iPSC-derived cortical neurons to study sporadic Alzheimer disease: A transcriptome comparison with post-mortem brain samples.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by the progressive impairment of cognition and memory loss. Sporadic AD (sAD) represents approximately 95 % of the AD cases and is induced by a complex interplay between genetic and environmental factors called "Alzheimerogens". Heavy metals (e.g. copper) and pesticides (e.g. fipronil) can affect many AD-related processes, including neuroinflammation (considered as AD-inducing factor). Research would benefit from in vitro models to investigate effects of Alzheimerogens. We compared transcriptomics changes in sAD induced pluripotent stem cell (iPSC) derived cortical neurons to differentially expressed genes (DEGs) identified in post-mortem AD brain tissue. These analyses showed that many AD-related processes could be identified in the sAD iPSC-derived neurons, and furthermore, could even identify more DEGs functioning in these processes than post-mortem AD-brain tissue. Thereafter, we exposed the iPSCs to AD-inducing factors (copper(II)chloride, fipronil sulfone and an inflammatory cytokine cocktail). Cytokine exposure induced expression of immune related genes while copper-exposure affected genes involved in lipid and cholesterol metabolism, which are known AD-related processes. Fipronil-exposure did not result in significant transcriptomic changes, although prolonged exposures or higher doses may be necessary. Overall, we show that iPSC-derived cortical neurons can be beneficial in vitro models to identify Alzheimerogens and AD-related molecular mechanisms.

Preliminary performance data of the RHE/IL-18 assay performed on SkinEthic™ RHE for the identification of contact sensitizers.

OBJECTIVE: The purpose of this study was to evaluate the performances of the RHE/IL-18 assay using the SkinEthic™ RHE model for the identification of contact sensitizers. METHODS: A set of 18 substances and mixtures was tested on this epidermal model, following the RHE/IL-18 protocol. The final results of the assay were obtained following 5 interpretation schemes, to determine the optimal prediction model for this assay with this specific test system. The data were analysed with a special focus on the basal level of IL-18 release and on the performance obtained with respect to three different gold standards: LLNA, HRIPT and an integrated reference, constructed from all available results. RESULTS: No important differences were found in the performance levels depending on the three gold standards. The performances obtained with the SkinEthic™ RHE model support that this model may be considered as an alternative to different reconstructed epidermis models (EpiDERM™ , EpiCS™ and VUMC-EE) for the performance of RHE/IL-18 assays. CONCLUSION: The prediction model to be used was refined, and more substances have to be tested in order to gather enough data for this evaluation and to determine the right criteria applicable for this assay using the SkinEthic™ RHE test system.

IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy.

BACKGROUND: Development and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational epitopes. OBJECTIVE: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. METHODS: Polyclonal sera from three individual patients, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library. Resulting epitope-mimicking sequences were aligned for identification of consensus sequences and localised on the surface of the Ara h 1 molecule by a computer-based algorithm. RESULTS: All epitope-mimicking sequences identified were found to correspond to conformational epitopes. Each individual patient had his/her own distinct IgE as well as IgG4 epitope recognition profile, though some important IgE epitopes were common to all patients. In general the IgG4 epitope pattern was more heterogeneous than the IgE pattern, did not coincide with IgE epitopes and had a lower affinity than IgE. CONCLUSIONS: This study demonstrated the usefulness of the phage-display technology in distinguishing between the epitope pattern of IgE and IgG4, giving detailed information on fine specificity and affinity. Competitive immuno-screening of phage-display random peptide libraries could be a future valuable tool to study the balance and dynamics of the IgE and IgG4 epitope recognition repertoire and provide a diagnostic tool giving information on the associated allergic phenotype.

IgE epitopes of intact and digested Ara h 1: a comparative study in humans and rats.

