Identification and potential use of a soluble tumor antigen for the detection of liver-fluke-associated cholangiocarcinoma induced in a hamster model

A liver-fluke-associated cholangiocarcinoma (CCA), comparable to that occurring in humans, was induced by exposing Opisthorchis viverrini-infected hamsters to dimethylnitrosamine (DMN). Tumor masses were removed and histopathologically identified, then one portion was extracted for antigens used in the production of monoclonal antibodies (MAbs). The remaining portions were used to establish CCA cell lines. The antigens produced and secreted by these cell lines, as well as those originally present in the tissue extracts, possessed a 200-kDa glycoprotein that appeared to be immunologically distinct from other tumor markers. A specific MAb called 6E5 was used to set up a sandwich ELISA for the quantification of this antigen in the serum and bile of tumor-bearing animals. The assay system was sensitive enough to detect the antigen at concentrations below 10 ng/ml. The serum and biliary levels of this antigen were markedly elevated in animals with progressive tumors when compared with untreated controls. The serum taken serially from each animal that subsequently developed CCA showed a gradual but significant elevation of the antigen as carcinogenesis progressed. A few isolated animals exhibited a slight elevation of the antigen at a time as early as the end of DMN treatment, when the CCA should not yet have developed, judging from microscopic examination. The data from this animal model suggested that the CCA-associated soluble antigen defined by MAb 6E5 was a useful marker for the detection of tumors at an early stage of development.     

Antigens, antibodies and immune complexes in cerebrospinal fluid of patients with cerebral gnathostomiasis

Methods for the detection of antigens, antibodies and immune complexes in the cerebrospinal fluid (CSF) of patients with neurological manifestations suggestive of cerebral gnathostomiasis were developed, in the hope that they may be useful in confirming the diagnosis of Gnathostoma spinigerum infection. Gnathostoma antigens were determined by a sandwich enzyme linked immunosorbent assay (ELISA) using antibodies from rabbits immunized with the excretory/secretory (ES) antigens obtained from the in vitro supernatant fluid in which the third-stage G. spinigerum larvae were maintained. With a biotin streptavidin procedure, the presence of G. spinigerum antigens as low as 2 ng in one ml of CSF could be detected. An indirect ELISA was used for the quantitation of IgG antibodies in the paired serum and CSF of these patients. A complement consumption method was used for the detection of immune complexes in the concentrated CSF specimens. Of the 11 patients with clinical signs and symptoms suggestive of having G. spinigerum infection involving the central nervous system, only one patient had antigens detected in the CSF and in this one patient no antibody could be demonstrated. One other patient had immune complexes in her CSF. All remaining patients had IgG antibodies demonstrable in the CSF specimens. These data suggest that the detection of IgG antibodies in CSF is more reliable than the other two methods in confirming the diagnosis of cerebral gnathostomiasis.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the diagnosis of Penicillium marneffei infection

A monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay was developed for the detection of Penicillium antigen in clinical specimens from patients with Penicillium marneffei infection. The IgM from clone 8C3, reactive with both yeast and mycelial antigens, was immobilized on a microtiter plate. The antigen in serum or urine was captured and detected with biotinylated polyclonal rabbit anti-P. marneffei antibody. The test was sensitive in detecting soluble yeast exoantigen at a concentration as low as 4.88 ng/mL. The reliability of the assay method was evaluated using sera at a dilution of 1:2 from 18 patients with culture-proven penicilliosis, 23 patients with other fungal and bacterial infections, and 125 healthy volunteers. The method exhibited a sensitivity of 72%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 97%, and an accuracy of 97%. However, the sensitivity could be increased to 90% when the specimens were used undiluted. The method was also useful for the detection of antigen secreted in the urine of the patients. The results showed that the newly described assay method can be used in the diagnosis of P. marneffei infection with a high degree of reliability.     

Detection of Opisthorchis viverrini by monoclonal antibody-based ELISA and DNA hybridization

Monoclonal antibody-based enzyme-linked immunosorbent assay and DNA hybridization techniques were developed and evaluated for their potential in the detection of Opisthorchis viverrini infection in humans. A mixture of three IgG1 monoclonal antibodies (MAb) specific for the 89 kDa metabolic product of O. viverrini was captured on a microtiter plate by rabbit anti-mouse IgG and used in a sandwich ELISA for the detection of parasite antigen. The 89 kD component bound to the MAb was detected with biotinylated rabbit IgG antibody to O. viverrini metabolic products. As little as 0.05-0.1 ng of the antigen could be detected by this technique. A specific O. viverrini DNA probe constructed from a repetitive DNA segment containing 340 base pairs was used in a dot blot hybridization for the detection of parasite DNA. The labeled probe constructed as such could detect DNA released from as few as five O. viverrini eggs. Both methods were specific for O. viverrini and their sensitivity was comparable with that of the classical parasitological technic.     

An Association between Thalassaemia and Autoimmune Haemolytic Anaemia (AIHA)

An association between thalassaemia and autoimmune haemolytic anaemia (AIHA) has not been previously recognized. 10 patients with thalassaemic diseases were found to give positive results to antiglobulin test with polyvalent antiserum. Tests with monovalent antisera revealed immunoglobulins on the red cells in all cases. From history of blood transfusion, auto-antibodies following alloimmunization were very unlikely. IgG, either alone or together with IgM or IgA, was the predominant antibody on the red cells. All cases had warm type AIHA and responded dramatically well with steroid treatment except 3 who eventually needed splenectomy which responded in 2 and failed in 1.     

Lipopolysaccharide heterogeneity among Burkholderia pseudomallei from different geographic and clinical origins

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.     

Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients

Burkholderia pseudomallei is the causative agent of melioidosis. One of the main risk factors for B. pseudomallei infection in endemic areas is diabetes mellitus. The present study investigated IL-17 mRNA and protein expression by peripheral blood mononuclear cells in response to B. pseudomallei infection in 10 diabetic patients in comparison to 10 healthy blood donors. The IL-17 expression in diabetic patients was significantly lower (p < 0.05) than in the controls. However, IL-23 mRNA expression of the 2 groups was comparable. The present findings suggest that melioidosis affects T cell IL-17 production and that patients with diabetes mellitus have a defective IL-17 production in response to this type of infection.