Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay

Staphylococcus aureus binds Immunoglobulin G (IgG) on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN) against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA) with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01); but the former were killed more efficiently than the latter (80-90% and 40%, respectively). It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.

Importance of immunoglobulin E (IgE) in the protective mechanism against gastrointestinal nematode infection: looking at the intestinal mucosae

This review discusses experimental evidences that indicate the IgE participation on the effector mechanisms that leads to gastrointestinal nematode elimination. Data discussed here showed that, for most experimental models, the immune response involved in nematode elimination is regulated by Th-2 type cytokines (especially IL-4). However, the mechanism(s) that result in worm elimination is not clear and might be distinct in different nematode species. Parasite specific IgE production, especially the IgE produced by the intestinal mucosae or associated lymphoid organs could participate in the intestinal elimination of Trichinella spiralis from infected rats. Intestinal IgE may also be important to the protective mechanism developed against other gastrointestinal nematodes that penetrate the murine duodenum mucosa tissue, such as Strongyloides venezuelensis and Heligmosomoides polygyrus. At least in Trichinella spiralis infected rats, the results indicated that intestinal IgE might work independently from mast cell degranulation for worm elimination.

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 ± 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT.

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

Diagnosis of rubella infection by detecting specific immunoglobulin M antibodies in saliva samples: a clinic-based study in Niterói, RJ, Brazil

This study was designed to investigate whether saliva could be a feasible alternative to serum for the diagnosis of recent rubella infection in a clinic setting. Forty-five paired blood and saliva samples collected 1 to 29 days after onset of illness were tested for specific immunoglobulin (Ig) M by antibody-capture radioimmunoassay (MACRIA). Rubella IgM was detected in all serum samples and in 38 (84.4%) saliva specimens. Forty-six serum and saliva samples from other patients with rash diseases were tested by MACRIA for control purposes and two saliva specimens were reactive. The saliva test had specificity of 96%. These results indicate that salivary IgM detection may be a convenient non-invasive alternative to serum for investigation of recent rubella cases, especially for disease surveillance and control programmes.

The secretory immunoglobulin A response to Mycobacterium tuberculosis in a childhood population

We report on the measurement of saliva anti-Purified Protein Derivative sIgA and 38kDa antibodies from 127 children, of whom 31 were strong tuberculosis suspects and 96 were healthy contact children. The results concerning the percentage of children with antibody reactivity to PPD and 38kDa antigens showed that, of these 2 antigens, 38kDa induced higher reactivity in patients positive and negative for the Tuberculin Skin Test (28% and 16.6%, respectively) in comparison to controls positive and negative for the TST (11.7% and 7.1%, respectively). There was a statistically significant difference between patients positive and controls negative for the TST. In relation to the Purified Protein Derivative antigen, while 14.2% of patients positive for the TST showed antibody reactivity to the PPD antigen, no patients negative for the TST had reactivity to this antigen. The findings suggest that these two antigens seem be associated with a different development of the mucosal defence mechanisms mediated by sIgA against Mycobacterium tuberculosis.

Immunoglobulin E-rheumatoid factor in juvenile rheumatoid arthritis

OBJECTIVES: To determine the presence of immunoglobulin E-rheumatoid factor in patients with juvenile rheumatoid arthritis and to correlate it with clinical and laboratory parameters. METHODS: A multicenter prospective study was carried out from January 1993 to January 1999 with the enrollment of 3 centers of pediatric rheumatology. Ninety-one children with juvenile rheumatoid arthritis diagnosed according to the American College of Rheumatology criteria were studied: 38 (42%) with systemic, 28 (31%) with pauciarticular, and 25 (27%) with polyarticular onset. Ages ranged from 2.1 years to 22.6 years (mean 10.5 ± 4.7), with 59 (65%) girls. The control group consisted of 45 healthy children. The detection of immunoglobulin E-rheumatoid factor was carried out utilizing an enzyme-linked immunosorbent assay. Associations of immunoglobulin E-rheumatoid factor with immunoglobulin M-rheumatoid factor (latex agglutination test), total serum immunoglobulin E, erythrocyte sedimentation rate, antinuclear antibody, and functional and radiological classes III or IV were analyzed. RESULTS: Positive immunoglobulin E-rheumatoid factor was found in 15 (16.5%) of the 91 children with juvenile rheumatoid arthritis: 7 (18.5%) with systemic, 5 (18%) with pauciarticular, and 3 (12%) with polyarticular onset. A significant correlation was observed between immunoglobulin E-rheumatoid factor and total serum immunoglobulin E in the juvenile rheumatoid arthritis patients. No correlation was found between immunoglobulin E-rheumatoid factor and positive latex agglutination slide test, erythrocyte sedimentation rate, antinuclear antibody, or the functional and radiological classes III or IV in any disease onset group. In 4 out of 45 control children (8.9%), immunoglobulin E-rheumatoid factor was positive but with no correlation with total serum immunoglobulin E levels. CONCLUSIONS: Immunoglobulin E-rheumatoid factor could be detected in 16.5% of juvenile rheumatoid arthritis patients, particularly in those with high levels of total serum immunoglobulin E, and immunoglobulin E-rheumatoid factor appears not to be associated with disease activity or severity.