BACKGROUND: Allergen epitope characterization provides valuable information useful for the understanding of proteins as food allergens. It is believed that IgE epitopes in general are conformational, nevertheless, for food allergens known to sensitize through the gastrointestinal tract linear epitopes have been suggested to be of great importance. OBJECTIVE: The aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. METHODS: Sera from five peanut allergic patients and five Brown Norway rats were used to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule to mimic epitopes using a computer-based algorithm. RESULTS: Patients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which by far accounted for most of the eluted peptide sequences. Epitope patterns were rather similar for both intact and digested Ara h 1 as well as for humans and rats. CONCLUSIONS: Individual patient specific epitope patterns have been identified for the major allergen Ara h 1. IgE binding epitopes have been suggested as biomarkers for persistency and severity of food allergy, wherefore recognition of particular epitope patterns or motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergy.

Vaccination for birch pollen allergy: comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance.

BACKGROUND: Allergen-specific immunotherapy (SIT) is associated with increased levels of allergen-specific IgG in serum. However, it is not clear to what extent qualitative changes in the allergen binding capacity of IgG may be induced as well. OBJECTIVE: The purpose of this study was to investigate the influences of SIT on antibody affinity. METHODS: The binding affinity of purified serum IgG1, IgG4 and IgE to the major allergen in birch (Betula verrucosa) pollen, Bet v 1, was analysed by surface plasmon resonance. The antibodies were obtained from 10 birch pollen-allergic patients receiving SIT and from 10 patients with no SIT. RESULTS: The patients having received SIT have a significant higher titre of anti-Bet v 1 antibodies in their blood, but the affinity to Bet v 1 of allergen-specific IgE, IgG1 and IgG4 does not differ between the two groups. For IgG1 and IgG4, correlations between less allergic symptoms and affinity of the antibodies were observed both in the SIT group and to a smaller extent in the non-SIT group. CONCLUSION: SIT has no effect on antibody affinity of allergen-specific IgE, IgG1 or IgG4. Allergic patients with high-affinity IgG1 and IgG4 antibodies report less symptoms than patients with low-affinity antibodies.

Monoclonal antibodies against Haemophilus ducreyi lipooligosaccharide and their diagnostic usefulness.

Monoclonal antibodies against the lipooligosaccharide of Haemophilus ducreyi were produced. Two of them, MAHD6 and MAHD7, were found to be relatively, although not absolutely, specific and reacted with nearly all strains of Haemophilus ducreyi tested: 59 of 60 and 60 of 60, respectively. The diagnostic usefulness of MAHD7 was assessed. Clinical specimens collected in Zambia from patients with genital ulcers were tested using indirect immunofluorescence (IF), enzyme immunoassay (EIA), the polymerase chain reaction (PCR) and bacterial culture. Compared with culture, IF had a sensitivity of 100%; compared with PCR, sensitivity was 89%. The corresponding figures for the EIA were 83% and 74%, respectively. The sensitivity of culture compared with PCR was 63%. The results suggest that IF on genital smears using MAHD7 might be an excellent tool for the diagnosis of chancroid in high-prevalence populations. However, further evaluation of the specificity of this test is needed.

Localisation and immunological properties of a 24-kDa surface protein of Haemophilus ducreyi.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Monoclonal antibodies against Haemophilus lipopolysaccharides: clone DP8 specific for Haemophilus ducreyi and clone DH24 binding to lacto-N-neotetraose.

