Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

BACKGROUND: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed. RESULTS: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature. CONCLUSION: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.

OBJECTIVES: Cancer patients generally exhibit circulating tumor-reactive immunoglobulins; however, these antibodies fail to eradicate tumors or prevent their progression. This study identifies and characterizes an aberrant tumor-reactive IgG population present in women with ovarian cancer. METHODS: In this pilot study, IgG was isolated from the sera of women with advanced-stage ovarian cancer (stages III and IV, n = 62) and age-matched female volunteers (n = 50) by affinity chromatography. These IgGs were characterized on the basis on their aberrant binding to concanavalin A affinity columns. Subsequently, the concanavalin A-binding moiety was localized following IgG fragmentation, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and characterized by oligosaccharide profiling. RESULTS: The level of concanavalin A-binding IgG in our control population was 8.9 +/- 2.9%, whereas in ovarian cancer patients, the level of concanavalin A-binding IgG was 38.8 +/- 7.4%. In the patients with ovarian cancer, 87.5 +/- 5.7% of the tumor-reactive IgG was demonstrated to be concanavalin A-binding. Based on oligosaccharide profiling of the fragmented concanavalin A-binding IgG, the aberrant lectin binding appeared to be the consequence of altered glycosylation of one of the two Fc chains. CONCLUSIONS: While our previous studies have identified the presence of circulating IgG reactive with specific tumor-associated antigens and its association with poor prognosis, this report demonstrated the presence of an aberrantly glycosylated IgG population in cancer patients. This altered IgG appeared to be the primary class of tumor-reactive antibodies in these women.

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei.

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.

Enhanced expression of Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P < 0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 micrograms/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis.

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha.

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.

Clinical and prognostic significance of concanavalin-A-induced suppressor cell activity in malignant cervical neoplasia.

Peripheral blood lymphocytes from patients with different stages of cancer of the uterine cervix were analysed for concanavalin-A-induced suppressor cell activity. All cancer patients had high levels of suppressor activity, the increase corresponding to tumour load. Radiotherapy resulted in further increase of suppressor activity. About six months after radiotherapy, patients who remained disease free returned to pretreatment levels of suppressor cell activity and later showed even lewer levels of suppression. On the other hand, patients who developed recurrent disease maintained sustained high levels of suppression and for all stages they always had higher suppressor activity than the patients who remained disease free in the corresponding stage. These results stress the importance of a possible deranged immune system in these patients and also shows the clinical and prognostic significance of the assay.

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes.

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.

Nephritogenic glycoprotein. XIII. Isolation and its immunochemical characterization of the antigenic substance of membranous glomerulonephritis induced by a single injection of crude nephritogenoside in homologous animals.

Studies on the immunochemical characterization were made on the antigenic substance of membranous glomerulonephritis (MG) that is induced, in homologous animals, by a single footpad injection of rat crude nephritogenoside (CNG). In rats injected with rat CNG, typical MG with immunofluorescent granular pattern (immune complex type nephritis) was produced 3-4 months after the injection. Since CNG nephritis is a model which induces MG only in homologous animals, the development of MG in this model should be explained by antigen-autoantibody immune complex mechanism. Thus, the features of the antigenic substance contained in CNG was immunochemically investigated, utilizing the serum (autoantibody) of rats with MG as well as rabbit antiserum to rat tubular brush border antigen (FXIA) (heteroantibody). As a result, two different fractions (fr. 31 and fr. 36) were separated from the antigenic substance contained in CNG: that is, fr. 31 reacts only with rabbit antiserum to rat tubular brush border antigen (FXIA) (heteroantibody) but does not react with the serum (autoantibody) of rats with MG. Fr. 36 does not react with rabbit antiserum to FXIA (heteroantibody) and reacts only with the serum (autoantibody) of rats with MG (which was induced by a single footpad injection of CNG or soluble FXIA). These results suggest immunologically that fr. 36 is the main antigen responsible for the development of MG which is induced in homologous animals by a single footpad injection of CNG.

Studies on the 240-kDa Con A-binding glycoprotein of rat cerebellum, a putative marker of synaptic junctions.