Endemic pemphigus foliaceus (fogo selvagem) and pemphigus vulgaris: immunoglobulin G heterogeneity detected by indirect immunofluorescence

Pemphigus are autoimmune intraepidermal blistering diseases in which immunoglobulin G (IgG) autoantibodies are directed against desmosomal glycoproteins. The aim of this study was to determine the IgG subclass profile of endemic pemphigus foliaceus (fogo selvagem) and pemphigus vulgaris utilizing indirect immunofluorescence. PATIENTS AND METHODS: Twenty-five patients with pemphigus vulgaris, 25 with endemic pemphigus foliaceus (fogo selvagem), and 25 healthy controls were analyzed by indirect immunofluorescence for circulating autoantibodies (total IgG and its subclasses). RESULTS: Our data revealed a significant correlation (P <.05) of disease activity and autoantibody levels in both forms of pemphigus, i.e., negative titers related to clinical remission, whereas positive results related to active disease. Immunoglobulin G subclass analysis in fogo selvagem demonstrated that in patients in remission, 56% showed positive immunoglobulin G4; in active disease, immunoglobulin G4 was the predominant subclass (100% positive in all cases). The IgG subclass profile in pemphigus vulgaris showed that in patients in remission, only 10% were positive for immunoglobulin G4; in active disease, positivity for immunoglobulin G4 was present in 78% to 88% of the cases. CONCLUSION: Subclass characterization of immunoglobulin G autoantibodies is a useful tool for pemphigus follow-up, since immunoglobulin G4 (IgG4) is the subclass that is closely related to recognition of pathogenic epitopes, and consequently with disease activity. Careful monitoring should be performed for fogo selvagem in clinical remission with a homogeneous IgG4 response, since this may indicate more frequent relapses.

The immunopathology of human schistosomiasis-III: immunoglobulin isotype profiles and response to praziquantel

Immunoglobulin (Ig) isotype (IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD and IgE) levels were investigated, both pre- and post-treatment with praziquantel (PZQ), in 43 adults and children chronically infected with Schistosoma mansoni , by means of a two-site, isotype-specific immunoenzymometric assay. The patients were classified as responders (R) or non-responders (NR) on the basis of their circumoval precipitin test (COPT) results 12 months after treatment. In comparison with controls, pre-treatment R children showed significantly higher levels of IgG, IgG1, IgG4 (p<0.001) and IgE (p<0.01), and diminished IgG2 (p<0.05), while NR children showed significantly elevated levels only of IgE (p<0.05). Twelve months after therapy, R children maintained significantly lower levels of IgG2, but showed significantly decreased levels of IgG, IgG1, IgG4, and IgE, while the Ig isotype profile of NR children was unaltered. Adult R and NR showed similar isotype profiles before chemotherapy, with the exception of significantly elevated IgM levels in R. Twelve months after therapy, R adults showed significantly decreased levels of IgG, IgG1, and IgG4, while NR adults showed only diminshed IgG4 levels. These results reveal different Ig isotype profiles in untreated adults and children chronically infected with S. mansoni. The results further show that the pre-treatment Ig isotype profile may be significantly modified after an effective R to chemotherapy, accounted for by down regulation of the IgG1 isotype in association with negative seroconversion of the COPT in R patients. The COPT reaction has been associated with the highly specific egg glycoprotein antigen w1, which shows a significant reduction in reactivity six months after treatment. IgG1 may thus play a main role in the response against the w1 antigen.

An immunoenzymatic assay for the diagnosis of hepatitis A utilising immunoglobulin Y

The detection of anti-hepatitis A virus (HAV) antibody levels by diagnostic kits in the convalescent period of disease generally use immunoglobulin G (IgG), which is expensive. An alternative to IgG is immunoglobulin Y (IgY), an immunoglobulin antibody encountered in birds and reptiles. The aim of this study was to develop a competitive immunoenzymatic assay to measure total anti-HAV antibody levels using anti-HAV IgY as the capture and conjugated immunoglobulins. For this purpose, anti-HAV IgY was conjugated to horseradish peroxidase (HRP) and the optimal dilution of HRP-conjugated antibodies was evaluated to establish the competitive immuneenzymatic assay. The results obtained from our "in-house" assay were plotted on a receiver operator curve, which showed a sensitivity of 95% and a specificity of 98.8%, demonstrating that a competitive anti-HAV IgY immunoenzymatic assay developed "in house" could be used as an alternative to commercial assays that utilise IgG.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Immunoglobulin G4-related disease: autoimmune pancreatitis and extrapancreatic manifestations

Abstract We present a case of immunoglobulin G4 (IgG4)-related disease with pancreatic and extrapancreatic involvement, including the biliary and renal systems. Given the importance of imaging methods for the diagnosis of IgG4-related disease and its differentiation from pancreatic adenocarcinoma, we emphasize important abdominal computed tomography and magnetic resonance imaging findings related to this recently recognized systemic autoimmune disease.