Retrospective study on the diagnostic value of IgG ELISA, dot immunoassay and indirect hemagglutination in septicemic melioidosis.

Three serological methods for diagnosis of melioidosis were compared with the culture method currently used as the "gold standard". The diagnostic values of the serological methods were evaluated retrospectively in 306 patients residing in an endemic area. The enzyme-linked immunosorbent assay (ELISA), using affinity purified antigen for detecting specific IgG antibody, showed a slightly higher specificity (86.0%) than the dot immunoassay (DOT) (84.0%) and both were superior to indirect hemagglutination (IHA) (72.0%). The sensitivity of DOT (96.4%) and ELISA (85.7%) were considerably higher than that of IHA (50.0%). The primary benefit of the high negative predictive value of both ELISA (96.4%) and DOT (99.0%) in an area of high prevalence is the ability to rule out most of the non-melioidosis patients.

Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei (BP) causes melioidosis, a potentially fatal human infection in the tropics. Clinical isolates from different geographical locations have similar morphological and biochemical characteristics. Although BP has been reported to possess 2 types of lipopolysaccharide (LPS) differing in the chemical structure of their O-polysaccharide (O-PS) component, earlier report demonstrated that the clinical strains exhibited identical LPS moieties. Recently, we reported antigenic similarity between the pathogenic (Ara-) and nonpathogenic (Ara+) biotypes. However, a few clinical isolates showed atypical SDS-PAGE profiles. In this study, LPS from 739 BP isolated from patients and animals in different geographical areas were extracted by proteinase K digestion method. Their SDS-PAGE profiles and their immunoreactivities with patients' sera and monoclonal antibody (MAb) to LPS were analyzed. The isolates showed 3 LPS patterns differing in the number and electrical mobility of bands in silver-stained gel. A majority of BP (711) isolates exhibited identical typical ladder pattern, 21 isolates showed atypical ladder pattern and 7 isolates did not exhibit ladder appearance. However, all LPS preparations exhibited similar endotoxic activity as determined by Limulus amebocyte lysate assay. On the other hand, there were no immunological cross reactivity between typical and atypical LPS, as judged from Western blot against homologous and heterologous sera from melioidosis patients from whom the typical and atypical LPS were isolated. Nevertheless, a Western blot profile of the typical LPS showed some variations when probed with MAb against BP LPS (9D5). Heat-killed bacteria from all LPS groups could similarly activate mouse macrophage cell line to produce nitric oxide (NO) and inducible NO synthase (iNOS).

TNF-alpha induces caspase 3 (CPP 32) dependent apoptosis in human cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countires especially in some areas of Southeast Asian countries such as Thailand. This tumor is relatively resistant to chemotherapy. In this study, molecular mechanism of killing of this tumor by TNF-alpha was investigated. Human cholangiocarcinoma cell line (HuCCA-1) was developed and used as a model for treatment. Activation of HuCCA-1 with TNF-alpha in the present of actinomycin D (1 microg/ml) caused death of the tumor cells. Western blotting analysis of the cells extracted demonstrated the cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 hours following TNF-alpha treatment indicating apoptotic death. The cleavage of PARP was inhibited when the cell line was pretreated with peptide inhibitor, Ac-DEVD-CHO, suggesting that apoptosis induced by TNF-alpha of this cell line involves activation of caspase II subfamily. The procaspase 3 (proCPP-32), one of the caspase group II subfamily was degraded after the HuCCA- I cell line was treated with TNF-alpha. Furthermore, Gelsolin, an 83 kDa protein which is identified as caspase 3 substrate, was cleaved to 43 kDa fragments after the cells were treated with TNF-alpha. These results indicate that apoptosis of human cholangiocarcinoma cell line as induced by TNF-alpha treatment is mediated through caspase 3.

Ultrastructural characteristics of liver fluke associated human cholangiocarcinoma cell lines.

The ultrastructure of a cholangiocarcinoma cell line (HuCCA-l) originally established from an intrahepatic bile duct tumor of a patient seropositive for a liver fluke infection was studied by scanning (SEM) and transmission (TEM) electron miscroscopy. With the SEM, the surface of HuCCA-1 cells were found to be covered with microvilli. The size of these microvilli varied from cell to cell and they were irregularly distributed. The TEM clearly revealed the presence of cytokeratin filaments, an intracytoplasmic lumen, tight junctions at the apices and desmosomes at the lateral surfaces of neighboring cells, all of which are characteristics of adenocarcinoma cell origin. However, the tumor mass that developed in a nude mouse following subcutaneous injection of these cells was found to exhibit some morphological changes. Specifically, about 20-30% of the tumor cells, particularly those lining the base of the tumor tubules, exhibited electron dense tonofilaments typical of squamous cells. However, this alteration was reversible as the cell line (HuCCA-1Nu) derived from this nude mouse-passage did not exhibit any characteristics reminiscent of squamous cells. These observations are consistent with those occasionally found in human cases reported previously by other investigators. Altogether, the data showed that squamous transformation of adenocarcinoma cells can occur under appropriate conditions. It further showed that reversion to adenocarcinoma cells can occur when the microenvironment is changed.

