A potential mechanism by which aspiration of duodenogastric fluid augments the risk for bronchiolitis obliterans syndrome after lung transplantation.

OBJECTIVE: Aspiration of duodenogastric refluxate may damage the respiratory epithelium of lung allografts in transplant recipients. We sought to define a mechanism by which aspiration of duodenogastric fluid augments the risk of bronchiolitis obliterans syndrome after lung transplant in a murine model. METHODS: We analyzed the immunological effects of acute aspiration of duodenogastric fluid (0.5 mL/kg) on transplant naive (strain DBA/2J) and transplanted mice (strain B6D2F1/J to strain DBA/2J). Serum antibodies to the lung self-antigens (SAgs) K-alpha1 tubulin and collagen-V were determined by enzyme-linked immunosorbent assay. Exosomes were isolated from serum, and immunoblot membranes were probed for antibodies to lung SAgs. Lung sections were assessed for fibrotic burden and obliterative bronchiolitis lesions by histologic and immunohistochemical analyses, including trichrome staining. RESULTS: Transplanted mice that received duodenogastric fluid developed higher levels of antibodies to the lung SAgs K-alpha1 tubulin and collagen-V and exosomes with lung SAgs on posttransplant days 14 and 28 than transplanted mice with sham aspiration or transplant naive mice (with and without aspiration). All lung allografts demonstrated severe grade A4 rejection on posttransplant day 14, with the highest mean fibrotic burden and mean number of obliterative bronchiolitis-like lesions per microscopic field on day 28 in recipients with aspiration. CONCLUSIONS: This study links aspiration of duodenogastric fluid after lung transplant to higher autoimmune responses to lung SAgs and the release of circulating exosomes with lung SAgs, which together promote sustained immune responses leading to extensive lung parenchymal damage and, ultimately, severe obliterative bronchiolitis-the histologic hallmark of bronchiolitis obliterans syndrome.

A comparative proteomic analysis of the soluble immune factor environment of rectal and oral mucosa.

OBJECTIVE: Sexual transmission of HIV occurs across a mucosal surface, which contains many soluble immune factors important for HIV immunity. Although the composition of mucosal fluids in the vaginal and oral compartments has been studied extensively, the knowledge of the expression of these factors in the rectal mucosa has been understudied and is very limited. This has particular relevance given that the highest rates of HIV acquisition occur via the rectal tract. To further our understanding of rectal mucosa, this study uses a proteomics approach to characterize immune factor components of rectal fluid, using saliva as a comparison, and evaluates its antiviral activity against HIV. METHODS: Paired salivary fluid (n = 10) and rectal lavage fluid (n = 10) samples were collected from healthy, HIV seronegative individuals. Samples were analyzed by label-free tandem mass spectrometry to comprehensively identify and quantify mucosal immune protein abundance differences between saliva and rectal fluids. The HIV inhibitory capacity of these fluids was further assessed using a TZM-bl reporter cell line. RESULTS: Of the 315 proteins identified in rectal lavage fluid, 72 had known immune functions, many of which have described anti-HIV activity, including cathelicidin, serpins, cystatins and antileukoproteinase. The majority of immune factors were similarly expressed between fluids, with only 21 differentially abundant (p<0.05, multiple comparison corrected). Notably, rectal mucosa had a high abundance of mucosal immunoglobulins and antiproteases relative to saliva, Rectal lavage limited HIV infection by 40-50% in vitro (p<0.05), which is lower than the potent anti-HIV effect of oral mucosal fluid (70-80% inhibition, p<0.005). CONCLUSIONS: This study reveals that rectal mucosa contains many innate immune factors important for host immunity to HIV and can limit viral replication in vitro. This indicates an important role for this fluid as the first line of defense against HIV.

Enhanced mucosal and systemic immune responses obtained by porous silica nanoparticles used as an oral vaccine adjuvant: effect of silica architecture on immunological properties.

