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.

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.

Secretory epithelial cell marker on gastrointestinal tumors and in human secretions defined by a monoclonal antibody.

A new marker for human secretory epithelial cell types (Exo-1) has been defined by a mouse monoclonal antibody (Pa-G14). The antibody was raised against a human exocrine pancreatic tumor cell line (Capan-1) and tested against 46 cultured human cell types and 228 freshly frozen human tissue sections. It reacted specifically with 28 normal and 55 secretory neoplastic epithelial tissues tested. Eleven different secretory epithelial cell types expressed this antigen, as well as human fetal tissues of the gut and bronchi. One hundred and twenty samples of normal tissues, cells, and tumors of nonexocrine origin were Exo-1 negative. In normal secretory tissues staining was most pronounced at the apical poles and as shown by immunoelectron microscopy in the case of the duodenum, at the microvilli. In cultured Exo-1 positive tumor cells the antigen was not demonstrable on the cell surface but in the cytoplasm after acetone/methanol fixation only. The antigen was identified biochemically as a polar neutral glycolipid and detected in human salivary, bronchial, pancreatic, and intestinal secretions by an enzyme-linked immunosorbent assay, but was not found in sera of healthy controls or patients with gastrointestinal and other tumors. Antigen Exo-1 represents a novel common antigen for normal and tumorous glandular epithelial cells.

A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions.

Mouse intestinal secretions can be readily obtained without harm to the mice by administering a lavage solution to them intragastrically followed by pilocarpine intraperitoneally. These secretions are rich in proteases but this enzyme activity can be blocked by addition of a mixture of inhibitors. Both total and specific IgA antibody could be measured in these secretions using ELISA techniques. The total IgA recovered was found to vary considerably, even in the same group of mice sampled on multiple occasions. Specific IgA anti-cholera toxin antibody was easily demonstrable in the intestinal secretions of mice fed cholera toxin but not of mice fed an irrelevant antigen. Expression of the specific IgA antibody per unit of total IgA recovered is desirable in order to correct for the variable recovery of IgA.

An overview of intestinal immunity and malabsorption.

Intestinal immune responses are adapted to function at external mucosal surfaces. Specialized forms of antibody, secretory immunoglobulin A (IgA) and immunoglobulin M (IgM), provid humoral immunity but little is known of local cell mediated immune reactions. Antigens in the intestinal lumen gain preferential access via Peyer's patches in which sensitised lymphocytes proliferate before entering the lymphatic system. These lymphoblasts return to the intestinal mucosa via the bloodstream to provide predominantly IgA antibody responses. Secretory IgA antibody can neutralize viruses, bacteria and toxins, and appears to block the entry of some food antigens into the lamina propria. Disturbances of intestinal immunity may result in malabsorption. Immunodeficiency states are often associated with malabsorption due to Giardia lamblia infestation. In alpha chain disease there is a malignant expansion of plasma cells in the intestinal mucosa which secrete an abnormal heavy chain fragment of IgA. Arthus type hypersensitivity reactions to milk proteins and gluten may contribute to the mucosal injury in patients suffering from milk allergy and coeliac disease.

Cancer associated antigen CA19-9 in colonic effluent of patients with neoplasia of the colon and inflammatory bowel disease.

We measured colonic effluent samples from 10 patients with colorectal cancer, 13 with adenomatous polyps, 14 with normal colons and compared them to 10 patients with inflammatory bowel disease by measuring this CA19-9 content. Results showed considerable overlap between the different pathologic categories, making differentiation impossible. A lower level of CA19-9 in the effluent samples from patients with adenomas was noted. These differences were reproducible for assays performed several months apart. CA19-9 may originate from the upper gastrointestinal tract since large amounts are present in pancreatico-biliary secretions. This antigen is therefore not useful in the diagnosis of neoplasia or inflammatory bowel disease using colonic effluent samples as the test material.

A simple assay of intrinsic factor-vitamin B12 complex employing the binding intrinsic factor antibody.

The reaction between binding intrinsic factor antibody and intrinsic factor-vitamin B(12) complex has been studied. Initially in the zone of antibody excess, the relationship between the amount of antigen present and the amount of antigen-antibody complex adsorbed onto zirconium phosphate gel was linear. With increasing amounts of antigen, the curve departed from linearity and reached a plateau. The linear portion of the reaction forms the basis of a simple and reproducible assay for quantitating intrinsic factor to which vitamin B(12) has already been bound. The assay provides a method for studying the fate of intrinsic factor-vitamin B(12) complex during digestion and absorption. In two normal subjects given radioactive vitamin B(12) orally, aspiration of ileal contents showed that only 50 to 70% of the radioactivity was bound to intrinsic factor at that level.

Mucosal immunity.

