Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

Determinants of hepatitis A vaccine immunity in a cohort of human immunodeficiency virus-infected children living in Switzerland

Vaccination in HIV-infected children is often less effective than in healthy children. The goal of this study was to assess vaccine responses to hepatitis A virus (HAV) in HIV-infected children. Children of the Swiss Mother and Child HIV Cohort Study (MoCHiV) were enrolled prospectively. Recommendations for initial, catch-up, and additional HAV immunizations were based upon baseline antibody concentrations and vaccine history. HAV IgG was assessed by enzyme-linked immunosorbent assay (ELISA) with a protective cutoff value defined as ≥10 mIU/ml. Eighty-seven patients were included (median age, 11 years; range, 3.4 to 21.2 years). Forty-two patients were seropositive (48.3%) for HAV. Among 45 (51.7%) seronegative patients, 36 had not received any HAV vaccine dose and were considered naïve. Vaccine responses were assessed after the first dose in 29/35 naïve patients and after the second dose in 33/39 children (25 initially naïve patients, 4 seronegative patients, and 4 seropositive patients that had already received 1 dose of vaccine). Seroconversion was 86% after 1 dose and 97% after 2 doses, with a geometric mean concentration of 962 mIU/ml after the second dose. A baseline CD4(+) T cell count below 750 cells/μl significantly reduced the post-2nd-dose response (P = 0.005). Despite a high rate of seroconversion, patients with CD4(+) T cell counts of <750/μl had lower anti-HAV antibody concentrations. This may translate into a shorter protection time. Hence, monitoring humoral immunity may be necessary to provide supplementary doses as needed.

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.

Post-surgical follow-up (by ELISA and immunoblotting) of cured versus non-cured cystic echinococcosis in young patients

The study was designed to determine comparatively the prognostic value of immunoblotting and ELISA in the serological follow-up of young cystic echinococcosis (CE) patients exhibiting either a cured or a progredient (non-cured) course of disease after treatment. A total of 54 patients (mean age 9 years, range from 3 to 15 years) with surgically, radiologically and/or histologically proven CE were studied for a period up to 60 months after surgery. Additionally, some of the patients underwent chemotherapy. Based on the clinical course and outcome, as well as on imaging findings, patients were clustered into 2 groups of either cured (CCE), or non-cured (NCCE) CE patients. ELISA showed a high rate of seropositivity 4 to 5 years post-surgery for both CCE (57.1%) and NCCE (100%) patients, the difference found between the two groups was statistically not significant. Immunoblotting based upon recognition of AgB subcomponents (8 and 16 kDa bands) showed a decrease of respective antibody reactivities after 4 years post-surgery. Only sera from 14.3% of CCE patients recognized the subcomponents of AgB after 4 years, while none (0%) of these sera was still reactive at 5 years post-surgery. At variance, immunoblotting remained positive for AgB subcomponents in 100% of the NCCE cases as tested between 4 and 5 years after surgical treatment. Immunoblotting therefore proved to be a useful approach for monitoring post-surgical follow-ups of human CCE and NCCE in young patients when based upon the recognition of AgB subcomponents.

Recombinant major urinary proteins of the mouse in specific IgE and IgG testing

Background: Recombinant allergens are preferred over natural allergen extracts in measuring antibodies. We tested the use of recombinant variants of the major mouse allergen Mus m 1 in detection of mouse-specific antibodies in sera of laboratory animal workers and children. Methods: Six recombinant major urinary proteins (MUPs) were produced and antibody-binding capacity was compared to natural Mus m 1 and to mouse urine extract. In a specific subset, cross-reactivity of MUP with Mus m 1 and between the different recombinant MUPs was determined. Results: For IgE antibodies, MUP8 showed high cross-reactivity with Mus m 1. MUP8-specific IgE was found in 55% of the mouse urine IgE-positive sera. Specific IgG and IgG4 antibodies against natural Mus m 1 correlated strongly with antibodies against recombinant MUP8 and were cross-reactive. IgG4 levels against MUP8 and mouse urine extract correlated, but detection of mouse urine-specific IgG4 in the absence of MUP-specific IgG4 was not uncommon. Cross-reactivity of IgG antibodies between MUP8 and Mus m 1 as well as between the different MUPs was high and inhibition varied between 54 and 99%. Conclusion: The mouse allergen Mus m 1 can be replaced in antibody testing by recombinant MUP8. Other MUPs, except MUP4, are interchangeable with MUP8. However, mouse urine extract showed better detection of both mouse-specific IgE and IgG4 levels. Other components in the mouse urine, like mouse albumin and other yet unidentified components, also induce IgE and IgG(4) antibodies.

Expression of Plasmodium falciparum 3D7 STEVOR proteins for evaluation of antibody responses following malaria infections in naïve infants

Clinical immunity to Plasmodium falciparum malaria develops after repeated exposure to the parasite. At least 2 P. falciparum variant antigens encoded by multicopy gene families (var and rif) are targets of this adaptive antibody-mediated immunity. A third multigene family of variant antigens comprises the stevor genes. Here, 4 different stevor sequences were selected for cloning and expression in Escherichia coli and His6-tagged fusion proteins were used for assessing the development of immunity. In a cross-sectional analysis of clinically immune adults living in a malaria endemic area in Ghana, high levels of anti-STEVOR IgG antibody titres were determined in ELISA. A cross-sectional study of 90 nine-month-old Ghanaian infants using 1 recombinant STEVOR showed that the antibody responses correlated positively with the number of parasitaemia episodes. In a longitudinal investigation of 17 immunologically naïve 9-month-old infants, 3 different patterns of anti-STEVOR antibody responses could be distinguished (high, transient and low). Children with high anti-STEVOR-antibody levels exhibited an elevated risk for developing parasitaemia episodes. Overall, a protective effect could not be attributed to antibodies against the STEVOR proteins chosen for the study presented here.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease

AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes. METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates (M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice. RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively). CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.