Three different kinds of silica (S2, S1 and SBA-15) with different particle sizes (130, 430 nm and 1-2 µm) and different pore characteristics (i.e. pore size and shape) were developed as oral vaccine immunological adjuvants and the relationship between the silica architecture and immunological properties was investigated. The silica particles were characterized using SEM, TEM and nitrogen adsorption. Model antigen bovine serum albumin (BSA) was successfully entrapped into the silica pores to produce a sustained release vaccine delivery system. Compared with the responsiveness induced by parenteral administration of BSA emulsified in Freund's complete adjuvant (FCA), oral immunization with the silica/BSA formulation produced a stimulated humoral and mucosal (sIgA) response. The IgG and IgA titers induced by loading BSA was as follows: S1>S2>SBA-15. The highest IgG and IgA titers of S1 were attributed to its large honeycombed pores and the optimal particle diameter of 430 nm. The corresponding IgG1 and IgG2a titers were also investigated to confirm that BSA loaded in nanoparticles by oral immunization can induce both T-helper 1- and T-helper 2- (Th1 or Th2) mediated responses. We believe that the results of our research will open up new avenues for the formulation of oral vaccines.

Secretory IgA: arresting microbial pathogens at epithelial borders.

Secretory IgA (SIgA) is the predominant class of antibody found in intestinal secretions. Although SIgA's role in protecting the intestinal epithelium from the enteric pathogens and toxins has long been recognized, surprisingly little is known about the molecular mechanisms by which this is achieved. The present review summarizes the current understanding of how SIgA functions to prevent microbial pathogens and toxins from gaining access to the intestinal epithelium. We also discuss recent work from our laboratory examining the interaction of a particular protective monoclonal IgA with Salmonella and propose, based on this work, that SIgA has a previously unrecognized capacity to directly interfere with microbial virulence at mucosal surfaces.

Kinetic analysis of antibody responses to Blastocystis hominis in sera and intestinal secretions of orally infected mice.

The kinetics of antibody production against Blastocystis hominis, an emerging infectious protozoan parasite which causes intestinal disorder in humans and animals, was studied. Sera and intestinal secretions were collected from B. hominis-immunized Balb/C mice for 8 weeks. Flow cytometry was used to monitor the levels of immunoglobulins A (IgA), G (IgG), and M (IgM) from both types of biological samples. The kinetic profile derived from flow cytometry analysis revealed that IgM led the early immune action against B. hominis infection in immune sera while IgA was the predominant antibody isotype in intestinal secretions. Western blotting revealed an array of antigens recognized by both serum and intestinal secretion antibodies. Immunoreactive B. hominis soluble proteins with molecular weights ranging from 28.2 to 77.6 kDa were detected by serum antibodies and 15.1 to 117.5 kDa by secretory antibodies. These antigens may be cytoplasmic or membrane-bound as determined through indirect fluorescent antibody test. Moreover, two immunogens (39.8 and 77.6 kDa) were commonly recognized by serum antibodies, one (70.8 kDa) by secretory antibodies and two (55.0 and 56.2 kDa) by both serum and secretory antibodies, suggesting a possible target in the further understanding of B. hominis pathogenicity, discovery of virulence factors, and development of immunology-based diagnostic protocols and alternative modes of treatment.

Enhancement of ovalbumin-specific IgA responses via oral boosting with antigen co-administered with an aqueous Solanum torvum extract.

An experiment was conducted with the objective to enhance mucosal immunity against ovalbumin (OVA) by co-administration of OVA with an aqueous extract from the fruit of Solanum torvum (STE). Five groups of female ICR mice aged approximately 8 weeks at the commencement of the experiment were caged in groups of eight and received various treatments. The treatments included OVA alone, OVA with cholera toxin (CT), and OVA with various doses of STE. Mice were primed intraperitoneally with 500 microg of OVA alone or co-administered with 0.1 microg CT, or with 1 microg STE. All mice were boosted orally via gastric intubation 14 days after priming with 10 mg OVA alone, or co-administered with 10 microg CT or with 10 mg, 1 mg or 0.1 mg STE. One week later all mice were killed and organs obtained for analysis of the immune response. Intestinal, faecal and pulmonary OVA-specific sIgA concentration was significantly increased (p<0.05) in mice that received booster combinations of OVA/CT and OVA with all extract doses (p<0.05). Specific serum IgG titres did not differ significantly between groups. It is concluded that STE can significantly enhance secretory immunity in the intestine to OVA with mucosal homing to the lungs. The adjuvant effect of STE is comparable to that of CT.

Contribution of serum immunoglobulin transudate to the antibody immune status of murine intestinal secretions: influence of different sampling procedures.