A Con A-binding glycoprotein of Mr 240,000 was isolated from the remaining residue of rat cerebella after sequential extraction with buffers supplemented with or without neutral detergents. It was further purified by affinity chromatography on Con A-Sepharose in the presence of sodium dodecyl sulfate and preparative gel electrophoresis. This glycoprotein partially resists Triton X-100 extraction and is soluble in N-lauryl sarcosinate. The 240-kDa glycoprotein was not detected in kidney, liver, heart, forebrain and was specifically seen in cerebellar homogenate. The isolated glycoprotein appears to be similar, not necessarily identical with the GPA--a synaptic junction 240-kDa Con A-binding glycoprotein isolated from cerebellum earlier (Groswald and Kelly, J. Neurochem., 42 (1984) 534-546). Monospecific antibodies obtained against the purified 240-kDa protein were used for developmental study in normal and hypothyroid rats. There was observed an increase in the amount of 240-kDa glycoprotein, dependent on the age of the rat and this rise was in correlation with the synapse formation in rat cerebellum. The amount of 240-kDa glycoprotein is considerably reduced in hypothyroid rats.

Schistosoma mansoni: stimulation of artificial granuloma formation in vivo by carbohydrate determinants.

A subset of Schistosoma mansoni egg glycoproteins that share a common carbohydrate epitope recognized by monoclonal antibody 128C3 was shown to induced formation of hepatic granulomata when conjugated to Sepharose beads and injected into the portal circulation of naive mice. Concanavalin-binding egg glycoproteins exhibited more granuloma-inducing activity than did total egg extract, although deglycosylated egg proteins also induced granulomata; thus, both amino acid and carbohydrate epitopes appeared to be involved. Glycoproteins derived from adult male worms also were active, indicating that immunological processes responsible for granuloma formation may not be absolutely stage specific.

Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoproteins: evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping.

Two major rat thymocyte surface glycoproteins, the leucocyte-common (L-C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX-1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing. TRITC-phalloidin staining of the isolated caps demonstrated the presence of F-actin within these structures, and lectin-affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L-C antigen and LSGP caps, but absent from the detergent-insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross-linked glycoproteins with the cytoskeleton during capping.

Specificity of antibodies to the purified Con A acceptor glycoproteins of cultured tumour cells.

Con A acceptor glycoproteins from the human Molt 4 (T cell leukaemia) and HeLa (endocervical adenocarcinoma) cell lines were purified by affinity chromatography and used for the preparation of rat antisera. Cross-absorption analysis showed that each antiserum contained antibodies which recognised cell surface antigens preferentially expressed by the donor cell line. Molt 4-associated antigens were fully expressed on T cell tumour lines and normal thymocytes, but not on non T cell tumour lines, peripheral blood lymphocytes or other blood cells. Immunofluorescence studies showed that the antigens were preferentially expressed on a sub-population of immature thymocytes. HeLa-associated antigens were only fully expressed on one other epithelial tumour cell in a panel of 17 cell lines. Immunofluorescence studies showed that the HeLa-associated antigens were expressed on normal endocervical adenoepithelium but not on ectocervical, endometrial or intestinal epithelia. Thus purified Con A acceptor glycoproteins of cultured tumour cell lines are potent immunogens for the generation of antibodies recognising lineage-associated differentiation antigens. These antigens should be useful in tumour classification and in the study of normal differentiation.

Recognition of asparagine-linked oligosaccharides on murine tumor cells by natural killer cells.

MDW4, a wheat germ agglutinin resistant mutant of the murine tumor line MDAY-D2, expresses abnormal asparagine-linked oligosaccharides, is less metastatic when injected intravenously, and is hypersensitive to natural killer (NK) lysis in vitro. To determine whether these phenotypes may be related, variants of the YAC-1 lymphoma and a YAC-1 X MDAY-D2 hybrid line were compared for sensitivity to four different lectins and to NK cell lysis in vitro. A relationship between sensitivity to concanavalin A (Con A) and NK cell lysis in vitro was observed. Although no single plasma membrane glycoprotein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with 125I-labeled Con A correlated with NK and Con A sensitivities of the cell lines, a relationship between these phenotypes and the collective 125I-Con A staining intensity on the gels was apparent. In a more direct test of carbohydrate recognition by NK cells, specific glycopeptide structures isolated from tumor cells and added to the NK cell assay in microM quantities were found to inhibit tumor cell lysis. Thus, a subset of asparagine-linked oligosaccharides, including high mannose and some incomplete complex structures on a number of cell surface glycoproteins, appears to be recognized as part of the target structures for NK cell lysis. The administration of polyinosinic:polycytidylic acid stimulated splenic NK activity in vivo but had no effect on the growth of the NK-resistant MDAY-D2 cells. However, the low tumorigenicity of MDW4 cells injected intravenously was reduced further by pretreating the mice with polyinosinic:polycytidylic acid, which indicated a role for NK cells in the elimination of circulating tumor cells expressing high mannose and/or incomplete complex asparagine-linked oligosaccharides.

The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes.

Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).