Antibodies In Saliva Research Articles

Detection of trypanosome‐specific antibodies in saliva, towards non‐invasive serological diagnosis of sleeping sickness

The detection of trypanosome-specific antibodies in saliva is technically feasible, and, if clinically validated, could become an attractive option for non-invasive diagnosis of sleeping sickness. We wanted to optimize the test format of an enzyme-linked immunosorbent assay (ELISA)-based antibody detection system. Different ELISA formats for antibody detection in serum and saliva were developed and standardized. Saliva and serum samples were collected from 78 patient and 128 endemic control samples, and sensitivity and specificity of saliva ELISAs, serum ELISAs and the card agglutination test for trypanosomiasis (CATT), were evaluated. All ELISA formats showed sensitivity and specificity above 90%. Saliva ELISAs showed a similar test performance as serum ELISAs and the CATT on whole blood or serum. This study confirms the potential of trypanosome-specific antibody detection in saliva.

Cellular and humoral systemic and mucosal immune responses stimulated in volunteers by an oral polybacterial immunomodulator “Dentavax”

The oral polybacterial immunomodulator Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates was created for immunoprophylaxis and therapy of oral mucosa and parodont inflammations. The stimulating effect of the preparation was evaluated in twelve volunteers immunized for 10 consecutive days. On days 7, 14, 21, 28 and 49 after the last immunization peripheral blood (PB) lymphocyte subsets, T lymphocyte activation and PB phagocytic activity, were studied by flow cytometry. PB lymphocyte proliferative responses to PHA, rIL-2, LPS and D were evaluated radiometrically. The production of TNF-alpha in supernatants of in vitro stimulated lymphocytes and specific IgA, IgM and IgG antibodies in serum and saliva was determined by ELISA. Ultrastructural morphologic changes in T and B lymphocyte populations were also investigated. Although no significant changes in the levels of basic lymphocyte subsets were detected, the early/late (CD57+/CD57−) CD8 T effectors ratio was increased at the end of the studied period, as were the percentage of PHA-responding (CD69+) T cells and PB phagocytizing cells. The most prominent lymphoprolipherative responses were measured upon costimulation with LPS+D and PHA+D on day 21. Electron-microscopic studies demonstrated a significant effect of D on both T and B cell activity. TNF-alpha concentration increased progressively from day 7 till the end of the investigation. Maximal concentrations were observed after stimulation with D and LPS. An increased level of specific salivary and serum antibodies against the components of D was found, with highest levels between days 7 and 21. Specific secretory IgA predominated in saliva as compared to IgM and IgG. Our results demonstrate the stimulating effect of Dentavax on PB lymphocyte functional activity and the specific humoral systemic and mucosal immunity.

Oral transmission of HIV, reality or fiction? An update

Human immunodeficiency virus (HIV) and many other viruses can be isolated in blood and body fluids, including saliva, and can be transmitted by genital-genital and especially anal-genital sexual activity. The risk of transmission of HIV via oral sexual practices is very low. Unlike other mucosal areas of the body, the oral cavity appears to be an extremely uncommon transmission route for HIV. We present a review of available evidence on the oral-genital transmission of HIV and analyse the factors that act to protect oral tissues from infection, thereby reducing the risk of HIV transmission by oral sex. Among these factors we highlight the levels of HIV RNA in saliva, presence of fewer CD4+ target cells, presence of IgA antibodies in saliva, presence of other infections in the oral cavity and the endogenous salivary antiviral factors lysozyme, defensins, thrombospondin and secretory leucocyte protease inhibitor (SLPI).

In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT

In vitro immune-stimulating activities of Bordetella bronchiseptica dermonecrotoxin (BBD)-loaded in chitosan microspheres (CMs) were reported with a mouse alveolar macrophage cell line (RAW264.7). Based on the report, in vivo activity of immune-induction was investigated by intranasal administration of the BBD-loaded CMs into mice. BBD was loaded into the CMs prepared by an ionic gelation process with tripolyphosphate. Mice were immunized by direct administration of the BBD-loaded CMs into the nasal cavity. After immunization of the mice, BBD-specific immune responses (IgG and IgA titers) were measured in sera, nasal wash, and saliva by ELISA. BBD-specific IgA titers in the nasal cavity were time- and dose-dependently increased by the administration. Similar phenomena were observed in the analysis of systemic IgA and IgG in sera. However, the antibody in saliva was undetectable by ELISA. These results suggested that direct vaccination via the nasal cavity was effective for targeting nasal-associated lymphoid tissues, and that CMs were an efficient adjuvant in nasal mucosal immunity for atrophic rhinitis vaccine.