Serum immunoglobulin transudation into the murine gut after intragastric immunization with the model antigen ovalbumin and cholera toxin adjuvant was investigated with regard to the mucosal sampling technique applied. The levels of serum-derived immunoglobulin A (IgA) turned out to be lowest in feces, intermediate in gut lavage fluid specimens, and highest in filter wick-collected samples. However, these levels did not exceed 2% of total and specific IgA in any mucosal sample type, except after the administration of very high antigen doses (> or =1 mg of antigen per g of body weight), when transudation rates of up to 31% could be measured in filter wick-collected samples from individual animals. Luminal IgG was plasma transudate and/or bile borne and appeared to be reabsorbed at the mucosa to some extent.

[Effect of rhubarb on intestinal immune associated secretion in healthy mice and in burn mice].

OBJECTIVE: To explore the therapeutic mechanism of rhubarb (sennosides) in protecting intestinal muco-membranous barrier by observing the change of intestinal immune associated secretion (IIAS) in mice before and after burn and the enhancing effect of rhubarb on it in healthy and in burn mice. METHODS: Bal b/c mice were divided into 4 groups. Group A (n = 8), untreated healthy mice; Group B (n = 11), healthy mice treated with 10% rhubarb decoction 12 ml/kg, once every 6 hrs; Group C (n = 7), the mice with 30% back full-thickness burn and no treatment was given; Group D (n = 9), the mice with 30% back full-thickness burn and treated with 10% rhubarb decoction 12 ml/kg, once every 6 hrs. The animals were killed 24 hrs after burn, the intestinal juice was collected after intestinal lavage, and centrifuged for determination of IgA, total protein, C3 and high density lipoprotein. RESULTS: IgA content in intestinal juice of Group A, B, C and D were 93.5 +/- 13.7 mg/L, 150.8 +/- 44.6 mg/L, 43.4 +/- 21.5 mg/L and 59.8 +/- 19.3 mg/L respectively, suggesting that it was significantly reduced by burn (P < 0.05), while rhubarb decoction could increase it in either healthy or burn mice (P < 0.05). The other immune associated substance, such as total protein, C3 and high density lipoprotein in intestinal juice were also increased significantly after rhubarb decoction treatment. CONCLUSION: Rhubarb could increase the reduced intestinal juice IgA content in mice caused by burn, it may be an important mechanism of rhubarb in protecting muco-membranous barrier.

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice.

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.

The relationship between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection.

Specific serum and intestinal immunoglobulin (Ig)G1 and IgA responses to Heligmosomoides polygyrus were measured in a panel of seven inbred mouse strains which exhibit 'rapid' (<6 weeks (SWRxSJL)F1), 'fast' (<8 weeks, SJL and SWR), 'intermediate' (10-20 weeks, NIH and BALB/c) or 'slow' (>25 weeks, C57BL/10 and CBA) resolution of primary infections. Mice with 'rapid', 'fast' or 'intermediate' response phenotypes produced greater serum and intestinal antibody responses than those with 'slow' phenotypes. The F1 hybrids ((SWRxSJL)F1) of two 'fast' responder strains showed the earliest antibody response with maximum titres evident within 6 weeks of infection. There was a negative correlation between the serum IgG1 responses and worm burdens in individual mice within a number of mouse strains, and also between serum IgG1 and IgA responses and worm burdens in the 'rapid' ((SWRxSJL)F1) responder strain. The presence of IgG1 in the gut was found to be due to local secretion rather than plasma leakage. Using Western immunoblotting, serum IgG1 from 'rapid' and 'fast' responder but not 'slow' responder mice was found to react with low molecular weight antigens (16-18 kDa) in adult worm excretory/secretory products.

Local and systemic complement activity in small intestinal bacterial overgrowth.

It is unknown whether bacteriolysis due to luminal complement activation contributes to local defense mechanisms against small intestinal bacterial overgrowth, particularly with gram-negative bacteria. This study addressed this issue. Thirty adult subjects were investigated with culture of luminal secretions adherent to proximal small intestinal mucosa. Luminal and plasma concentrations of C3 and C3d and C3d/C3 ratios were determined. Activated terminal complement complex was sought in surface epithelium to which aspirated secretions had been adherent. Small intestinal bacterial overgrowth with gram-negative bacteria was present in 12/30 (40.0%) subjects. C3, C3d, and C3d/C3 profile indicated that increased local but not systemic C3 activation occurs in this group. Conversely, no activation of terminal complement complex was evident in this circumstance. Thus, complement-mediated bacteriolysis is unlike to contribute to local defense mechanisms against small intestinal bacterial overgrowth, even when overgrowth flora includes gram-negative bacteria. Factors preventing full local activation of the complement cascade in this circumstance require investigation.

