The nature of changes in the antigenic spectrum of the causative agent of melioidosis during animal passage

The comparative analysis of the antigenic spectra of Pseudomonas pseudomallei museum and subcultured strains, carried out by the method of immunoelectrophoresis, has revealed that, along with an essential increase in the virulence of P. pseudomallei for white mice and changes in the morphology of colonies, a decrease in the amount of detected precipitinogens occurs in the process of subculturing. The immunoelectrophoregrams of the subcultured variants show the absence of antigens 5, 6 and the simultaneous increase of the production of antigen 8, one of the components of mucoid (in the pseudocapsule).     

Development of a diagnostic system for Burkholderia pseudomallei infections

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.     

Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10

Three human thymic cell-surface antigens T6, T9 and T10, previously defined by monoclonal antibodies, were analyzed using immunoprecipitation techniques. The antigen T6 was found to be a 49,000 dalton glycoprotein, which is associated with β2-microglobulin, the small subunit (12,000 daltons) of the HLA-A, -B, and -C antigens. The target antigen for the monoclonal reagent anti-T9 was found to be a glycoprotein of 94,000 daltons, which appears as a disulfide-linked dimer of 190,000 daltons on the cell surface. The antigen precipitated by the anti-T10 antibody is a 45,000 dalton glycoprotein. We present preliminary evidence that all three cell-surface proteins may be integral membrane proteins. These findings, in addition to the distribution patterns, suggest that the T6 antigen is the human homolog of the murine thymus leukemia (TL) antigen.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.     

Nature of Pseudomonas pseudomallei antigens common to some species of microorganisms

An antigen, common for the infective agents of glanders , tularemia, plague, pseudotuberculosis, cholera and brucellosis, has been obtained from the aqueous saline extract of P. pseudomallei by means of affinity chromatography on antibody sorbent. The isolated antigen has proved to be of a protein-polysaccharide-lipid nature, to have insignificant cathode mobility and to constitute a component of O-antigen. It has also been found to be antigenically related to the tissues of melioidosis-sensitive animals and localized in the surface structure of the bacterial cell.     

Pathogenicity of Burkholderia pseudomallei: extracellular and surface antigen functions

The data of literature and the results of investigations carried out by the authors on the analysis of B. pseudomallei pathogenicity factors. They include mucoid, endotoxin, lecithinase, proteases, hemolysins, etc. In addition to fimbriae and pili, the adhesive properties of B. pseudomallei are supposed to be associated with surface capsule-like structures whose composition includes Ag8. The characterization of exoproteases, hemolysins and lethal toxins is given. The virulence and immunogenicity of B. pseudomallei were shown to be linked with the components of surface glycoprotein Ag8. The conclusion has been made that the analyzed pathogenicity factors are of importance for deciphering the pathogenesis of melioidosis.     

Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra antigen or EA-D in Raji cells, indicating that infectious virus is required for the observed effects of HHV-6 on these EBV products. These results show that HHV-6, another lymphotropic human herpesvirus, can activate EBV replication and may thus contribute to the pathogenesis of EBV-associated diseases.     

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

Antigenic properties of the electrophoretic fractions of the causative agent of melioidosis

Antisera to the antigens of 5 fractions, isolated as the result of the separation of P. pseudomallei aqueous saline extract by continuous electrophoresis in the vertical block of granulated gel, have been obtained. Immunoelectrophoresis with the use of P. pseudomallei aqueous saline extract has revealed that antisera to electrophoretic fractions contain antibodies mainly to the antigens of the corresponding fractions, which shows that this technique ensures the effective separation of P. pseudomallei biopolymers by their electrophoretic motility and molecular weight. These antisera differ in their species specificity. Thus, antisera to antigens with anode motility have been found to contain antibodies mainly to P. pseudomallei antigens and antisera to electroneutral antigens or to those with cathode motility, to P. pseudomallei and P. mallei antigens.     

Serosurveillance for double infection with Pseudomonas pseudomallei in tuberculous patients.