Cellular and Humoral Systemic and Mucosal Immune Responses Stimulated by an Oral Polybacterial Immunomodulator in Patients with Chronic Urinary Tract Infections

An oral polybacterial immunomodulator Urostim (U), composed of killed cells and their lysates from E. coli expressing type 1 and Pili, E. coli Rc mutant, P. mirabilis, K. pneumoniae and E. faecalis was created for immunoprophylaxis and immunotherapy of urinary tract infections (UTIs). In experimental animal models, the stimulating effect of U on lymphocyte functional activity, macrophage phagocytosis and antibody producing cells, was established. In this study the immuno-modulating effects of U on the proliferating capacity and ultrastructural morphologic changes of lymphocytes, cytokine production and specific systemic humoral and mucosal immune responses in patients with UTIs have been evaluated. Patients enrolled in the study, received orally 50 mg U daily for a period of three months. On days 0, 30 and 90 a quantitative analysis was performed on lymphoproliferative responses to polyclonal mitogens, IL-2 and the specific antigen U, the production of specific serum and saliva IgA, IgM and IgG antibodies to all components of U and the concentration of pro-inflammatory cytokines. There was significant improvement of non-specific and specific lymphoproliferative responses on days 30 and 90 after the onset of treatment with U, confirmed by electronmicroscopic studies. The highest concentrations of serum proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 were registered at baseline followed by a decrease until the end of the observation period. This finding correlates with the gradual decrease of immune activation as measured by the spontaneous lymphocyte proliferation. Data from the production of specific antibacterial antibodies in serum and saliva show two types of reactions. The first type was registered in patients with low pre-treatment levels in whom the concentration of specific antibodies increased on days 30 and 90. The second type of reaction was observed in patients with high pre-treatment levels, which dropped on day 30 and were usually followed by an increase at the end of the study. These results provide evidence for the immuno-modulating effect of U. Our data show that the oral administration of the polybacterial immunomodulator Urostim stimulates adequate cellular and humoral systemic and mucosal immune responses in patients with chronic UTIs.

Immunoglobulin-A antibodies in Upper Airway Secretions may Inhibit Intranasal Influenza Virus Replication in Mice but not Protect Against Clinical Illness

Mice immunized intranasally with a formalin-inactivated A/PR/8/34 (H1N1) influenza whole virus vaccine adjuvanted with cholera toxin, outer membrane vesicles from group B meningococci or formalin-inactivated whole cell Bordetella pertussis were protected against replication of the homologous virus in the nasal cavity. Only some mice were protected against clinical illness measured as weight loss and lowered body temperature. All mice immunized subcutaneously with one-tenth the intranasal vaccine dose without adjuvant were protected against clinical illness but not against local mucosal viral replication. Replicating virus was primarily found in animals with low concentrations of immunoglobulin (Ig)-A antibodies in saliva regardless of concentrations of IgG antibodies in serum. Clinical illness was seen only in those with low serum antibodies regardless of antibody levels in saliva. Nonreplicating nasal vaccines may not be sufficiently protective unless they also have a substantial influence on systemic immunity.

Serum and saliva antibodies do not inhibitCandida albicansSap2 proteinase activity using a BSA hydrolysis assay

The opportunistic fungal pathogen Candida albicans possesses 10 members of a secreted aspartyl proteinase (Sap) family, which are associated with fungal virulence. The C. albicans proteinases are known to induce antibody responses in humans, but the direct inhibition of Sap activity by antibody has not yet been demonstrated. The aim of this study was to determine whether antibodies in saliva or serum could inhibit C. albicans proteinase activity. A two-step Sap-inhibition assay based on bovine serum albumin (BSA) hydrolysis was developed. First, serum and saliva were incubated with Sap2 to allow antibodies to bind to the enzyme, and then a Sap activity assay was performed to determine whether or not the bound antibodies were capable of inhibiting Sap activity. Inhibition of Sap2 activity was investigated using nine sources of sera or saliva: mouse Sap1, Sap2 and Sap3 antisera; rabbit Sap2 antiserum; two pooled human serum samples from HIV-positive and HIV-negative patients with oral C. albicans infection; and three pooled saliva samples from patients with oral C. albicans infection, C. albicans carriers, and Candida-culture-negative individuals. Pooled saliva samples did not inhibit Sap2 activity, whereas mouse, rabbit, and human sera demonstrated inhibition of Sap2 activity by 20-45%. Further analysis of different serum fractions, purified total IgG, and Sap2-specific antibodies demonstrated that non-protein, non-antibody components of serum appeared to be responsible for the partial inhibition of Sap2 activity. Therefore, no evidence was found to demonstrate that specific or non-specific antibodies in serum or saliva could inhibit C. albicans Sap2 activity.

