated that antigen detection tests are useful for the diagnosis of candidemia; however, none is satisfactory for the serodiagnosis of candidemia. We suggest that a combination of two assays may increase the accuracy of diagnosis of candidiasis.     

Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro

Aluminium adjuvants (alum) have been the only widely approved adjuvants for use in human vaccines since the 1920s, however, the mechanism of action of these adjuvants remains elusive. Due to increasing demand for novel adjuvants, a clearer understanding of the mechanisms that allow these important agents to affect adaptive immune responses will make a significant contribution to the rational design of future vaccines. Using a novel approach to tracking antigen and antigen presentation, we demonstrate that alum induces higher antigen accumulation and increased antigen presentation by dendritic cells (DCs) in vitro. Antigen accumulation was 100-fold higher and antigen presentation 10-fold higher following alum treatment when compared with soluble protein alone. We also observed that alum causes an initial reduction in presentation compared with soluble antigen, but eventually increases the magnitude and duration of antigen presentation. This was associated with reduced protein degradation in DCs following alum treatment. These studies demonstrate the dynamic alterations in antigen processing and presentation induced by alum that underlie enhanced DC function in response to this adjuvant.     

Role of nonspecific cross-reacting antigen, a CD66 cluster antigen, in activation of human granulocytes.

Nonspecific cross-reacting antigen (NCA) is the name of a family of highly glycosylated bacterial-binding receptors found on human granulocytes and other tissues. These glycoproteins are members of the immunoglobulin supergene family and are related structurally to carcinoembryonic antigen. In this study, we demonstrate that ligation of granulocyte NCA results in the activation of the cells, as measured by degranulation and the flux of intracellular calcium. These studies further the proposition that NCA has a function in the immune response of granulocytes against bacterial infections.     

华支睾吸虫膜抗原/排泄分泌抗原酸性磷酸酶的克隆、表达、生物学特征分析及组织定位

 目的:对华支睾吸虫(Clonorchis sinensis, Cs)成虫酸性磷酸酶 (acid phosphatase, AP)进行克隆、表达、生物学特征分析、组织定位及膜抗原/排泄分泌抗原鉴定。方法:对CsAP进行生物信息学、分子生物学、免疫组化及明胶酶谱分析。结果:从Cs cDNA文库中筛选出编码AP新基因,全长1 410 bp,重组并由大肠杆菌表达、纯化,得到分子量为55 kD的重组蛋白CsAP。Western blotting分析表明,CsAP既是膜抗原又是分泌排泄抗原;免疫组化显示,CsAP荧光显示于成虫的表皮层和肠支,在囊蚴也有显示,在雷蚴和尾蚴未显示荧光;ELISA分析表明CsAP识别华支睾吸虫病人和日本血吸虫病人存在吸虫间的交叉免疫反应,CsAP及粗抗原识别轻、中、重度感染程度华支睾吸虫病人的差别不明显。重组蛋白免疫大鼠后,总IgG抗体滴度于3周达较高峰,抗体效价大于1∶25 600。明胶降解实验表明：CsAP具降解胶原能力。结论: 上述结果表明,CsAP在大肠杆菌中高效表达,具有较好的免疫原性,但血清诊断价值不理想;CsAP可能既是膜抗原,又是排泄分泌抗原。     

Efficient antigen presentation of soluble,but not particulate,antigen in the absence of Wiskott-Aldrich syndrome protein

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.     

Target antigen, age, and duration of antigen exposure independently regulate immunoglobulin G subclass switching in malaria

The isotype/subclass of immunoglobulin determines antibody function, but rather little is known about factors that direct class switching in vivo. To evaluate factors that might influence the maturation of the antibody response during infection, we conducted a seroepidemiological study of the immunoglobulin G (IgG) subclass response to four merozoite-associated antigens of Plasmodium falciparum in a mountainous region of northeastern Tanzania, where malaria endemicity declines with increasing altitudes. We found that IgG1/IgG3 class switching is independently affected by the nature of the antigen, cumulative exposure to the antigen, and the maturity of the immune system (i.e., the age of the individual). These observations provide insights into the effects of immune system maturity, the duration and intensity of antigen exposure, and inherent characteristics of individual antigens on the process of class switching in human B cells. Our data also throw light on the consequences of class switch decisions on the gradual acquisition of antimalarial immunity.     

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen,recombinant antigen and PCR

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.     

Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.     