The serosensitivity to Pseudomonas pseudomallei antigen in pulmonary tuberculous patients was surveyed in Ubon Ratchathani Province, a melioidosis-endemic and tuberculosis-prevalent area in Thailand. Indirect hemagglutination assay (IHA) and indirect immunofluorescent assay (IFA) for IgM and IgG were employed for the measurement of antibody levels, with cut-off points set at 1:160, 1:8, and 1:32, respectively. Retrospective protocol survey of clinical data was also conducted. From these studies, however, no evidence was obtained to show that tuberculous patients have a disposition to acquire double-infected with P. pseudomallei and to develop melioidosis. The serosensitivity was never higher than that of the general population of the province as represented by healthy blood donors, nor related with the clinical severeness. Tuberculosis and melioidosis appear to be mutually independent diseases without showing interrelated prevalence in the endemic area.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

单克隆抗体对鼻疽和类鼻疽病原抗原及其血清学鉴别诊断的实验研究

应用抗鼻疽杆菌和抗类鼻疽杆菌的单克隆抗体(McAb),以间接ELISA,IFA以及免疫组织化学(下简称免疫组化)等技术方法,对来自不同地区的鼻疽杆菌(Ps.mallei)和类鼻疽杆菌(Ps.pseudmallei)的表面抗原进行了分析。在此基础上,又对鼻疽(Mallcus)和类鼻疽(Melioidosis)之间的血清学鉴别诊断等问题进行了研究。试验结果表明：(1)鼻疽杆菌和类鼻疽杆菌各自表达了不同的表面抗原反应类型,其闻并有一定的交叉关系;(2)鼻疽杆菌和类鼻疽杆菌各株均与McAb 2D4发生反应,说明表位2D4很可能为二菌所共有;(3)McAb4D4和lA9的类似抗体在鼻疽和类鼻疽血清都表现了较高的出现频率,说明其可能为二种血清的共有抗体成份;(4)McAb 3A1是仅同类鼻疽杆菌各株发生反应的特异性抗体。应用该McAb,以相应的实验技术,有可能解决长期以来存在的鼻疽和类鼻疽菌体间和血清间的免疫学鉴别诊断问题。     

Inflammation patterns induced by different Burkholderia species in mice

Burkholderia pseudomallei , which causes melioidosis, a severe, mainly pulmonary disease endemic in South-East Asia, is considered to be the most pathogenic of the Burkholderia genus. B. thailandensis , however, is considered avirulent. We determined differences in patterns of inflammation of B. pseudomallei 1026b (clinical virulent isolate), B. pseudomallei AJ1D8 (an in vitro invasion-deficient mutant generated from strain 1026b by Tn5-OT182 mutagenesis) and B. thailandensis by intranasally inoculating C57BL/6 mice with each strain. Mice infected with B. thailandensis showed a markedly decreased bacterial outgrowth from lungs, spleen and blood 24 h after inoculation, compared with infection with B. pseudomallei and the invasion mutant AJ1D8. Forty-eight hours after inoculation, B. thailandensis was no longer detectable. This was consistent with elevated pulmonary cytokine and chemokine concentrations after infection with B. pseudomallei 1026b and AJ1D8, and the absence of these mediators 48 h, but not 24 h, after inoculation with B. thailandensis . Histological examination, however, did show marked pulmonary inflammation in the mice infected with B. thailandensis , corresponding with substantial granulocyte influx and raised myeloperoxidase levels. Survival experiments showed that infection with 1 × 10³ cfu B. thailandensis was not lethal, whereas inoculation with 1 × 10⁶ cfu B. thailandensis was equally lethal as 1 × 10³ cfu B. pseudomallei 1026b or AJ1D8. These data show that B . pseudomallei AJ1D8 is just as lethal as wild-type B. pseudomallei in an in vivo mouse model, and B. thailandensis is perhaps more virulent than is often recognized.     

Identification of cell structure antigens of the causative agent of melioidosis by a 2-dimensional immunoelectrophoresis method

The antigenic composition of some cell structures of P. pseudomallei has been studied and the chemical nature of the antigens has been determined by the method of two-dimensional electrophoresis. In some cell components common antigens have been detected; at the same time these components have been found to possess their own characteristic antigenic complexes. The place of the cell structure antigens in the total antigenic structure of P. pseudomallei has been determined.     