Noninvasive Diagnostic Tests for Helicobacter Pylori Infection in Children

Noninvasive tests can be used for the initial diagnosis of Helicobacter pylori infection and to monitor the success of eradication therapy. In populations with a low prevalence of H. pylori infection (children living in North America and Europe), a high sensitivity is required to make the test valuable for clinical practice. The 13C-urea breath test has been validated in children of different age groups in a significant number of infected and noninfected children in several countries and, thus far, is the only noninvasive test that fulfills sensitivity and specificity quality standards. In studies to date, enzyme immunoassays using monoclonal antibodies to detect H. pylori antigen in stool provide excellent results, but the number of children tested, particularly post-treatment, is not sufficient to recommend the test. All other noninvasive stool tests or methods based on the detection of specific antibodies in serum, whole blood, urine or saliva have limited accuracy in comparison with the 13C-urea breath test. Therefore, these tests cannot be recommended for clinical decision making in pediatric patients.

Parenteral Influenza Vaccination Influences Mucosal and Systemic T Cell–Mediated Immunity in Healthy Adults

We sought to determine whether palatine tonsils (PTs) harbor naturally acquired influenza-specific T cell immunity and whether routine parenteral immunization with influenza vaccine influences mucosal and systemic T cell reactivity. We demonstrate that tonsillar and peripheral blood mononuclear cells (PBMCs) proliferate strongly to influenza antigens, suggesting that naturally acquired immunity exists within both the mucosal and systemic compartments. Influenza vaccination induced significantly stronger T cell responses in both PTs and blood, in addition to increasing titers of anti-influenza antibodies in serum and saliva. More-rapid proliferative responses of PTs after vaccination were associated with a shift from a response involving both CD45RA+ and CD45RO+ T cells to an entirely CD45RO+-dependent response. Interestingly, the ratio of interferon- gamma to interleukin-5 was dramatically higher in cultures of PT T cells responding to influenza than in PBMCs. Our data indicate that parenteral influenza vaccination influences both mucosal and systemic naturally acquired T cell immunity.

Role of peptide antigen for induction of inhibitory antibodies to Streptococcus mutans in human oral cavity.

The alanine-rich repeating region (A-region) in the surface protein antigen (PAc) of Streptococcus mutans has received much attention as an antigenic component for vaccines against dental caries. The PAc (residue 361-386) peptide in the A-region possesses a multiple binding motif (L- -V-K- -A) to various HLA-DR molecules and a B-cell core epitope (- Y- - -L- -Y- - - -) that recognizes the inhibiting antibody to S. mutans. In the present study, we investigated the immunogenicity of the PAc (361-386) peptide in humans and regulators of induction of the anti-PAc (361-386) peptide IgA antibody (aPPA) in saliva. The PAc (361-386) peptide was confirmed as an ideal peptide antigen for induction of the inhibiting antibody to S. mutans in 151 healthy human subjects (36.6 +/- 12.6 years old) by quantitative analyses of oral bacteria and ELISA, as the aPPA titre in human saliva decreased significantly in an age-dependent manner. Homozygous DRB1*0405 and 1502, and heterozygous DRB1*0405/1502 showed a negative association with production of aPPA and tended to reduce the number of total streptococci in saliva. In contrast, the DRB1*1501 allele was significantly correlated with a high level of induction of the antibodies, and also tended to reduce lactobacilli and mutans streptococci. Further, peptide immunogenicity was confirmed in NOD-SCID mice grafted with human peripheral blood mononuclear cells. Our results indicate that the interplay between regulators such as age, DRB1 genotype, cytokines, and peptide immunogenicity may provide a potential means for developing a vaccine useful for the prevention of dental caries as well as their diagnosis.

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age ( P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth ( P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.

Increasing risk behaviour and high levels of undiagnosed HIV infection in a community sample of homosexual men

Objectives: To estimate changes in sexual behaviour over time. To examine the proportion of undiagnosed HIV infection in a community sample of homosexual men. To explore the relation between HIV...

