Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C.?concisus DNA and higher levels of antibodies specific to C.?concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C.?concisus immunoreactive antigens. Proteins from C.?concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C.?concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C.?concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C.?concisus proteins that are immunoreactive within patients with Crohn's disease.     

Persisting antibody reaction in paragonimiasis after praziquantel treatment is elicited mainly by egg antigens

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.     

Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis

Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM). In the present study, released antigen preparations (exoAg) obtained from P. brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients. Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly. A total of 28 different components were detected, 11 of them shared by all isolates. The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40). Six bands reacted with more than 20% of sera. Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently. Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms. The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment. Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa. The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P. brasiliensis infection.     

Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.     

Complement components and terminal complement complex in oesophageal smooth muscle of patients with achalasia.

This study investigates whether patients with achalasia exhibit autoimmune reactions with subsequent complement activation within oesophageal smooth muscle, vessels and neurones. Oesophageal muscular biopsies from 8 patients undergoing surgery for achalasia and from 6 patients operated for oesophageal cancer were investigated by immunofluorescence for the presence of the complement components C1q, C4, C3c, C3d, C9 and the C9 neoantigen of the terminal C5b-C9 complement complex. Tissues were also investigated for the expression of immunoglobulins (G,A,M) and of the antigens of rubella and varicella zoster viruses. In addition, sera of both patient groups were tested for the presence of autoantibodies against Auerbach's plexus. The terminal complement complex C5b-C9 was found within muscle cells from all patients with achalasia but in only one specimen from a patient with cancer. Two patients with achalasia also exhibited the terminal complement complex as well as IgM within ganglion cells. Muscle cells stained positive for the complement component C9 in all five patients with achalasia in whom this test was performed but in none of the control tissues. In addition, sera from four patients with achalasia contained antibodies against Auerbach's plexus. Studies for the complement components C1q, C4, C3c and for antigens of rubella and varicella zoster viruses revealed negative results in all patients and controls. The results of this study suggest that a complement activation is involved in the autoimmune pathogenesis of achalasia. However, the triggering mechanism of this phenomenon remains to be determined.     

Metabolic labeling of Entamoeba histolytica antigens: characterization of a 28-kDa major intracellular antigen

The in vivo incorporation of radiolabeled amino acids into antigens of Entamoeba histolytica, HM-1:IMSS, is reported. Immunoprecipitation with sera from patients with invasive amebiasis revealed a 28-kDa antigen present in whole cell lysates of E. histolytica. This antigen was of cytoplasmic origin, as indicated by cell fractionation and Triton X-114 detergent-phase separation. Immunoprecipitation, using sera from patients with invasive amebiasis and symptomless cyst passers, revealed the 28-kDa antigen as the major antigen recognized by the sera tested. Immunoprecipitation analysis using radiolabeled-released proteins instead of whole cell lysates showed a number of bands, including the 28-kDa antigen. The data suggest that the 28-kDa antigen is of cytoplasmic origin or is released from the cytoplasmic compartment.     

Complex of GM1- and GD1a-Like Lipo-Oligosaccharide Mimics GM1b,Inducing Anti-GM1b Antibodies

Objective

Molecular mimicry between Campylobacter jejuni lipo-oligosaccharides (LOSs) and human gangliosides GM1 and GD1a induces the production of anti-GM1 and anti-GD1a antibodies, and the development of Guillain-Barré syndrome. Complexes of two different gangliosides form new molecular shapes capable of enhancing recognition by anti-ganglioside antibodies. To test the hypothesis that the complex of GM1-like and GD1a-like LOSs of C. jejuni induces the development of anti-GM1b antibodies in Guillain-Barré syndrome patients.

Methods

Mass spectrometry analysis determined the LOS outer core structures, with which mice were immunized. IgG antibodies to single gangliosides and complex of gangliosides were tested in sera from Guillain-Barré syndrome patients from whom C. jejuni LOS had been isolated.

Results

Two isolates from GBS patients who had anti-GM1b antibodies, but neither anti-GM1 nor -GD1a antibodies, expressed both GM1-like and GD1a-like LOSs, but not GM1b-like LOS. Anti-GM1b antibodies were induced in one of the mice immunized with the C. jejuni bearing GM1-like and GD1a-like LOS. Sera from 20 patients had antibodies to the complex of GM1 and GD1a, all of which carried anti-GM1b reactivity. Five of these sera harbored neither anti-GM1 nor anti-GD1a antibodies. IgG antibodies to the complex were absorbed by GM1b, but by neither GM1 nor GD1a.

Conclusions

GM1-like and GD1a-like LOSs form a GM1b epitope, inducing the development of anti-GM1b antibodies in patients with Guillain-Barré syndrome subsequent to C. jejuni enteritis. Here, we present a new paradigm that the complex of two different structures forms a new molecular mimicry, inducing the production of autoantibodies.     

