Biochemical characterization and localization of Fasciola hepatica 26-28 kDa diagnostic coproantigen

We have previously reported the usefulness of a 26-28 kDa coproantigen of Fasciola hepatica for diagnosis of infection. In this study, the 26-28 kDa coproantigen was biochemically characterized with the aid of monoclonal antibodies (MoAb) in an effort to better understand the biology of the antigen. Differential staining of chromatographically-purified 26-28 kDa coproantigen on SDS-PAGE, under reducing and non-reducing conditions, indicated that the coproantigen was a monomeric, highly glycosylated glycoprotein. Alkaline treatment of the purified coproantigen resulted in an 8 kDa protein core which still contained the epitope recognized by the MoAb. No protease activity was associated with the 26-28 kDa coproantigen. The coproantigen could be cleaved by trypsin without altering the reactive epitope recognized by the MoAb, but was resistant to pepsin digestion. Further, the coproantigen was stable under several different storage conditions. Indirect immunofluorescence on tissue sections of adult flukes indicated that the coproantigen was present in gut cells and tegument. Taken together these results confirm the stability of the 26-28 kDa coproantigen and its usefulness in diagnostic tests for F. hepatica infections.     

Fulminant Amebic Colitis in a Homosexual Man

This report describes a case of fulminant amebic colitis leading to perforation and death in a 35-yr-old homosexual man. Although Entamoeba histolytica may be isolated from stool specimens in 20 to 30% of selected homosexual populations, reports of severe or invasive disease are rare. Some workers have suggested that amebae are only passive colonizers of the colon in homosexual men. In our patient, pathological examination confirmed the presence of extensive colitis with penetration of amebae through the bowel wall. Pertinent data concerning pathogenicity of E. histolytica in homosexuals are reviewed and the public health implications of a virulent strain of amebae are discussed.     

Passive protection against fasciolosis in mice by immunization with a monoclonal antibody (ES-78 MoAb)

Recently, we reported a partial characterization of the epitope recognized by the ES-78 monoclonal antibody (MoAb). This monoclonal antibody was obtained from spleen lymphocytes of a mouse immunized with excretory-secretory antigens of Fasciola hepatica adult worms. In the present study, we report the results obtained in experiments of passive protection using this MoAb in BALB/c mice infected with 15 Fasciola hepatica metacercariae. The monoclonal antibody was able to reduce the parasite burden when administered 24 h before challenge but not when delivered 7 days after challenge. The antibody recognition of digestive tract structures in 3-week-old parasites was demonstrated by immune histochemical techniques. The antigens purified by affinity chromatography using this antibody had molecular weights of 14-20, 25-29 and 36-45 kDa and demonstrated proteinase activity similar to cathepsin L. These results suggest that the antigens carrying the epitope recognized by the ES-78 MoAb may be used as target in the protection against fasciolosis.     

The role of gamma interferon in the generation of human macrophages cytotoxic for Entamoeba histolytica trophozoites

Upon exposure to Entamoeba histolytica antigen, lymphocytes from patients treated for amebic liver abscess produce lymphokines which activate monocyte-derived macrophages to kill E. histolytica trophozoites. We now demonstrate that gamma interferon (IFN-gamma) is produced by these stimulated lymphocytes and is sufficient but not exclusively necessary to activate monocyte-derived macrophage amebicidal activity. Supernatants from mononuclear cells of 7 patients when stimulated with amebic antigen contained more IFN-gamma than comparable supernatants derived from control cells (1,862 U/ml vs. 174 U/ml geometric means, P less than 0.01); IFN-gamma levels were similar in patient and control supernatants following concanavalin A stimulation. Macrophages activated solely by partially purified IFN-gamma or recombinant human IFN-gamma (300 U/ml) killed 47% of virulent amebae by 6 hr at 37 degrees C. Monocyte-derived macrophages stimulated with lymphokines elicited by amebic antigen or concanavalin A killed 48% and 57% of axenic E. histolytica trophozoites, respectively, over 6 hr at 37 degrees C (P less than 0.001 for each compared to control). Macrophages incubated with the identical lymphokines, but in the presence of monoclonal antibody to IFN-gamma, were only able to kill 18% and 27% of amebae, respectively, at 6 hr (P less than 0.05 to control or when antibody to IFN-gamma was not present). If antibody to IFN-gamma was added to the stimulating lymphokine, more macrophages died during interaction with amebae (P less than 0.05). In summary, IFN-gamma has a major but not exclusive role in activating human monocyte-derived macrophages in vitro to kill virulent E. histolytica trophozoites.     

When should we look for amebae in patients with inflammatory bowel disease?