Secretion of IgA by rat parotid and submandibular cells in response to autonomimetic stimulation in vitro

The major antibody in saliva is IgA, which is actively transported by pIgR expressed by parenchymal cells within the salivary glands. The rate of IgA secretion into saliva is regulated by the autonomic nerves supplying the glands in vivo. This study examined the mechanism of increased IgA secretion into saliva with autonomimetic stimulation. In vitro stimulation of IgA secretion from cells prepared by digestion of rat salivary glands found submandibular cell preparations responded to α- and β-adrenergic stimuli whereas the parotid cells responded only to β-adrenergic stimulation, although cells from both glands responded similarly to cholinergic stimulation. The additional responsiveness of submandibular cells to α-adrenergic stimulation probably reflects the presence of granular duct cells (absent in parotid glands) which are known to secrete protein in response to high frequency sympathetic stimulation. The increased secretion of IgA was not dependant upon increased plasma cell activation since isolated salivary gland plasma cells did not respond to agonists. Further evidence for the regulating role of parenchymal cells in IgA secretion into saliva was revealed by analysis of polymeric immunoglobulin receptor (pIgR) levels expressed on cells. Following in vivo nerve stimulation, there was an increased amount of pIgR expressed on the membrane surface. This was functionally demonstrated in vitro by increased uptake of human IgA by acutely prepared rat salivary cells following stimulation by adrenaline, indicating increased mobilisation of pIgR with stimulation. This study confirms that salivary cells increase the delivery of IgA into saliva by a pIgR-mediated mechanism in response to autonomic stimulation.

Testing for anti-human transglutaminase antibodies in saliva is not useful for diagnosis of celiac disease.

Undetected celiac disease (CD) can cause serious complications and excessive mortality (1)(2)(3). Noninvasive tests could be useful in physicians’ offices to identify patients for intestinal biopsies (4). Serum IgA anti-endomysium (AEA) and anti-human transglutaminase (anti-htTG) antibody assays are the most widely used laboratory tests for CD (5). Autoantibody determinations have been simplified by the use of rapid testing (6)(7), but when saliva specimens were evaluated for AEA testing, they were found to be not suitable (8). To evaluate the potential utility of saliva as a sample for anti-htTG testing, we compared salivary IgA anti-htTG (measured by ELISA with a dot-blot method and an AEA test) with serum IgA anti-htTG antibodies (measured by ELISA and an AEA test) in patients with CD and in controls. Salivary IgA anti-htTG activity was analyzed for the IgA secretory chain by use of a monoclonal antibody. The recognition patterns of the saliva samples were compared with those for the serum samples and with the pattern for a gut-derived IgA monoclonal antibody to htTG from one CD patient, using two transglutaminase deletion mutants, one recognized and one not by the patients’ sera (9). We studied 47 untreated CD patients (29 females and 18 males; median age, 19 years; range, 2–52 years) diagnosed in 2002–2003 according to ESPGHAN criteria (10) and 47 patients with celiac disease (26 females and 21 males; median age, 19 years; range, 4–50 years) on a gluten-free diet (GFD) for at least 12 months. We recruited 51 healthy controls with no history of gastrointestinal or autoimmune diseases (25 females and 26 males; median age, 14 years; range, 6–50 years) and identified samples from 49 individuals suffering from various gastrointestinal diseases (18 with Crohn disease, 10 with ulcerative colitis, 10 with milk allergy, 6 with acute …

Immunisation schedules for non-replicating nasal vaccines can be made simple by allowing time for development of immunological memory

Mice immunised intranasally with multiple doses of outer membrane vesicles (OMVs) from group B meningococci developed antibody responses that depended on the interval between doses. High levels of antibodies in saliva and extracts of faeces were induced within 4 weeks after an OMV vaccine had been given at weekly intervals, whereas the antibody responses in these samples were negligible when given four times at 1-day or 1-h intervals, or as one large dose. Only modest responses were obtained in serum after 4 weeks, however, whether the vaccine had been given repeatedly at any schedule, including the 1-week interval, or as one dose. On the other hand, two large doses given 8 weeks apart induced booster antibody responses in both serum and secretions that matched the responses from a second series of the four smaller doses. Intranasal immunisations may thus stimulate immunological memory more rapidly in secretions than in serum. In order to secure adequate systemic responses by a minimum of doses, nasal vaccines should therefore be given at intervals longer than 4 weeks, in harmony with the intervals recommended for injectable vaccines.

Diagnosis of Militarily Relevant Diseases Using Oral Fluid and Saliva Antibodies: Fluorescence Polarization Immunoassay

This laboratory is developing fluorescence polarization (FP) methods as diagnostic tools to assay antibodies in saliva and other oral fluids. FP provides quantitation of molecular interaction, such as antigen-antibody binding, of a single, small-volume sample in real time and without prior separation of components such as blood cells. There is potential for wide-spread use of these homogeneous assays as noninvasive tests, especially as more compact, simplified fluorescence polarimeters become available. FP tests can be designed that are applicable to a wide spectrum of microorganisms and may be used in a clinic or far-forward deployed setting to aid in diagnosis of disease or verification of vaccination. Rapid salivary diagnostics, including FP, have been identified by the Office of Naval Research as requirements for future naval capabilities in basic and applied medical research for warfighter protection in casualty prevention. The applications of FP salivary diagnostics for determination of tuberculosis exposure and of anthrax immunization status are discussed as examples.