Monoclonal antibodies against protein antigens of salmonellae causing paratyphoid fever and their diagnostic application.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.

Production of monoclonal antibody specific to Campylobacter jejuni and its potential in diagnosis of Campylobacter enteritis.

A monoclonal antibody (MAb 3G6) specific for Campylobacter jejuni and Campylobacter coli was produced by immunizing BALB/c mice with a local strain of C.jejuni (28.1). No cross-reactivity was observed with Enterobacteriaceae controls. By immunoprecipitation, MAb 3G6 identified a major protein band of molecular weight 45 kDa and also gave a slight reactivity with 30 and 55 kDa proteins. Using an indirect enzyme-linked immunosorbent assay, MAb 3G6 was able to detect C.jejuni suspended in stool without cross-reactivity to 14 other enteropathogenic bacteria suspended, normal flora in fecal suspension, or fecal debris. In the analysis of fifty clinical specimens, MAb 3G6 detected most positive samples with the exception of one which possessed very low Campylobacter concentration and gave no reactivity to negative samples, demonstrating its high specificity to C. jejuni and C. coli. MAb 3G6 may be suitable as a new tool for the development of diagnostic method for Campylobacter infection.

Establishment and characterization of cell lines from liver fluke-associated cholangiocarcinoma induced in a hamster model.

Cholangiocarcinoma (CCA) is a relatively rare tumor that occurs primarily in tropical countries and particularly in those with a high incidence of liver fluke infection. A hamster model for a liver fluke-associated CCA has been described previously. In the present study, hamster cholangiocarcinoma cell lines were established and characterized in order to obtain information regarding diagnostically useful tumor marker which could shed light for a future investigation for human cholangiocarcinoma. Two related cell lines, one from the original intrahepatic bile duct tumor and one from an allotransplanted tumor, were established. The established cell lines were found to have population doubling times of 31 and 26 hours respectively, and were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum for over 80 passages. The cell monolayers were subjected to scanning and transmission electron microscopic study and found to have ultrastructural characteristics, including cytoplasmic lumens, consistent with those of adenocarcinoma cells of epithelial origin. An immunoperoxidase study using monoclonal antibodies (MAbs) specific for tumor antigens showed the cytoplasm and membrane of both cell lines to be positive. These antigens were also secreted in soluble form into the culture medium, judging from polyacrylamide gel electrophoresis in the presence of SDS and from immunoblot analyses. Different lines of evidence presented suggested that a 200 kDa glycoprotein produced and secreted by the tumor cell lines could be considered a cholangiocarcinoma-associated marker which has diagnostic potential.

Identification of tumor markers for cholangiocarcinoma and evaluation of their diagnostic potential.

Results obtained from studies using experimental animal model clearly showed that (1) A marker(s) for CCA does exist; 2) This marker is a glycoprotein with a molecular weight of 200 kDa; (3) It is produced and secreted in vitro by tumor cell lines; (4) It is highly immunogenic in mice and the MAb specific for this antigen is directed against the carbohydrate moiety; (5) This tumor antigen can be detected in serum and bile of tumor-bearing animals by a sandwich ELISA employing this MAb; (6) Kinetic studies show a gradual elevation of this antigen during tumor development; and (7) The elevation of this antigen can be detected at a time when no pathological changes have yet taken place, as judged by microscopic examination. Preliminary work from the human counterpart using human cholangiocarcinoma cell line showed promising results. CCA-specific antigen could be similarly identified and the MAbs produced were highly specific for this 160 kDa antigen.

Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer.

A new human cholangiocarcinoma cell line (HuCCA-1) was established from cholangiocarcinoma (CCA) tissue fragments surgically removed from a Thai patient with intrahepatic bile duct cancer. The growth medium used for the primary cell culture was Ham's F12 supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml epithelial growth factor (EGF). Approximately one month later, the cells were subcultured in Ham's F12 supplemented with only 10% FBS. The population doubling time was approximately 55 hr. Staining of the cells for cytokeratin and mucin confirmed that the cells were mucin-secreting tumor of epithelial cell origin. The supernatant fluid secreted a number of non-specific tumor markers including CA125 and traces of MCA and AFP. The ability of the HuCCA-1 cell line to synthesize specific marker that may have potential in the diagnosis of cholangiocarcinoma is now being investigated.

