Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus.

The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and high-molecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecular-weight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.     

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.     

A study on the body fluid antigen of Clonorchis sinensis using immunogold labeling method

In order to observe the antigenic localization in the tissues of the adult Clonorchis sinensis, immunogold labeling method was applied using serum immunoglobulins (IgG) of either worm-infected rabbits (group I) or antigen-immunized rabbits (group II) (by the body fluid obtained from the adult worms). The electron micrographs of the sectioned worm tissue antigens, embedded in Lowicryl HM 20 medium and stained with protein A-gold complex (particle size: 12 nm), were compared between the group I and group II. The gold particles were observed in the interstitial matrix of the worm parenchyma, the epithelial lamellae of the cecum, and the cecal lumen both in group I and II. But the particles were in general more concentrated in group II. The gold particles were not observed on the basal lamina of the tegument or on vitelline glands in group I, while they were highly concentrated on those areas in group II. There were also differences in the antigenicity of interstitial matrix(reacted with group I IgG) and head part(reacted with group II IgG) of the sperm cells in the seminal receptacle. Conclusively, it is suggested that the substances comprising the basal lamina of the tegument or vitelline glands act as specific antigens reacting with antigen(body fluid) immunized rabbit IgG. On the other hand, the substances in the cecal lumen and cecal epithelial lamellae are thought to be the specific antigen that react with the worm-infected rabbit IgG.     

Serodiagnosis of Echinococcosis by ELISA Using Cystic Fluid from Uzbekistan Sheep

According to increase of travel, the cases of imported echinococcosis have been increasing in Korea. The present study was undertaken to develop a serodiagnostic system for echinococcosis in Korea. For diagnosis of echinococcosis, the fluid of Echinococcus granulosus hydatid cysts was collected from naturally infected sheep in Uzbekistan. Also serum samples of infected patients who were surgically confirmed were collected in a hospital in Tashkent, Uzbekistan. According to the absorbance of 59 echinococcosis positive and 39 negative control serum samples, the cut-off value was determined as 0.27. The sensitivity and specificity of ELISA with hydatid fluid antigen were 91.5% and 96%, respectively. The antigen cross-reacted with the serum of some cysticercosis or clonorchiasis patients. However, immunoblot analysis on the cystic fluid recognized antigenic proteins of 7-, 16-, and 24-kDa bands in their dominant protein quantity and strong blotting reactivity. In conclusion, the present ELISA system using hydatid cyst fluid antigen from Uzbekistan sheep is sensitive and specific for diagnosis of echinococcosis cases.     

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.     

A comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid

Measurements of specific antibody titres in uterine fluid of mice immunized by different routes indicated that two immunizations in the pelvic presacral space using aluminium hydroxide as adjuvant was a simple and effective way to elicit a significant IgA and IgG response. Higher IgA and IgG titres were produced in uterine fluid by subcutaneous immunization with antigen in Freund's complete adjuvant followed by intravaginal boosting without adjuvant, but this immunization involved both a toxic adjuvant and repeated applications of large doses of antigen in the vagina. Intragastric immunization produced an IgA response in the uterus but no IgG. Local intravaginal priming and boosting with large doses of antigen without adjuvant produced an IgA response in uterine fluid, but was less effective for IgG and was inefficient in terms of time and the amount of antigen used. Hysterectomy reduced the concentration of specific IgA in vaginal fluid of immunized mice to no more than 5% of normal, indicating that most of the IgA in vaginal fluid originates in the uterus. In contrast, IgG titres were not significantly different in hysterectomized and intact mice. IgA titres in vaginal fluid were at least partly restored to normal levels in sham-hysterectomized mice.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.     

Evidence for a gonad-specific receptor for H-Y antigen: binding of exogenous H-Y antigen to gonadal cells is independent of beta 2-microglobulin.

This report addresses the question whether two different types of binding exist for the reaction of H-Y antigen with the cell surface. Anti-H-Y antiserum in the presence of complement was cytotoxic only for gonadal cells expressing their own H-Y antigen, but not to ovarian cells loaded with H-Y antigen. H-Y antigen was co-redistributed with beta 2--microglobulin on newborn testicular cells, but some residual H-Y activity was found on similarly treated testis cells from 15 day old rats. After beta 2--microglobulin redistribution, testis cells maintained their binding capacity for exogenous H-Y antigen prepared from epididymal fluid or Daudi cell culture supernatants. This result suggests that exogenous H-Y antigen is bound via a gonad-specific receptor which is independent of beta 2--microglobulin and that this type of binding for H-Y antigen is different from the beta 2--m-associated expression of H-Y antigen on the cell surface.     

Turnover and fate of I-Ak antigen on the murine macrophage cell surface

The macrophage plasma membrane is a major site of the cell's activities, including phagocytosis, antibody-dependent cellular cytotoxicity, and antigen presentation. To present antigen, the expression by the macrophage of immune region-associated (Ia) antigen is required. The turnover and fate of this cell surface constituent was studied in macrophages cultured with lymphokine or recombinant interferon-gamma. Surface-labeled subregion I-Ak antigen was lost from the cell surface at a rapid rate, with a half-life of approximately 24 hours. However, the shedding of I-A antigen into the culture fluid was not detected. Therefore, the loss of I-A antigen from the macrophage surface is most likely by its degradation. Upon removal of lymphokine or interferon from macrophage cultures, I-A antigen expression declined, with an apparent half-life of 2 days.     