Comparative Detection of Measles Specific IgM Antibody in Serum and Saliva by an Antibody-Capture IgM Enzyme Immunoassay (EIA).

Laboratory diagnosis of acute measles is usually achieved by serology assays for measle-specific IgM antibody. For comparison of measle-specific IgM antibody in saliva and serum, 95 paired blood and saliva samples were collected 1-14 days after the onset of rash. The specimens were tested for specific IgM antibody by an IgM antibody-capture Enzyme Immunoassay (EIA). Measles IgM antibody was detected in 89 (93.7%) of serum samples and in 85(89.5%) of saliva specimens. Of the 6(6.3%) serum samples that were IgM antibody-negative, 2 (2.1%) of the paired saliva samples were IgM antibody-positive. The sensitivity and specificity of saliva testing compared with serum was 95.5% and 66.7% respectively. Positive predictive value (PPV) and negative predictive value (NPV) of saliva testing were 97.7% and 50.0% respectively and the accuracy of saliva testing was 93.7%. Our results indicate that saliva samples provided Enzyme Immunoassay results that were in good agreement with results from serum samples. Salivary IgM antibody detection is a suitable non-invasive method for diagnosing recent measles infections and epidemiological studies, especially in children.

Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis

In this study, we have assessed the efficacy of the 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis as a nasal vaccine for the prevention of adult periodontitis. Mice nasally immunized with 40k-OMP and cholera toxin as mucosal adjuvant displayed significant levels of 40k-OMP-specific serum IgG1, IgG2b and IgA as well as mucosal IgA antibodies (Abs) in saliva and nasal secretions. Ab-forming cell (AFC) analysis confirmed the antibody titers by detecting high numbers of 40k-OMP-specific AFCs in spleen, salivary glands and nasal passages. Because 40k-OMP-specific IgG inhibited coaggregation of P. gingivalis vesicles and S. gordonii, it may be an important tool for the prevention of adult periodontitis.

Towards saliva-based screening for sleeping sickness?

A pilot study was carried out on the detection of trypanosome-specific antibodies in saliva for diagnosis of sleeping sickness. All twenty-three saliva samples of parasitologically confirmed Trypanosoma brucei gambiense patients tested positive in an indirect enzyme-linked immunosorbent assay, whereas all 14 saliva samples of a negative control group remained negative. Trypanosome-specific antibody levels in patient saliva correlated with antibody levels in serum, but were about 250-fold lower. Eight of 23 undiluted saliva samples of sleeping sickness patients tested positive in CATT/T. b. gambiense and two of 23 in LATEX/T. b. gambiense. All fourteen saliva samples of the negative control group were also positive in CATT/T. b. gambiense, as were four of 14 in LATEX/T. b. gambiense. CATT and LATEX were thus inappropriate for antibody detection in saliva. These results indicate that trypanosome-specific antibody detection in saliva is possible. This could lead to the development of a simple, non-invasive, reliable saliva field test for diagnosis of sleeping sickness.

Early acquisition of serum and saliva antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins by infants living in an endemic area.

Enteropathogenic Escherichia coli (EPEC) is the most common etiological agent of acute diarrhea among infants living in poor social conditions in Brazil and other developing countries. This infection is rare in breast-fed infants, as well as in children older than 2 years. Over the past few years, our group has attempted to identify antibodies to EPEC virulence proteins in human milk and to establish the in vitro protective role of these antibodies. In the present study, we report the identification of antibodies to EPEC virulence proteins in sera and saliva from children of different ages, living in slums in the city of São Paulo, Brazil. Using EPEC and bacterial constructs (pET) for immunoblotting (IB) analysis, antibodies reacting to the main adhesins (intimin, bundle-forming pilli) and cell-signaling proteins (EPEC secreted proteins - Esp A, Esp B) were detected in sera from adults and children older than 1 year. Almost all children older than 1 year presented recognition patterns similar to those of adults in IB assays for serum IgG and secretory IgA antibodies, using EPEC outer membrane and other antigenic preparations. As previously observed for human milk, all samples from adults and older children recognized the 94 kDa molecular weight adhesin intimin strongly. In most children, previous EPEC symptomatic diarrhea could not be confirmed; however, almost all of them have presented one or more diarrhea episodes during their lifetime. These results suggest that reduction of EPEC infection frequency after 2 years of age may be associated with the development of anti-EPEC antibody repertoires.