The expression of complement protein 4 and IgG3 in luminal secretions.

BACKGROUND: Factors regulating proximal small-intestinal luminal concentrations of IgG3, the predominant IgG subclass at this site, are unclear. This study determined whether luminal IgG3 concentrations are related to those of complement protein 4 (C4), an acute-phase reactant predominantly derived from local mucosa. METHODS: Proximal small-intestinal luminal and peripheral blood IgG subclass and C4 concentrations were measured by radial immunodiffusion in 30 adult subjects without predisposition to disturbed mucosal immunity. Mucosal C4 immunoreactivity and the presence or absence of small-intestinal bacterial overgrowth were determined in all subjects. Caecal luminal concentrations of IgG3 and C4 were measured in a separate cohort of eight asymptomatic subjects. RESULTS: Proximal small-intestinal luminal C4 and IgG subclass concentrations were not significantly influenced by the presence of absence of small-intestinal bacterial overgrowth (P > 0.2). Nor did plasma C4 levels significantly influence C4 concentrations in small-intestinal luminal secretions (P > 0.2). Mucosal immunoreactivity for C4 was present in every subject. A significant correlation was found between C4 and IgG3 concentrations in proximal small-intestinal luminal secretions (P < 0.0005) and also in caecal secretions (P < 0.05) but not in peripheral blood (P > 0.1). CONCLUSIONS: Common factors, not including the presence or absence of small-intestinal bacterial overgrowth, regulate luminal concentrations of C4 and IgG3. Local investigation is mandatory when assessing mucosal immune mechanisms.

Reversible effects on B and T cells of the gut-associated lymphoid tissues in rats malnourished during suckling: impaired induction of the immune response to intra-Peyer patches immunization with cholera toxin.

To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry. After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01). These alterations remained even after 3 weeks of refeeding with stock diet. The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur. At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus. When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery. When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001). These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids. When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low. When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values. These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.

IgM, IgA, IgG1 and IgG2 specific responses in blood and gut secretion of calves fed soyabean products.

Calves fed soya proteins may develop severe gastrointestinal disorders. Whether these are predominantly associated with particular Ig subclasses and (or) dietary proteins remains unclear. Therefore, antibody responses to soyabean protein were analysed by dot- and blot-immunobinding in plasma and intestinal mucous secretions. One-month-old calves were fed for 2.5 months liquid diets based on skim milk powder (SMP) or a mixture (2:3, protein basis) of whey and soyabean products including a low antigenic hydrolysed soya protein isolate (HSPI) and a highly antigenic heated soya flour (HSF). Specific antibodies (Abs) of the main isotypes (IgM, IgA, IgG1, IgG2) were characterised by immunostaining of samples which had been previously incubated with nitrocellulose sheets coated with SMP, HSPI or HSF extracts. Plasma collected before feeding experimental diets showed very little specific Abs. By contrast, 2.5 months later, a three-fold increase (P < 0.05) in IgG1 and IgA titres against HSF antigens was observed in calves fed HSF compared with those fed the control or HSPI diet. IgG1 immunoblotting revealed many protein bands from soya in the molecular range of 22-32 and 38-42 kDa. Immunorecognition of specific proteins from SMP and HSPI remained low and similar among animal groups. Specific IgM, IgA and IgG1 titres against HSF, and to a lesser extent HSPI, were significantly higher (P < 0.05) in jejunal mucous secretion of calves fed HSF compared with other groups. Secretions from calves fed HSF bound to many soyabean proteins in the range of 17-23 and 26-38 kDa, with similar patterns for IgA and IgG1. By contrast, only weak bands were found for IgM and IgG2 in all groups of calves. Thus, calves fed antigenic HSF do present specific Abs including IgG1 and IgA isotypes, both systemically and locally. Therefore, IgG1 and (or) IgA rather than IgM and IgG2 Abs may be preferred for assessing the immunogenicity of soyabean products in calves. Interestingly, soyabean immunogenicity was drastically reduced by adequate proteolysis.

Intestinal immune response induced in mice by staphylococcal enterotoxin B.