Immunodiagnosis of opisthorchiasis.

Monoclonal antibody-based enzyme-linked immunosorbent assay and DNA dot blot hybridization techniques were developed and evaluated for their potential in the detection of Opisthorchis viverrini. A mixture of IgG monoclonal antibodies 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 soluble parasite antigen in the feces of patients with opisthorchiasis. As little as 0.1 ng of the antigen could be detected. A specific O. viverrini DNA probe was used in a dot blot hybridization of parasite DNA. The labeled probe could detect DNA released from as few as five O. viverrini eggs. Both approaches were highly specific for O. viverrini and their sensitivity appeared to be comparable with that of the classical parasitological method. Preliminary data obtained from a field trial showed that these two methods have potential in the diagnosis of opisthorchiasis. Moreover, the limited data currently available showed that it is possible to use these methods to detect the presence of O. viverrini metacercariae in naturally infected fish.

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.

Effect of praziquantel treatment on antibody levels and lymphoproliferative responses in patients with opisthorchiasis.

The purpose of this study was to explore alternative method(s) to monitor the efficacy of anthelmintic treatment of patients with opisthorchiasis. Therefore, in our initial attempt, we studied the changes in antibody levels and lymphoproliferative responses in O. viverrini infected patients before and 2 months after successful praziquantel treatment. The results showed that although a substantial reduction of the antibody levels occurred after such a treatment, it did not occur in all patients. In those showing reduction, the final level were still above 2 standard deviations of the normal mean value. The reduction was more profound for IgG antibody. With regard to the IgA antibody isotype, the reduction was not as marked. In contrast, IgE antibody levels in most patients not only failed to decline, but instead, showed a tendency to be elevated after praziquantel treatment. Unlike the antibody levels, there was no alteration in the lymphoproliferative response to PHA stimulation and therefore this parameter is not useful for our intended objective. It was suggested that studies of a more specific O. viverrini component may be more reliable than the current method of parasitological examination of eggs in the feces of suspected individuals.

Gel-filtration chromatography of Salmonella protein antigens and their implication in immunodiagnosis.

Crude Barber protein (Bp) antigens were prepared from Salmonella typhi, S. krefeld and S. derby by an original method that has been described previously. These antigens were subjected to gel-filtration chromatography using Sephadex G-200. A sharp peak that eluted together with the void volume was thus separated from a broad second peak that eluted from the column at positions equivalent to 118,000 to 12,000 daltons. The proteins eluted in the latter peak were arbitrarily divided into 5 fractions and, together with the first peak, subjected to polyacrylamide gel electrophoresis and immunoprecipitation with both homologous and heterologous rabbit antisera. The extent of immunological cross reactivities was determined by enzyme-linked immunosorbent assay. The preliminary results obtained by this technique showed species-specific protein antigens to have molecular weights ranging between 36,000 and 68,000 daltons.

Analysis of Opisthorchis viverrini antigens: physicochemical characterization and antigen localization.

Antigens of Opisthorchis viverrini were identified and characterized by enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis following radioimmuno-precipitation, and indirect immunofluorescence. The immunoreactive protein with a relative molecular weight (Mr) of 89 kD was the predominating component of the parasite metabolic products. An immunofluorescence study showed it to be associated with parasite eggs, linings of the reproductive system and secretions therein. Protein of the surface tegument had Mr of greater than 116, 108, 64, 38, 34, 20 and 16-17 kD. The 16-17 kD surface molecule was the predominant protein, representing approximately 50% of the total protein in crude aqueous extracts of adult worms. However, it was poorly immunogenic when compared with the 89 kD molecule. Together with data presented previously, it appears that in addition to the 89 kD protein, several tegumental molecules are also specific for O. viverrini and have immuno-diagnostic potential.

Immunization of hamsters against Opisthorchis viverrini infection.

Attempts were made to induce acquired immunity against Opisthorchis viverrini infection in hamsters by immunizing them with aqueous somatic extract and metabolic products of adult worms, crude adult worm homogenates and metacercarial somatic extracts via either the intraperitoneal or combined intraperitoneal and oral routes. These procedures failed to stimulate significant protective response in animals that had never been exposed to O. viverrini. However, the protective response reached a significant level (30% worm reduction) in hamsters that had been infected with a small member of flukes prior to immunization with aqueous somatic extract of adult worms. Although these findings indicate that it may be possible to reduce reinfection in people in the endemic area by immunization, it appears that a better method currently available for the control of O. viverrini infection is health education aimed at changing food habits and improving sanitation and personal hygiene.