B cell expression of endogenous retroviral envelope antigen: biochemical and immunofluorescence characterization

Plasma cells of line 15I5 chickens have been shown to express antigen that is cross-reactive with endogenous retroviral envelope glycoprotein. In the present study, we isolated this antigen and showed that it is structurally homologous to envelope glycoprotein. As assayed by immunofluorescence, most, if not all, of the plasma cell-associated envelope glycoprotein is present on the cell surface although it is not bound to surface Ig or Ia antigen. Although envelope antigen was not detectable by immunofluorescence on the surface of 15I5 bursal cells or peripheral B lymphocytes, immunochemical assays established that retroviral envelope glycoprotein is present on the surface of the bursal cells but at much lower levels than on the surface of the plasma cells. Both bursal and plasma cells synthesize envelope glycoprotein. These findings indicate that an increased level of expression of endogenous retroviral envelope glycoprotein is a concomitant of the differentiation of B lymphocytes to plasma cells in 15I5 chickens.     

Antigen 8 biosynthesis during cultivation of Burkholderia pseudomallei and B. mallei

The dynamics of the antigen 8 synthesis in Burkholderia pseudomallei and B. mallei under conditions of their submerged was studied. Differences in the intensity of this antigen synthesis in two pathogenic Burkholderia species were established and the producer strains, most effective with respect to this sign, were selected.     

Boar sperm membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell membrane

A monoclonal antibody, designated mAb P86/5, was generated by immunization of female Balb/c mice with a membrane vesicle fraction composed of the outer acrosomal membrane and plasma membrane (PM-OAM). As determined by fluorescence microscopy and electron microscopy P86/5 recognizes a sperm plasma membrane antigen that is restricted to the sperm head. In intact spermatozoa the P86/5-antigen is distributed over the surface of the sperm head with the exception of the rostral region. By comparing the antibody binding pattern generated at 4 degrees C and 25 degrees C, it could be shown that the P86/5-antigen is capable to diffuse freely within the cell membrane overlying the acrosome whereas its lateral mobility is restricted to the post-acrosomal region. The P86/5-antigen had a molecular weight of about 78 kDa as revealed by SDS-PAGE and western blotting. The glycoprotein nature of the P86/5-antigen was established by lectin affinity chromatography.     

Use of antibody/peptide constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation.

Human T cells can express MHC-class II products and were shown to be potential antigen-presenting cells. However, they are unable to capture the antigen and only antigens, which bind to T cell membranes such as the gp120 glycoprotein of HIV, are internalized, processed, and presented by T cells. To better understand the role of T cells as antigen-presenting cells, we established a method which overcomes the lack of antigen capture by T cells. Antigen (tetanus toxoid, TT) or an antigenic peptide of TT (residue 830-843, P2) was coupled to antibodies directed to T cell surface molecules such as CD2, CD4, CD8. Antibody/TT and antibody/P2 constructs stimulated P2-specific T cell clones in the absence of accessory cells, if the antibody recognized a T cell surface structure. Compared to the peptide alone, a 100-500 times lower molar concentration of the antibody/peptide construct was required to achieve a similar proliferative response. T cell stimulation via the constructs involved intracellular processing, as nonspecific, glutaraldehyde fixed T cell lines pulsed with the constructs could present the peptide and processing inhibitors like Leupeptin or Chloroquine inhibited the development of a proliferative response to the constructs. Our data underline the ability of T cells to function as antigen-processing and -presenting cells and show that antibody/antigen or antibody/peptide constructs are able to direct a certain antigen or peptide to a T cell. Antibody/peptide constructs may be interesting tools to better understand antigen processing and to study the consequences of antigen presentation by different cells.     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

Ecological and biological maintenance of sapronoses exemplified by melioidosis

The growth and death of Pseudomonas pseudomallei, the causative agent of melioidosis, in the soil and the antigenic properties of this microorganism in the soil, in culture media, and in the body of animals have been studied. As revealed in this study, P. pseudomallei can grow in nonsterile soil substrates without the loss of virulence and changes in its antigenic structure. In the body of animals this microorganism rapidly adapts its virulence to host species by the transformation of its antigenic structure. The pathogenicity factors of P. pseudomallei are mainly thermolabile antigens, probably exoenzymes. This microorganism has been shown to have close ecological relations with abiotic environmental objects. The author suggests that the type of relationship between saprophytic microorganisms acting as causative agents of diseases and warm-blooded hosts should be characterized as pseudoparasitic.