Sero-epidemiology of Neisseria meningitidis infections in parts of Eastern Nigeria.

The prevalence of antibodies to meningococci in the Nsukka area, East Central Nigeria, was studied by the indirect hemagglutination test employing tanned chicken erythrocytes. Of the 1332 apparently healthy persons, whose sera were tested, 982 samples (73.8%) were positive at 1:8 dilution of the sera. Fifty-six per cent of the population had antibodies to Group B, followed by 13% of Group C and 5% to Group A. Twenty-five sera from patients with meningococcal infections, from whom Group B meningococci were isolated, 100% were positive. Percentage reactivity in the 2-5-year age group was low (15%), whereas it increased rapidly from 78% (6-10-year group) to 95% (31 years and above).     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement-fixation in diagnosis

The combined use of complement fixation (CF) and latex agglutination (LA) tests is reported on sera from 6328 patients with suspected hydatid disease; 191 were confirmed positive at operation ('known positives'). Results by LA are related to CF titres. Both tests were negative in 90% of specimens. Nine patients were subsequently found infected of whom 3 became positive in tests after operation. Of sera positive in both tests, 75% were from 'known positives'. The remainder were almost certainly from infected patients. Half the patients whose sera were LA positive/CF less than or equal to 1/4 were follow-up 'known positives' in whom CF titres had waned; 2 were early infections. Only 3% of the cases with an LA negative/CF titre of greater than or equal to 1/16 were 'known positives' and 6% where the CF titre was 1/8. The remaining CF results in the group were false positives and accounted for 1.2% of all sera tested. Findings show that a CF titre greater than or equal to 1/8 with positive LA indicates past or present infection; a negative CF test with positive LA usually indicates past infection; rarely, infection is present when a CF titre is greater than or equal to 1/8 and LA is negative. A rising CF titre and positive LA indicates current infection; reliable prognosis following treatment is given by CF.     

Fluorescent-Antibody Studies with Antisera Against Heated and Unheated Poliovirus Type 1

Fluorescent-antibody (FA) reagents were prepared from sera of guinea pigs immunized with either native infectious poliovirus type 1 or poliovirus type 1 which had been heated at 56 C for 30 min. Conjugates made from sera of animals immunized with heated virus gave higher direct FA staining titers on air-dried, acetone-fixed, infected cells than conjugates made from sera of animals immunized with native infectious virus. Evidence was obtained that complement-fixing antibody reactive with heated antigen was responsible for the FA staining. Two conjugates prepared from sera of guinea pigs immunized with heated poliovirus type 1 were successfully used to identify 21 type 1 viruses isolated from a group of 44 stool suspensions studied as unknowns. These conjugates did not stain any of 23 heterologous enteroviruses present in the remainder of the stools and gave minimal non-specific staining.     

Rapid decay of Salmonella flagella antibodies during human gastroenteritis: a follow up study

An indirect enzyme-linked immunosorbent assay (ELISA) based on Salmonella re-polymerized flagella was employed to measure levels of immunoglobulin (Ig) G, IgM and IgA antibodies in sera from 303 Danish patients diagnosed with either Salmonella enteritidis or Salmonella typhimurium. The antibody-levels were assessed at one, three and six months after onset of salmonellosis, and sera from a control-group of 170 healthy blood donors were additionally analysed in order to establish cut-off values for the analysis. Cross-reactions to other Salmonella serotypes, as well as to Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli and Helicobacter pylori were observed. At one month after onset of symptoms, 70% of the patients recovering from a S. enteritidis infection carried detectable levels of anti-flagella antibodies, as did 77% of the patients recovering from S. typhimurium infection. Three months after onset of symptoms these detection rates had decreased to 46% and 40%; and six months after onset of symptoms the detection rates were 34% and 38%. This rapid decrease in the serum levels of flagella antibodies is in conflict with the "common knowledge" statement of a long-lasting anti-flagella immunoresponse. The present study suggests that such a tenacious statement is (or may be) inaccurate.     

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Specificity of antigens from pathogenic Aspergillus species. II. Studies with line immunoelectrophoresis

Somatic (mycelial) and metabolic (culture filtrate) antigens of Aspergillus flavus, A. fumigatus, A. nidulans, A. niger and A. terreus were compared by line immunoelectrophoresis with sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma, or from immunized animals. Number of lines observed when tested with human sera were similar for somatic and metabolic preparations of A. fumigatus, but up to 33 lines were present when both types of antigens were tested simultaneously. Cross-reactions between heterologous antigens and sera from patients with aspergilloma or ABPA were uncommon. In contrast, cross-reactions were common when standard antisera prepared in animals against heterologous species of Aspergillus were tested against A. fumigatus antigens. Lines of identity between homologous antigens and those from A. fumigatus were observed in 5 of 9 lines obtained with A. flavus, 4 of 16 lines of A. nidulans, 4 of 9 lines of A. niger and 4 of 8 lines of A. terreus.     