Expression of the 5.1 H11 antigen, a fetal muscle surface antigen, in normal and neoplastic tissue

The monoclonal antibody 5.1 H11 recognizes an antigen on human fetal muscle and on rhabdomyosarcoma cell lines and xenografts that has been shown to be homologous to the neural cell adhesion molecule. To evaluate its range of expression, we used immunoperoxidase staining of fresh frozen-tissue sections to determine monoclonal antibody 5.1 H11 reactivity in normal and neoplastic tissue. Among normal tissue specimens, intense antibody staining was seen in brain and peripheral nerve, and weaker staining in ganglionic elements of colon. In addition to 26 of 29 rhabdomyosarcoma specimens, 5.1 H11 antibody showed reactivity to 9 of 10 Wilms' tumors, 6 of 6 neural tumors, and 4 of 4 gliomas, and with single specimens of ectomesenchymoma, clear-cell sarcoma of kidney, undifferentiated sarcoma of liver, ovarian fibroma, and neurofibroma. We conclude that the monoclonal antibody 5.1 H11 recognizes an antigen present not only on fetal muscle but on normal brain and nerve as well. In addition to rhabdomyosarcoma, a variety of other tumors, most of which have been previously shown to express neural cell adhesion molecule, also appear to express the antigen recognized by 5.1 H11. Our results thus offer additional confirmatory evidence that an epitope of neural cell adhesion molecule is the antigen for 5.1 H11.     

Close association of human mixed lymphocyte culture antigen, Ia-like antigen and Fc receptor.

The effects of aggregated human IgG, human anti-Ia-like antibody, and anti-beta2-microglobulin on mixed lymphocyte culture (MLC) were studied. Aggregated human IgG inhibited both stimulatory and responding activities in MLC. When Fc-receptor-bearing cells were removed from responding cells, the inhibitory activity of aggregated IgG was markedly reduced. The result suggested that the inhibition of MLC by aggregated IgG is primarily based on the blocking of Fc-receptor-bearing cells contained in the stimulator cells. In addition, the removal of Fc-receptor-bearing cells from stimulatory cells resulted in the loss of MLC response. Anti-Ia-like antibodies contained in anti-HLA sera and B-cell-specific human alloantisera also inhibited stimulatory activities in MLC. Rabbit antiserum against human beta2-microglobulin showed inhibitions of both stimulatory and responding activities. These results suggested the close association of human MLC stimulator site with Fc receptor and Ia-like antigen and also some relation of beta2-microglobulin with MLC reaction.     

Cervical carcinoma antigen, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) in appraisal of uterine cervix smears

The distribution of cervical carcinoma antigens (AgCaCx), CEA, and NCA in different pathologic states of the uterine cervix was studied in cytologic smears by an immunofluorescence method (IF) using specific immune sera against perchloric acid (PCA) extract of cervical squamous cell carcinoma, anti-CEA, and anti-NCA. After excluding cross-reactivity with CEA and NCA, the presence of AgCaCx was demonstrated in the majority of cervical carcinomas, severe dysplasias, and only in one-fourth of squamous metaplasias, especially when accompanied by mild or moderate dysplasias. The intensity and percentage of IF-positive cells varied from case to case. The preparations of uterine cervix without pathologic changes usually were negative. Similar results were obtained with anti-CEA serum. NCA was present in the majority of smears independent of histologic diagnosis. The most intense fluorescence was observed in upper layers of the epithelium. NCA could be a differentiation antigen of stratified squamous cell epithelium.     

Hodgkin's disease,lymphocyte predominance type,nodular--a distinct entity? Unique staining profile for L&H variants of Reed-Sternberg cells defined by monoclonal antibodies to leukocyte common antigen,granulocyte-specific antigen,and B-cell-specific antigen.

Using monoclonal antibodies to leukocyte common antigen, granulocyte-related antigen, and B-cell specific antigens, L&H variants of Reed-Sternberg (R-S) cells in Hodgkin's disease, lymphocyte predominance type (nodular), exhibited a unique staining profile as compared with R-S cells of other histologic types. L&H variants were strongly immunoreactive for leukocyte common antigen, as defined by monoclonal antibodies PD6/27 and 2B11; whereas other types of R-S cells were negative or rarely positive. R-S cells and variants in 69 cases of Hodgkin's disease of nodular sclerosis (41), mixed cellularity (25) or lymphocyte depletion (3) types, were consistently strongly immunoreactive for Leu-M1, a granulocyte-related antigen, while L&H variants were uniformly nonreactive (4 cases). B-cell specific antigens, detected by three pan-B-cell monoclonal antibodies, were observed only for L&H variants. These observations suggest that L&H variants of R-S cells represent a distinct type of transformed cell, possibly of B-cell origin, and do not share a common lineage with other types of R-S cells. These studies provide further evidence that Hodgkin's disease, lymphocyte predominance type, nodular, may represent a distinct entity.     