Mouse monoclonal antibodies (MAbs) DP8 [immunoglobulin G1(kappa)] and DH24 [immunoglobulin M(kappa)], which are specific for Haemophilus ducreyi lipopolysaccharide (LPS), were generated by fusing mouse myeloma NS0 cells with spleen cells of BALB/c mice immunized with a total membrane preparation of H. ducreyi. MAb DP8 reacted in whole-cell enzyme immunoassay (EIA) and colony dot immunoblotting with all 50 strains of H. ducreyi but not with any other bacteria tested, which suggests an exposed and species-specific epitope on the H. ducreyi cell surface. This conclusion was supported by the finding that DP8 bound to all six H. ducreyi LPSs tested but not to any of the Haemophilus influenzae or enterobacterial LPSs or synthetic glycoconjugates. The MAb DH24 bound to 43 of 50 strains of H. ducreyi and to few strains of H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as evaluated by whole-cell EIA and colony dot immunoblotting. The MAb DH24 reacted with five of the six H. ducreyi LPSs tested and with the lacto-N-neotetraose (Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc) series of synthetic glycoconjugates, as determined by EIA. By using polysaccharides obtained after both mild acidic hydrolysis and strong alkali treatment and dephosphorylated samples as inhibitors of the MAbs binding to H. ducreyi LPS antigens, it could be shown that phosphate groups were essential for the binding of DP8 to LPS but that they did not affect antigenic recognition by DH24. None of the MAbs bound to isolated lipid A, but aggregation caused by the fatty acids of lipid A was essential for epitope recognition.

Human T cell lymphotropic virus type I infection among female sex workers in Peru.

Four hundred female sex workers attending a sexually transmitted disease clinic in Lima, Peru, were interviewed for demographic information and medical, contraceptive, and sexual practice histories. Cervical cultures were done for Neisseria gonorrhoeae and Chlamydia trachomatis, and serum was tested for antibodies to human immunodeficiency virus, human T cell lymphotropic virus type I (HTLV-I), Treponema pallidum, C. trachomatis, herpes simplex virus type 2 (HSV-2), and Haemophilus ducreyi. The prevalence of HTLV-I increased with duration of prostitution from 3.6% (< 3 years) to 9.3% (3-6 years) to 15.9% (> 6 years; P < .01). After adjustment for duration of prostitution, reduced risk of HTLV-I was significantly correlated with condom use for more than half of all sexual exposures for > 3 years (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.13-0.89). Further adjusting for condom use, HTLV-I seropositivity was associated with C. trachomatis (OR, 3.7; 95% CI, 1.4-13.2) and with antibody to HSV-2 (OR, 3.7; 95% CI, 0.5-29.6). Thus, duration of prostitution, lack of consistent condom use, and past infection with C. trachomatis were significantly associated with HTLV-I seropositivity.

Enzyme immunoassays (EIAs) for the detection of anti-Haemophilus ducreyi serum IgA, IgG, and IgM antibodies.

BACKGROUND AND OBJECTIVES: Chancroid is a risk factor for heterosexually acquiring HIV. Controlling its spread may reduce HIV transmission. GOAL OF THE STUDY: To develop EIAs for assessing antibody levels and for seroepidemiologic studies. STUDY DESIGN: Anti-Haemophilus ducreyi IgA, IgG and IgM EIAs were standardized using a crude cocktail antigen. Evaluation was on sera from Kenya, Rwanda, Thailand and The Gambia. The two-tailed student's t test was used to compare results. RESULTS: The specificity of IgA was 97% (95% confidence interval (CI): 95-99%), of IgG was 92% (95% CI: 89-95%), and of IgM was 99% (95% CI: 98-100%). The sensitivity of IgA was 88% (95% CI: 83-93%), of IgG was 93% (95% CI:89-97%), and of IgM was 78% (95% CI:71-85%) in patients having an ulceration for more than eight days. Thus, 95% (95% CI:92-98%) of the chancroid patients were seropositive for at least one antibody type. The IgG and IgA EIAs were more sensitive in patients older than 24 years of age. Higher IgG rates were found in HIV infected chancroid patients. CONCLUSION: The EIAs should be useful for studying the kinetics of antibody levels and the epidemiology of H. ducreyi infection.