Variability of the lytic susceptibility of Neisseria gonorrhoeae to human sera.

The bactericidal activity of human sera for Neisseria gonorhoeae was studied. Sera were obtained from a group of patients with gonococcal infections who had acute urethritis, acute pelvic inflammatory disease, disseminated gonococcal infection, or who were asymptomatic carriers. The homologous and heterologous strains were tested with these sera. The development of serum bactericidal antibodies was not a universal event. With few exceptions, the susceptibility of a particular strain to human antibody and complement appeared to be largely independent of the particular person from whom the serum was obtained and was due instead to antigenic properties intrinsic to each individual strain. Lipopolysaccharide appeared to be the predominant antigen against which bactericidal antibodies were directed. The principal bactericidal antibody class was IgM. Blocking antibodies were not found to account for the lack of lytic activity. A correlation of bactericidal antibodies with protection from developing gonococcal infection could not be demonstrated in three pateints.     

Prevalence of GB virus C/hepatitis G virus in Hungary

In 1995 a new flavivirus, GB virus C/hepatitis G virus (GBV-C/HGV), was discovered. The aim of this study was to determine the prevalence of the virus in healthy persons and hepatitis patients in Hungary. The sera of 408 healthy persons older than 60 years were tested for the presence of GBV-C/HGV antibodies, and 113 were positive (28%). Eight of the 71 healthy persons younger than 60 years and twenty of the 51 sera (39%) taken from patients suffering from hepatitis of unknown origin proved to be positive for GBV-C/HGV antibodies. Ten of the 124 sera (8%) of healthy persons and 36 of the 247 sera (14.6%) of hepatitis patients proved to be positive for GBV-C/HGV RNA. Eleven PCR products were sequenced, and the sequences were found to be different from each other and from the previously published ones. However, three sequences taken from the same patient at different times were identical. These results show that GBV-C/HGV is present in Hungary and cannot be considered rare.     

Fucosylated glycoproteomic approach to identify a complement component 9 associated with squamous cell lung cancer (SQLC)

Human lung cancer is a major cause of cancer mortality worldwide. Understanding the pathophysiological features and the development of novel biomarkers for diagnosis as well as treatment are major tasks. In the present study, sera from ten SQLC patients and healthy control (HEC) were collected and pooled, respectively. The pooled sera were depleted via an immunoaffinity method and further subjected to fucosylation enrichment. Enriched fucosylated glycoproteins were resolved by SDS-PAGE and subsequently analyzed by LC-ESI-MS/MS. From comparative proteomic analysis, we selected the C9 protein. C9 protein levels were validated by Western blot, protein arrays and the fucosylation levels of C9 by hybrid lectin ELISA (HLE) in the sera of 120 HEC and 118 SQLC patients. The C9 protein level was 6.4-fold higher in SQLC patients compared to HEC, as determined by Western blot analysis. The results were concurrently confirmed by a protein array that showed a C9 level significantly higher in SQLC patients, as compared to HEC, with a sensitivity of 53% and a specificity of 89%. C9 fucosylation levels were significantly higher in SQLC patients compared to HEC (p<0.05) when tested by HLE. These findings suggest that C9 and fucosylated form could serve as a useful marker for SQLC.     

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.     

Aeromonas sobria in bubaline (water buffalo) abortion: growth requirement, biochemical characters, antibiotic susceptibility, experimental pathogenicity and serology

A. sobria was isolated unexpectedly on Campylobacter selective medium from abortion specimens of 16 buffaloes. All strains (AS1-AS16) required Campylobacter growth supplement (sodium pyruvate, sodium metabisulphate and ferrous sulphate) and 10% C02 atmosphere on primary isolation. They were unusually sensitive to bile salt and failed to grow on MacConkey's agar. Biochemically, these strains were homogeneous but antibiogram profile and physiological/other characters revealed a moderate heterogeneity. In vitro antibiotic sensitivity pattern showed a number of antibiotics effective whereas only a few (penicillin and co-trimoxazole) ineffective against these strains. It took 9 days for 3 x 10(8) A. sobria organisms/ml in 5 ml quantity to terminate the pregnancy in an experimentally infected buffalo via iv route. Foetal and placental lesions were classical as that of field cases. The infection evoked serum antibody response. Apparently disease-free buffalo sera were devoid of such antibodies.