Epithelial markers in prostatic, bladder, and colorectal cancer: an immunoperoxidase study of epithelial membrane antigen, carcinoembryonic antigen, and prostatic acid phosphatase

Twenty prostatic adenocarcinomas, 20 transitional cell carcinomas of the bladder, and 20 colorectal adenocarcinomas were stained for epithelial membrane antigen, carcinoembryonic antigen, and prostatic acid phosphatase. Polyclonal affinity purified first and second antibodies and an indirect immunoperoxidase technique were used. All of the colorectal and bladder tumours and 16/20 prostatic tumours were positive for epithelial membrane antigen. All 20 colorectal, 7/20 bladder, and 5/20 prostatic tumours stained for carcinoembryonic antigen. All of the prostatic adenocarcinomas and none of the colorectal or bladder tumours were positive for prostatic acid phosphatase. These markers may be used to discriminate between tumours arising from these sites.     

Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen.

The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.     

Dissociation among Ia antigen expression,accessory cell function,and antigen processing in two acute monoblastic leukemia lines

To better understand the role of Ia antigen expression, accessory cell function, and antigen ingestion in antigen presentation and the initiation of T cell proliferation, we studied these events in two acute monoblastic leukemia (AMoL) lines. The cell lines were positive for surface Ia antigen; one stimulated proliferation of the allogeneic mononuclear cells in mixed lymphocyte culture and culture supernatants from the other line contained interleukin 1 (IL-1) when tested for comitogen activity in a standard mouse thymocyte assay. The AMoL cells also contributed accessary factors for mitogen-induced proliferative responses by T cells. High numbers of cells of one of the lines tended to suppress mitogen induced T cell proliferation. Irradiated trinitrophenylated AMoL cells were able to stimulate TNP-specific HLA-DR matched T cell blasts to proliferate. However, when irradiated AMoL cells were cultured with a protein antigen (tetanus toxoid or varicella zoster) plus antigen-specific parental T cell blasts, antigen presentation failed to occur. Diminished phagocytosis by the AMoL cells, together with reduced catabolism of labelled antigen, is a likely explanation for this finding. Our results demonstrate that the concurrent presence of a complex protein antigen and Ia-positive monocytic leukemia cells capable of accessory function is alone insufficient to maintain antigen-specific T cell proliferation. Moreover, these findings suggest that antigen processing, involving ingestion and reexpression of antigenic determinants, is an essential aspect of antigen presentation not tightly linked to Ia antigen expression or IL-1 production in these AMoL lines.     

Decomplementation antigen, a possible determinant of staphylococcal pathogenicity

We report the existence of an extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that causes rapid consumption of early-reacting complement components up to and including C5 in human serum. Complement activation occurs as a consequence of immune complex formation between DA and specific human immunoglobulin G antibodies and proceeds primarily via the classical pathway. The terminal components C7, C8, and C9 are not consumed during the process. Levels of DA production do not correlate with the expression of classical pathogenic factors, such as coagulase, clumping factor, protein A, or alpha-toxin. DA is a nondialyzable macromolecule eluting in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and displaying an apparent sedimentation coefficient of 3 to 4 S on sucrose density gradients. The molecule is remarkably stable and resists destruction upon boiling for 30 min or by treatment with pronase, lysostaphin, DNase, or RNase. We anticipate that DA protects staphylococci from complement attack through induction of abortive, complement-consuming reactions in the fluid phase.     

血清癌胚抗原、糖链抗原153与异常糖链糖蛋白在乳腺癌诊断中的应用

目的:探讨肿瘤异常糖链糖蛋白(tumor abnormal protein,TAP)、癌胚抗原(carcinoembryonic antigen,CEA)与糖链抗原153(carbohydrate antigen 153,CA153)在乳腺癌早期诊断中的临床价值.方法:收集45例乳腺癌患者为研究组,49例正常体检人群为对照组,比较各肿瘤志记物的敏感度、特异度、准确度等指标.结果:在乳腺癌组患者中,TAP,CEA,CA153检测敏感度分别为80.00％,8.89％,6.67％;特异度分别为100.00％,97.96％,100.00％,准确度分别为90.42％,55.32％,55.32％.结论:肿瘤TAP检测优于肿瘤标志物CEA和CA153检测,可作为乳腺癌的早期筛查指标.