PIP: In Belgium, the Department of Infection and Immunity of the Institute of Tropical Medicine in Antwerp modified an experimental enzyme immunoassay (EIA) for the detection of serum IgG to Hemophilus ducreyi to develop EIAs for detection of anti-H. ducreyi IgA and IgM antibodies. They tested the modified EIA on sera from people in Nairobi, Kenya; Kigali, Rwanda; Banjul, The Gambia; and Bangkok, Thailand, who had a sexually transmitted disease. The EIA was able to identify correctly those who did not have anti-H ducreyi IgA, IgG, and IgM antibodies in 97%, 92%, and 99% of cases, respectively. Among people with a genital ulceration for more than 8 days, it was able to identify correctly those who had IgA, IgG, and IgM antibodies in 88%, 93%, and 78% of cases, respectively. 95% of all culture-proven chancroid patients tested seropositive for at least 1 antibody type. The sensitivity of IgG and IgA EIAs was significantly enhanced in patients with culture-proven chancroid who were older than 24 years old (p .01). HIV seropositive people from Kigali who had culture-proven chancroid had higher anti-H. ducreyi IgG seropositivity rates (but not IgA and IgM seropositivity rates), than did HIV seronegative chancroid people from Kigali (p .05). The increased IgG seropositivity rate was not related to higher antibody titers, however, suggesting that HIV infection modifies the response to H. ducreyi. These results show that the 3 EIAs hold promise as a means to study the kinetics of antibodies and the epidemiology of chancroid.

Antigen detection and immunological typing of Haemophilus ducreyi with a specific rabbit polyclonal serum.

A rabbit polyclonal serum was raised against the 29-kDa species-specific marker, as well as the 30- to 34-kDa immunotype-specific markers of Haemophilus ducreyi described elsewhere (E. Roggen, S. De Breucker, E. Van Dyck, and P. Piot, Infect. Immun. 60:590-595, 1992). These antigens were purified from a cocktail of H. ducreyi isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immune serum reacted in enzyme-linked immunosorbent assay (ELISA) preferentially with H. ducreyi, at a titer as high as 50,000. To make it specific to H. ducreyi, nonspecific antibodies were removed by adsorption on a mixture of Haemophilus spp., Escherichia coli, Candida albicans, and Corynebacterium spp. In the 29- to 34-kDa region of immunoblot profiles from H. ducreyi isolates (n = 450), the adsorbed serum revealed essentially the same antigens as did a pool of well-characterized human sera. Yet, eight different immunotypes were observed. With this rabbit polyclonal serum, an ELISA-based antigen detection test was developed. The adsorbed serum reacted specifically with all H. ducreyi isolates tested (n = 450), but not with other bacterial species (n = 15). This test was evaluated with a limited number of clinical specimens from African patients with culture-proven chancroid and no evidence for any other ulcerating etiology (n = 10) and a number of chancroid-negative control patients from Belgium (n = 20). Within this context, the test yielded a sensitivity and specificity of 100%.

Antigenic diversity in Haemophilus ducreyi as shown by western blot (immunoblot) analysis.

The antigenic diversity within a panel of 63 Haemophilus ducreyi isolates was examined by Western blot (immunoblot) analysis with a pool of 238 well-characterized human antisera. When a serum pool adsorbed on a mixture of Haemophilus influenzae, H. parainfluenzae, and H. parahaemolyticus was used, the immunoprofiles suggested that prominent antigenic proteins involved in the human immune response have apparent molecular masses of 63, 42, 34 to 30, and 28.5 to 28 kDa. Preliminary subcellular localization revealed that these antigens are associated with the cellular membrane. Two subsets of antigens were discriminated by detergent extraction. There was no evidence that the antigen composition is altered by changing the growth conditions. With a serum pool adsorbed on the Haemophilus spp. mixture supplemented with Actinobacillus actinomycetemcomitans, Pasteurella ureae, Neisseria gonorrhoeae, and Escherichia coli, antigenic determinants more specific for H. ducreyi were identified. An immunodominant 28.5- to 28-kDa protein was expressed by all H. ducreyi isolates. In the range from 34 to 30 kDa, 56 isolates revealed a dominant protein with variable molecular mass. By using both proteins (28.5 to 28 kDa and 34 to 30 kDa) as immunotypic markers, seven different immunopatterns were identified. Antigenic diversity among isolates from different geographical origins as well as from a single area was observed.