Strongyloides ratti antigenic components recognized by IgE antibodies in immunoblotting as an additional tool for improving the immunodiagnosis in human strongyloidiasis

IgE antibody response in human strongyloidiasis was evaluated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) using Strongyloides ratti saline extract as heterologous antigen. A total of 50 serum samples of patients who were shedding S. stercoralis larvae in feces (group I, copropositive), 38 of patients with other intestinal parasites (group II), and 38 of subjects with negative results in three parasitologic assays (group III, copronegative) were analyzed. Levels of IgE anti-Strongyloides expressed in ELISA Index (EI) were significantly higher in patients of group I (1.32) than in group II (0.51) and group III (0.81), with positivity rates of 54%, 0%, and 10.5%, respectively. Fifteen S. ratti antigenic components were recognized in IB-IgE by sera of group I, with frequency ranging from 8% to 46%. In group II, only two antigenic bands (101, 81 kDa) were detected in a frequency of 10% and no reactivity was found in group III. Sera with EI values > 1.5 recognized five from 13 specific antigenic bands (70, 63, 61, 44, 7 kDa). It can be concluded that these five antigenic components recognized by IB-IgE using S. ratti antigen might be employed as an additional tool for improving the immunodiagnosis in human strongyloidiasis.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 ± 0.17). All nine sera from VL patients had such antibody (0.99 ± 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 ± 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 ± 0.14), and in all VL patients (0.65 ± 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 ± 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Evaluation of five screening tests licensed in Argentina for detection of hepatitis C virus antibodies

This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV). The tests analyzed were: Detect-HCV™ (3.0) Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK) and Serodia-HCV particles agglutination test (Japan) have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays.

Melan-A-specific cytotoxic T cells are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4.

PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Neutralizing and protective antibodies directed against vaccinia virus envelope antigens.

The infection mechanism of vaccinia virus is largely unknown. Neither the attachment protein of extracellular enveloped virus (EEV), the biologically relevant infectious form of the virus, nor its cellular receptor has been identified. Surprisingly, all former attempts using antibodies to block EEV infection of cells in vitro had failed. Here, we report the production of an anti-envelope hyperimmune serum with EEV neutralizing activity and show that a polyclonal antiserum against the extraviral domain of protein B5R also inhibited EEV infection. In vivo, mice vaccinated with B5R protein were protected against a lethal vaccinia virus challenge. This protectivity is likely to be mediated by neutralizing antibodies. Protein A33R, but not A34R and A36R, also proved to be protective in active and passive vaccination experiments. However, in contrast to B5R, A33R protectivity did not correlate with antibody titers. Because anti-A33R antibodies did not neutralize EEV in vitro, the protectivity mediated by A33R protein probably involves a mechanism different from simple antibody binding. Taken together, our results suggest that antibodies to a specific protective epitope or epitopes on protein B5R are able to prevent EEV infection. The protein encoded by the B5R gene is therefore likely to play a crucial role in the initial steps of vaccinia virus infection-binding to a host cell and entry into its cytoplasm.

Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAV IgM, 6 and 95% for anti-HBs, 13 and 100% for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV. The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

Antibodies directed against rubella virus induce demyelination in aggregating rat brain cell cultures.

To link the presence of intrathecal virus-specific oligoclonal immunoglobulin G (IgG) in multiple sclerosis patients to a demyelinating activity, aggregating rat brain cell cultures were treated with antibodies directed against two viruses, namely, rubella (RV) and hepatitis B (HB). Anti-RV antibodies in the presence of complement decreased myelin basic protein concentrations in a dose-dependent manner, whereas anti-HB antibodies had no effect. A similar but less pronounced effect was observed on the enzymatic activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase, which is enriched in noncompact membranes of oligodendrocytes. These effects were comparable to those in cultures treated with antibodies directed against myelin oligodendrocyte glycoprotein (MOG), previously found to be myelinotoxic both in vitro and in vivo. Sequence homologies were found between structural glycoprotein E(2) of RV and MOG, suggesting that demyelination was due to molecular mimicry. To support the hypothesis that demyelination was caused by anti-RV IgG that recognized an MOG epitope, we found that anti-RV antibodies depleted MOG in a dose-dependent manner. Further evidence came from the demonstration that anti-RV and anti-MOG IgG colocalized on oligodendrocyte processes and that both revealed by Western blot a 28 kDa protein in CNS myelin, a molecular weight corresponding to MOG. These findings suggest that a virus such as RV exhibiting molecular mimicry with MOG can trigger an autoimmune demyelination.

Anti-malaria humoral responses in children exposed to Plasmodium falciparum and Schistosoma haematobium

Antibody responses directed against the Plasmodium falciparum antigens, total extract, anti-merozoite surface protein-3 (MSP3b) and glutamate-rich protein (Glurp-R0) were studied in 42 children exposed to both Schistosoma haematobium and P. falciparum infections. The association between levels of the anti-malaria IgG subclasses and IgM with host age, sex, schistosome infection intensity and schistosome specific antibodies was studied before chemotherapeutic treatment of schistosome infections. This showed a significant negative association between schistosome infection intensity and levels of IgG1, IgG3, and IgG4 directed against malaria total extract antigen, and a positive association between levels of anti-schistosome soluble egg antigen IgG2, IgG3, and IgG4 and levels of the same subclasses directed against malaria total extract antigens. The effect of treating schistosome infections with praziquantel on malaria specific responses was also studied. This treatment resulted in increases in significant IgG4 levels against MSP3b and IgM against Glurp R0. Treatment also resulted in a significant decrease in IgG4 levels against Glurp R0. Host age, sex or pre-treatment infection intensity was not associated with the magnitude of change in the two IgG4 responses while males showed a significantly higher increase in levels of IgM. The results suggest cross reactivity between schistosome and malaria antigens in this population.