To investigate the induction of intestinal immunity to staphylococcal enterotoxins (SE) we have chosen the mouse as an experimental model. Since this species is devoid of emetic mechanism, SE can be administered orally without any loss. Mice were treated orally and/or parenterally with staphylococcal enterotoxin B (SEB). The anti-SEB response, either in serum or in the supernatant of in vitro cultured intestinal fragments was determined by enzyme immunoassay (EIA). The results showed that orally given SEB induced specific antibodies both in serum and intestinal secretions. Compared to oral route alone, parenteral followed by oral administration of SEB induced a higher intestinal response with IgA as predominant isotype. Although these results cannot directly be extrapolated to humans or animals with emetic reaction to SE, they do show the implication of intestinal immune system in response to this group of toxins.

Statistical analysis of clinical, immunological and nutritional factors in pediatric cryptosporidiosis in the Philippines.

A statistical analysis of clinical, nutritional, and immunological data gathered in a previous study suggest that nutritional factors, and in particular, iron status, appeared to be of significance in mounting an effective immune response to Cryptosporidium infection in young children. The primary protective mechanism seemed to be cell-mediated; humoral immunity was intact in all the study subjects, however, CMI was initially impaired but improved over six weeks.

Quantitation of salivary, urinary and faecal sIgA in children living in different conditions of antigenic exposure.

A sandwich-type ELISA was developed to quantify salivary, urinary and faecal secretory IgA (sIgA). The assay is based on binding of sIgA to microplates coated with anti-SC antibodies and reaction with peroxidase-labelled anti-IgA. The sensitivity of the technique was approximately 5 micrograms/L. Children, 1-6 years old (n = 142), were divided into two groups. Group 1 (n = 80) was composed of children living in a place with presumably low antigenic exposure conditions. Group 2 (n = 62) was composed of well-nourished (2A, n = 53) and malnourished children (2B, n = 9) living in a São Paulo slum with presumably high antigenic exposure. The subgroup 2A had salivary levels higher than group 1 and the ranges were similar to those found in the literature for older children and adults. The same subgroup presented a high incidence of undetectable faecal sIgA; their levels of urinary sIgA did not differ from group 1. The subgroup 2B did not have levels of salivary, urinary and faecal sIgA different from subgroup 2A. Our results suggest that environmental factors influence the ontogenesis of sIgA system.

Immunosenescence and mucosal immunity: significant effects of old age on secretory IgA concentrations and intraepithelial lymphocyte counts.

Concentrations of immunoglobulins (Ig) and levels of isotype specific antibodies to three dietary antigens in serum, pure parotid saliva, and in intestinal secretions obtained by whole gut lavage from groups of healthy elderly subjects (aged greater than 70 years) and of younger adult controls (aged 25-50 years) were measured. In addition, counts of lamina propria and intra-epithelial lymphoid cells were performed in histologically normal jejunal biopsy specimens from elderly and younger subjects. Elderly subjects had significantly higher concentrations of serum and salivary IgA and of salivary IgM (both, p less than 0.01), and of salivary IgA antibodies than did the younger subjects, but the amount of immunoglobulin and antibody in whole gut lavage fluid was similar in the two age groups. Jejunal biopsy specimen cell counts showed higher IgA plasma cell counts and lower intraepithelial lymphocyte counts in the elderly group (p less than 0.01), with similar counts of IgM and IgG plasma cells, eosinophils, and mast cells in the two groups. There is evidence of significant effects of old age on the mucosal immune system.

Use of a pilocarpine-based lavage procedure to study secretory immunoglobulin concentration in the alimentary tract of White Leghorn chickens.

A lavage procedure was used to study the kinetics of alimentary fluid IgA concentration in 15 specific-pathogen-free white leghorn chickens for 8 weeks post-hatch. Lavage solution was administered orally and collected from the distal alimentary tract following an intraperitoneal injection of pilocarpine. Concentrations of IgA, quantitated by enzyme-linked immunosorbent assay, were more than 0.04 mg/ml by 3 weeks and were negligible before this age. This level gradually increased over the next 5 weeks, peaking at nearly 0.4 mg/ml at 8 weeks of age. Alimentary lavage was easy to perform, required no necropsy or surgical manipulation, and facilitated repeated collection of alimentary fluid from live birds. Repeated lavage did not alter concentrations of IgA and IgG in alimentary fluid, and concentrations of IgA and IgG in alimentary fluid were stable during incubation at 37 C for 24-48 hr.