Amebic colitis can mimick Crohn's disease of the colon and ulcerative colitis. Inflammatory bowel disease (IBD) patients can also be carriers of amebae. Since steroids can provoke amebic activity and even cause a fulminating colitis, it is necessary to determine that amebae do or do not exist. Furthermore, amebae can be easily eliminated by drug therapy but it is hard to eradicate IBD. Despite the above, diagnostic modalities for IBD are as effective as those for amebiasis, particularly for Crohn's disease with ileal involvement. Problems in differential diagnosis arise with IBD grossly limited to the colon. In these cases, false negative stool studies are increased by diarrhea and preparation for examinations, and both stool studies and serologies are compromised by steroids. If the clinical course of IBD is downhill, the clinician is justified in starting steroids even if evaluation for amebiasis incomplete. If, however, the index of suspicion is high, concomitant treatment with Metronidazole would be reasonable.     

CASE REPORT: Colonic Perforation in Unsuspected Amebic Colitis

Unsuspected amebic colitis presenting as inflammatory bowel disease, as in our patient, has been previously reported (4, 7, 8). Misdiagnosis, delay in antibiotic treatment, and institution of immunosuppression were the result of failure to identify the parasite in stool specimens and have resulted in suffering, morbidity, mortality, and surgery. In all previously reported cases, routine stool studies failed to identify E. histolytica (4, 7, 8). The correct diagnosis was only established after reviewing the surgical specimen or biopsies obtained endoscopically. Because the erroneous diagnosis of inflammatory bowel disease can lead to disastrous complications, it is imperative to exclude amebic colitis prior to undertaking steroid therapy, especially in patients with a prior history of travel to or residence in areas with endemic E. histolytica (17). We recommend obtaining at least three stool specimens for microscopic examination, as well as testing for serum amebic antibody. Patients should submit fresh stool specimens directly to the laboratory to allow for prompt diagnostic evaluation. Such an approach might lead to the improved diagnosis of amebiasis.     

Two human monoclonal antiplatelet autoantibodies established from patients with chronic idiopathic thrombocytopenic purpura

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.     

Anticardiolipin antibodies possess anticoagulant activity which is blocked by a monoclonal antibody against a cross reacting idiotype of antiphospholipid antibodies

Anticardiolipin (aCL) antibodies were isolated from systemic lupus erythematosus serum using cardiolipin liposomes. In a sensitized activated partial thromboplastin time (aPTT) system, low concentrations of aCL were found to significantly prolong aPTT in a dose-dependent fashion. This effect was abolished by adding phosphatidylserine/phosphatidylcholine liposomes to the aPTT system, and could be significantly reduced by a monoclonal antibody against a cross-reacting idiotype of antiphospholipid antibodies (MoAb 5.2) previously incubated with aCL. These results suggest that the anticoagulant properties of aCL are mediated by an interaction with phospholipid components of the coagulation system. The functional block of this activity using monoclonal MoAb 5.2 antibody could be useful for investigating the mechanisms by which aCL is associated with clinical thrombosis.     

Idiotypic characteristics of immunoglobulins associated with systemic lupus erythematosus. Studies of antibodies deposited in glomeruli of humans

Indirect immunofluorescence with monoclonal antibodies to 6 different idiotypes was used to characterize immunoglobulins deposited in the glomeruli of renal biopsy samples from 32 patients with systemic lupus erythematosus (SLE) and 19 patients with nonlupus immune glomerulonephritis. IdGN2 was present in 75% of the biopsy specimens from SLE patients and in 6% of those from patients with non-lupus nephritis; IdGN1 occurred in 38% and 6%, respectively. The other idiotypes were not increased in biopsy samples from patients with SLE. Deposition of IdGN2 was associated with a subendothelial location of Ig and proliferative changes in the glomeruli. In studies of glomerular eluates from 4 immunosuppressed SLE patients, an average of 26% of total Ig and 37% of anti-DNA was composed of IdGN2. Compared with IdGN2- immunoglobulin, IdGN2+ immunoglobulin was enriched in IgG1 in all 4 eluates, and was enriched in high-avidity anti-DNA in 2 eluates. IdGN2 is a marker of antibody subsets that are characteristic of SLE and are associated with severe lupus nephritis.     

Amebic appendicitis with fulminating peritonitis: Report of a case

Summary A case of perforated amebic appendicitis in a 36-year-old man is reported. Microscopic examination of the appendix showed the presence of amebae in all layers of the appendix, in addition to the inflammatory process.     