Anti-human immunodeficiency virus type 1 humoral immune response and highly active antiretroviral treatment

Highly active antiretroviral treatment (HAART) of human immunodeficiency type 1 (HIV-1) infection is very effective in controlling infection, but elimination of viral infection has not been achieved as yet, and upon treatment interruption an immediate rebound of viremia is observed. A combination of HAART with an immune stimulation might allow treatment interruption without this rebounding viremia, as the very low viremias observed with successful HAART may be insufficient to permit maintenance of a specific anti-HIV-1 immune response. The objective of this study was to compare the humoral immune response of individuals undergoing successful HAART (NF=no failure) with that of individuals with evidence of failure of therapy (FT) and to verify if the viremia peaks observed in individuals with therapy failure would act as a specific stimulus for the humoral anti-HIV-1 immune response. Antibodies binding to gp120 V3 genotype consensus peptides were more frequently observed for FT, mainly against peptides corresponding to sequences of genotypes prevalent in the Rio de Janeiro city area, B and F. HIV-1 neutralization of HIV-1 IIIB and of four primary isolates from Rio de Janeiro was less frequently observed for plasma from the NF than the FT group, but this difference was more expressive when plasma from individuals with detectable viremia were compared to that of individuals with undetectable viral loads in the year before sample collection. Although statistically significant differences were observed only in some specific comparisons, the study indicates that presence of detectable viremia may contribute to the maintenance of a specific anti-HIV-1 humoral immune response.

Early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies.

Late treatment of invasive candidiasis (IC) results in severe complications and high mortality. New tools are needed for early diagnosis. We conducted a retrospective study to assess the diagnostic utility of mannan antigenemia (Mn) and antimannan antibodies (anti-Mn) in neutropenic cancer patients at high risk for candidiasis. Twenty-eight patients with IC (based on European Organization for Research and Treatment of Cancer and Mycoses Study Group definitions) and 25 controls were studied. Mn and anti-Mn were positive (> or = 0.25 ng/mL and > or = 5 AU/mL, respectively) in 25/28 (89%) patients with candidiasis and in 4/25 (16%) controls: sensitivity, 89%; specificity, 84%; positive predictive value, 86%; negative predictive value, 88%. In patients with hepatosplenic lesions, assessing Mn/anti-Mn shortened the median time of diagnosis of candidiasis when compared with imaging (9 versus 25 days after fever onset as first sign of infection; P < 0.001). Candidiasis was diagnosed before neutrophil recovery in 78% and 11% of cases with Mn/anti-Mn and radiology, respectively (P < 0.001). Mn and anti-Mn may be useful for early noninvasive diagnosis of IC.

Association of the presence of residual anti-Toxoplasma gondii IgM in pregnant women and their respective family groups in Miracema, Northwest Rio de Janeiro, Brazil

The seroprevalence of toxoplasmosis in 832 pregnant women in Miracema, Rio de Janeiro, was determined and 75.1% (625) and 2.0% (17) were anti-Toxoplasma gondii IgG and IgM positive, respectively. Out of the 17 IgM positive pregnant women, only one had low avidity IgG corresponding to the acute phase of the infection. All the other women presented with high avidity IgG and also presented with residual IgM anti-T. gondii. Of this sample, 106 received home visits (this includes 11 family nuclei of pregnant women with residual IgM anti-T. gondii, 68 nuclei of only IgG positive pregnant women and 27 nuclei of pregnant women with no antibodies to anti-T. gondii), resulting in 267 individuals visited. Out of these 267 individuals, 21 were positive for IgG and IgM anti-T. gondii and were candidates for the IgG avidity test. All of them presented with high avidity IgG and residual IgM. Five of these IgM+ individuals were (5/238; 2.1%) relatives of IgM negative pregnant women. The other 16 (16/29; 55.2%) were relatives of IgM+ pregnant women who were positive for residual IgM anti-T. gondii. This association was statistically significant (p = 0.0000). The analysis presented herein raises questions regarding the presence of residual IgM anti-T. gondii such as genetic determinants or even constant antigenic stimuli for the same family cluster.

Anti-inflammatory and anti-nociceptive effects of Zeyheria montana (Bignoniaceae) ethanol extract

In this work, the analgesic and anti-inflammatory activities of Zeyheria montana Mart. ethanol leaf extract were investigated at doses of 75, 150 and 300 mg/kg body weight. In the analgesic assay, against a chemical stimulus in mice, acetic acid-induced writhes were significantly inhibited by the extract at doses of 75 mg/kg (67.27%), 150 mg/kg (49.38%) and 300 mg/kg (82.87%). Also, a vigorous decrease in hyperalgesia was observed when measured after 2 h and 6 h of lipopolysaccharide stimulation of rats for all doses of extract tested. Z. montana extract, at doses of 75 and 300 mg/kg, caused very slight central analgesia in rats submitted to thermal stimulus, particularly noticeable at 30 min following treatment. The anti-inflammatory activity of Z. montana extract on carrageenan-induced oedema in rats was evaluated. The oedema development, measured at 180 min following carrageenan intraplantar injection, was significantly reduced by all tested doses: 75 mg/kg (33.30%), 150 mg/kg (45.80%) and 300 mg/kg (75.00%). The LD50 value was greater than 2000 mg/kg. These results demonstrated that the ethanol extract from Z. montana leaf possesses anti-nociceptive and anti-inflammatory activities, which could be of relevance for the pharmacological control of pain and inflammatory processes.