Cultivation of Neisseria meningitidis serogroup A on semisynthetic liquid nutrient media]

In cultivation of meningococcus of serological group A in fluid semisynthetic medium of simple composition prepared on the basis of purified acid casein hydrolysate with profound splitting there were obtained microbial cultures with a density of 4-5 x 10(9) microbial cells per 1 ml after 20-24 hours of cultivation with shaking. Alkalinity of the medium increased (to pH 8.0-8.2 during the stationary phase) with increase of the microbial cell concentration. A study of the accumulation of group-specific thermostable polysaccharide antigen in dynamics of meningococcus cultivation on semisynthetic medium tested showed the preparations obtained by alcoholic precipitation to be colourless and to contain much antigen (by inhibition of indirect hemagglutination), particularly at the phasees of negative growth acceleration and at the stationary phase. The suggested fluid semisynthetic medium of simple composition could be used for production of diagnostic and prophylactic meningococcus preparations belonging to the serological group A.     

Immunochemical study of the toxin of C1. botulinum type E]

Toxins extracted from the cells and the culture fluid of C1. butulinum type E had a similar mol wt and antigenic composition. Trypsin activation led to the appearance of an additional antigen. Coefficient of the cell toxin activation was much greater than the coefficient of activation of the toxin contained in the culture fluid.     

Activation of T Cells by Autoantigen Immobilized by Specific Antibodies

A convenient microtiter-plate assay that uses immobilized antibody to capture specific antigens for presentation to T cells has been developed. Initial experiments used KLH as the antigen, immune antisera and draining lymph node cells from immunized NOD mice as the source of antibody and T cells, and spleen cells from naive NOD mice as the source of antigen-presenting cells (APCs). The resulting proliferation of the T cells was shown to be antibody- and antigen-specific, suggesting that the APCs had internalized and processed the captured antigen, presenting it to the T cells in the form of peptide/MHC complexes. The approach was also tested for an autoimmune disease as part of an effort to identify autoantigens responsible for the proliferation of T cells in the synovial fluid of rheumatoid arthritis patients. When immunoglobulin from autologous synovial fluid was captured on plates coated with anti-human immunoglobulin antibodies, the addition of HLA-DR4 peripheral blood mononuclear cells as APCs and synovial fluid-reactive HLA-DR4-restricted T-cell clones resulted in significant proliferation, indicating that the specific antigen in the crude synovial fluid was human immunoglobulin. This response was also shown to be antigen-specific and HLA-DR4-restricted. This assay format should permit the definition of autoantigens by capturing with antibodies to crude autoantigen extracts, followed by the addition of the appropriate APC and T-cell populations.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Stability of rubella complement-fixing antigens

Various types of rubella complement-fixing (CF) antigen preparations derived from infected BHK-21 cell cultures were examined for stability to certain chemical and physical agents. Antigens studied included: infected culture fluids concentrated 100-fold, ether-treated fluid concentrates, alkaline buffer extracts of infected cells, and large- and small-particle CF antigens separated by Sephadex gel filtration. All preparations were stable at -20 C for months, and were unaffected by three cycles of freezing and thawing. Ether treatment rendered antigens highly susceptible to thermal inactivation at 56 C; untreated antigens were inactivated slowly and showed a persistent fraction of activity even after 2 hr. Large-particle antigen was found to be slightly more susceptible than small-particle antigen to thermal inactivation and ether treatment. CF activity in alkaline buffer extracts was more labile than that in untreated or ether-treated fluid concentrates upon dialysis, Sephadex gel filtration, and sucrose density gradient centrifugation.     

Immunohistological localisation of two hydatid antigens (antigen 5 and antigen b) in the cyst wall, brood capsules and protoscoleces of Echinococcus granulosus (ovine and equine) and E. multilocularis using immunoperoxidase methods.

Cyst wall, brood capsules and evaginated protoscoleces of E. granulosus (ovine and equine) and E. multilocularis were fixed in 10% formol-saline, embedded in paraffin and cut at 8 micrometer. Specific rabbit antisera to antigen 5 and antigen B of hydatid cyst fluid were used with immunoperoxidase methods to localise the antigens in the histological sections. Antigen 5 was found in all parasites and was associated with cells of the subtegumental area of the protoscolex, the brood capsule wall and the germinal membrane. The labelled antigen appeared as distinct granules in all areas. It is suggested that antigen 5 may be synthesised in all of these sites and that a source of the antigen in cyst fluid may be the germinal and brood capsule membranes. The laminated membranes of E. granulosus (ovine and equine) were, except for the superficial layers, free from antigen 5. Antigen B was present in all parasites. It was distributed diffusely throughout the laminated membrane, germinal membrane and brood capsule wall. There were areas of densely labelled antigen B on the surface of the distal cytoplasm of the protoscolex tegument and the surface of calcareous corpuscles. The distribution of antigen B in relation to PAS positive material and possible complement activating substances is discussed. The laminated membrane of E. granulosus was apparently more permeable to antigen B than to antigen 5. It is suggested that differences in the diffusion of these antigens through the laminated membranes of hydatid cysts in the same or different host species may account for variable serological responses during infection.     

Identification and hormonal control of reproductive-tract-specific antigens present in rabbit oviductal fluid

Utilizing the intra-abdominal flask technique to collect oviductal fluid, the presence of two or possibly three reproductive-tract-specific antigens have been observed in rabbit oviductal fluid. Two of these antigens may be accounted for by the two forms of uteroglobin. The other antigen has a molecular weight greater than 200,000 daltons and its concentration in oviductal fluid is under hormonal control. During pseudopregnancy (PSP), when progesterone concentrations are high, or upon progesterone administration, the concentration of this high molecular weight antigen doubles in oviductal fluid. This correlates well with the previously observed increase in release of secretory products from the oviductal epithelia.     

An enzyme-linked immunosorbent assay using a chaotropic agent (sodium thiocyanate) for serotype specific reaction between crude dengue viral antigen and anti-dengue mouse antibody.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti-dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement-fixing antigen (SCF) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted serotype specifically to anti-dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti-type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti-type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.