Isolation of tryptic fragment of antigen from mitochondrial inner membrane proteins reacting with antimitochondrial antibody in sera of patients with primary biliary cirrhosis

Most of the sera from patients with primary biliary cirrhosis contains antimitochondrial antibodies, which react with four proteins of the mitochondrial inner membrane. We reported in a previous paper that when beef heart mitochondrial inner membrane proteins were digested by trypsin, a new reactive 36 kDa fragment with antimitochondrial antibody was obtained. This 36 kDa fragment derives from original 70 kDa protein because the monoclonal antibody specific to 70 kDa protein reacts with the 36 kDa band equivalent to 70 kDa band. The 36 kDa fragment was purified using an affinity column conjugated with an immunoglobulin-rich fraction of primary biliary cirrhosis serum containing antimitochondrial antibody, preparative electrophoresis and high-performance liquid chromatography using a reverse phase column. The final preparation showed a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Its amino acid composition is in good agreement with that of the subunit binding domain of the pyruvate dehydrogenase complex E2 from bovine heart.     

A novel IgM class autoantibody to a hepatocyte-related 190 kDa molecule in patients with type 1 autoimmune hepatitis

It has been reported that autoantibodies to hepatocytes are frequently found in patients with autoimmune hepatitis (AIH). To elucidate the nature of these hepatocyte-specific autoantibodies, we attempted to generate a hepatocyte-specific monoclonal antibody (MoAb) from Epstein-Barr virus-transformed peripheral blood mononuclear cells obtained from a patient with AIH. We established a single clone, 2E3, that continued to produce an immunoglobulin M (IgM) antibody (lambda-type). This MoAb had the following properties: it reacted mainly with hepatocyte-derived cell lines, rather than with other cell lines, and it reacted with liver tissue but not with other tissues. By immunoblot analysis, we found that this MoAb recognized a 190 kDa molecule on hepatocytes. The MoAb was able to kill hepatocyte-derived cell lines in the presence of fresh human serum. This cytotoxic effect was completely abrogated by heat inactivation of human serum prior to its addition to cell lines. In addition, an IgM autoantibody that recognized a 190 kDa molecule was also found in patients with AIH but not in those with chronic hepatitis C; its titer correlated significantly with serum alanine aminotransferase (ALT) levels in patients with AIH. In conclusion, we generated a human MoAb that recognizes a 190 kDa molecule on hepatocytes. Because of its ability to mediate complement-dependent cytotoxicity and the presence of similar IgM autoantibody in patients with AIH, we hypothesize this autoantibody may play a role in the immunopathogenesis of AIH.     

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.     

Elution of cytomegalovirus antibodies from adenocarcinoma of the colon.

Eluates were prepared by high salt extraction from normal colonic mucosa and adenocarcinomatous tissue from 28 patients, eight more from unmatched colonic tissue and five from patients with other gastrointestinal disease. Immunoglobulins were detected by ELISA: IgG was present in 24% eluates from normal colon and 21% from carcinomas; IgA in 55% eluates from normal colon and 39% from carcinomas; IgM in 55% from normal colon and 37% from carcinomas. Cytomegalovirus-specific antibody was found in 15% eluates from normal colon and in 18% carcinomas. Out of the 28 matched specimens, cytomegalovirus-specific IgG was detected in one normal and four tumour eluates, specific IgA in two normal and four tumour eluates, and specific IgM in two normal and two tumour eluates. In two instances cytomegalovirus-specific antibody was present in the eluates prepared from the normal and tumour tissue of the same patient. Of those eluates which contained cytomegalovirus-specific antibodies by ELISA, two were positive by anti-complement immunofluorescence of human embryo fibroblasts infected with cytomegalovirus strain AD-169. It seems possible, therefore, that cytomegalovirus antigens on colonic cells may be masked by complexing with anti-cytomegalovirus antibodies, and may not therefore be detected by techniques such as immunofluorescence.     

Occurrence of human intestinal parasites in selected populations of Cracow region in the years 2000-2006 on the basis of parasitological stool examinations performed in the Laboratory of Parasitology of the District Sanitary-Epidemiological Center

BACKGROUND: Infections with intestinal parasites are the most frequent parasitic diseases in all human populations. According to available epidemiological data enterobiosis, giardiosis and ascariosis are the most prevalent in Poland. The aim of this study was to evaluate the prevalence of human intestinal parasites in three selected populations in Cracow between 2000-2006. MATERIAL AND METHODS: As many as 5383 stool samples were tested with the use of coproscopic methods and ELISA for the presence of Giardia intestinalis coproantigen. RESULTS: In 283 stool samples different species of intestinal parasites were detected. The prevalence of human intestinal parasites was minimal in 2002, with the ratio of 3.30%, while the maximal prevalence was noted in 2005 (8.86%). The mean prevalence of intestinal parasites in 2000-2006 was 5.26%. In this period of time the most prevalent intestinal parasite among children and adults was Enterobius vermicularis (2.35% and 1.84% respectively), and in the population of citizens returning from the tropics were Entamoeba coli (6.98%). Besides, a gradual trend of decrease in the number of patients referred directly to our laboratory for parasitic examination of stool samples was noted over the last years.     

Immunology and molecular biology of Opisthorchis viverrini infection

Opisthorchiasis is the major public health problems in Laos PDR and Thailand. The disease becomes chronic and persists for many years, leading to hepatobiliary disease and cholangiocarcinoma. Less severe manifestations include cholangitis, chronic cholecystitis and cholelithiasis. A significant degree of humoral and cell mediated immune responses to the parasite can be detected both in patients and animal models. The patients IgG levels appear to correlate with gall bladder size and dysfunction and correlated significantly with opisthorchis egg count and decrease after treatment. However, the possible significance of these immune responses to protective immunity is presently unknown. The development of immunodiagnostic method for Opisthorchis viverrini detection has been attempted. The components with molecular weight >116, 89, 78 and 20 kDa appear to be specifically associated with the somatic extract of adult fluke. The 89 kDa protein is the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract that can be a candidate with significant immunodiagnostic potential. Highly specific and sensitive monoclonal antibodies for O. viverrini antigens were prepared to detect parasite antigens in stool and antibody in serum. Information regarding the molecular approaches of O. viverrini is very limited. The genome of O. viverrini has neither CpG nor A methylated as found in other parasites. The total length O. viverrini ribosomal DNA is approximately 13 kb. and the presence of a highly repeated DNA specific for the parasite was demonstrated. A O. viverrini specific DNA probe was constructed and PCR based detection with high specificity for amplification of the repeated sequences is performed to detect the presence of eggs' DNA in stool samples in comparison with classical methods.     

Identification and diagnostic value of a major antibody epitope on the 12 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid

An IgG1 monoclonal antibody (MoAb), designated C9E7H8, has been produced against an epitope on the 12 kDa antigen of Echinococcus granulosus cyst fluid, believed to represent the smallest subunit of antigen B. This MoAb, raised against purified 12 kDa antigen eluted from a reducing SDS-PAGE gel, demonstrated strong binding to native sheep cyst fluid in ELISA and recognition of all three subunits of antigen B (at 12, 16, 23 kDa) by immunoblot under both reducing and non-reducing conditions. Immunoblot analysis also indicated that the complementary epitope is conserved amongst cyst fluids from different intermediate hosts of E. granulosus, including fluids from cysts of two distinct strains, and is present in cyst fluid from E. multilocularis. The monoclonal displays binding to a cDNA clone, EgPS-3, which we have previously shown expresses part of the 12 kDa molecule. EgPS-3, expressed as a glutathione-S-transferase fusion protein, was successful in positive detection of 74% of cystic hydatid patients, although cross-reactions were observed with 25% of sera from alveolar hydatid and 22% of sera from schistosomiasis japonica patients. Three peptides, based on the predicted amino acid sequence of EgPS-3. showed increased specificity but slightly reduced sensitivity in the detection of antibody from E. granulosus patients. The predominant epitope recognized by human antibody occurs in the Nterminal 27 amino acids (peptide 65) of EgPS-3 which also correlates with the location of the monoclonal antibody epitope.     

Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats.

Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-specific cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E. histolytica pathogenesis and for the future development of vaccines.     

Antibodies to somatic L3 antigen not protective against Brugia malayi infection

Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.     

Comparative demonstration of IgE (immunoglobulin E) in the gastric mucosa using monoclonal and polyclonal antibodies

It was the aim of this study to examine by using biopsy material the suitability of polyclonal and monoclonal antibodies against human IgE to support the clinical diagnosis of gastrointestinal allergy by means of morphological methods. A total of 190 gastric biopsies were collected from 56 ambulant patients; 95 specimens were shock-frozen in liquid nitrogen and treated with monoclonal antibodies (D epsilon 1, D epsilon 2); 95 biopsies were fixed in Bouin's solution, embedded in paraffin and treated with a polyclonal antibody. The slide preparations were examined semiquantitatively. The monoclonal (D epsilon 2) and the polyclonal antibody yielded different staining patterns. The polyclonal antibody mainly stained plasmocytes as constituent parts of the inflammatory infiltrate, which was regarded as a secondary phenomenon following the disruption of the mucus barrier. The monoclonal antibody mainly labeled mast cells within the glandular body of the antral and corpus-type mucosa; this finding we regarded as the most important one in the context of allergic reactions. The employment of the two different methods yielded a complete distribution of IgE-containing cells in the gastric mucosa. The immunohistochemical demonstration of IgE is therefore a useful method to support the clinical diagnosis of allergic processes in the gastrointestinal mucosa.