Mucosal defense is provided by a number of host factors countering the specific virulence factors of the many microorganisms infecting the mucous membranes. Secretory IgA antibodies presumably play an important role. Increase of the sIgA antibodies may most advantageously be attained by parenteral immunization, following mucosal priming. This was demonstrated in a rat model, where it was also noted that antigen injection into PP induced high milk IgA antibody levels. In man, parenteral vaccination against polio increased the sIgA antibody levels in the milk of mothers previously exposed naturally to the poliovirus. The response was relatively short-lived. In the previously unexposed, there was little or no response. By contrast peroral immunization with live poliovirus vaccine did not increase, or even decrease, the milk sIgA poliovirus antibody levels. Although salivary sIgA antibodies against antigens of colonizing E. coli appear during the first days of life, they are slow to increase. This deficiency is richly compensated for by all the sIgA antibodies that are provided the baby through the milk. No transfer of dimeric IgA into the milk could be shown in lactating rats, in contrast to what has been reported in mice. There is no evidence for a contribution to milk sIgA from serum in man. Close to parturition, human milk often contains some 7S IgA and various sizes of free SC, in addition to the dominating 11S sIgA. A few days later there is almost exclusively monomeric SC and 11S sIgA. IgG antibodies also play a role at the mucosal level. IgG2 antibodies against the bacterial polysaccharide capsule are as slow to appear as sIgA in ontogeny, possibly explaining the prevalence of infections with encapsulated bacteria and the poor response to polysaccharide vaccines in early childhood. Other defense factors preventing infections by way of mucous membranes may be important. Thus, oligosaccharides present in human milk seem to specifically prevent pneumococcal attachment to retropharyngeal cells. This anti-attachment capacity, in addition to that provided by milk and salivary IgA antibodies, may explain why breast-fed babies have less otitis media than formula-fed ones.

Immunity of cholera in man: relative role of antibacterial versus antitoxic immunity.

Purified cholera toxoid is antigenic when given enterally and orally. Purified toxoid fails to provide protection against experimental challenge. Clinical cholera confers formidable protection against homologous or heterologous rechallenge. Failure to culture vibrios from intestinal fluid or stool of re-challenge volunteers suggests that the predominant immune mechanism is antibacterial rather than antitoxic.

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.

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.

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.

Relationship of histamine in tissues and antiparasitic substances in gastrointestinal mucus to the development of resistance to trichostrongyle infections in young sheep.

The development of resistance to nematode infection and self-cure in a flock of young grazing sheep were examined in relation to changes in the levels of histamine in tissues and levels of antiparasitic substances in gastrointestinal mucus. Analysis of faecal egg counts showed that when the sheep were ranked according to individual mean monthly egg counts there was a significant trend to similar rankings in successive months. Sheep with high and low egg counts were slaughtered at monthly intervals for examination and comparison. Histamine levels in blood and intestinal content fluids were similar in both groups of sheep and were highest during maximum challenge by larval nematodes. Antiparasite activity of the intestinal mucus was significantly higher in sheep with low egg counts than those with high counts, between January and May, and was associated with significantly lower burdens of fourth stage larvae.

Isolation of turkey immunoglobulin-A.

Trukey biliary and gut IgA were isolated and monospecific antisera were prepared in rabbits. Isolation was accomplished by filtration of precipitated globulins on Sephadex G-200 followed by step-wise-elution DEAE ion-exchange chromatography. Using an immunodiffusion procedure, IgA was detected in turkey serum, saliva, lacrimal secretions, bile, gut washings, and tracheal washings. No IgA was detected in hatching-poult serum, egg white, or yolk. Two forms of IgA (high and low molecular weight) were detected in different body fluids.

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.

The effect of oral immunisation with heat stable Escherichia coli antigens on the sensitivity of pigs to enterotoxins.

It was found that oral immunisation of pigs with heat stable Escherichia coli antigens resulted in a decrease in the sensitivity of the porcine intestine to both the heat stable (ST) and the heat labile (LT) and the heat labile (LT) form of the enterotoxin produced by enterotoxigenic strains. Antitoxic factors capable of neutralising LT, but not ST, could be passively transferred in the intestinal secretions of the immunised animals.

Quantitation of the immunoglobulins in reproductive tract secretions of the mare.

IgG, IgA, IgM and albumin concentrations were measured in serum, follicular fluid and oviductal, uterine and intestinal secretions of the horse. Follicular protein concentrations were found to be dependent on serum concentration and molecular size. Of the immunoglobulins only IgG was detectable in oviductal secretions, but IgG:albumin ratios did not differ significantly from those in serum. IgG, IgA and IgM were measured in uterine secretions, with IgG predominant. Serum transudation into uterine secretions was minimal. In intestinal secretions, IgA levels were slightly higher than IgG, with albumin and IgM at low levels. In five mares with histories of chronic metritis, IgG, IgA and albumin concentrations were significantly elevated in uterine secretions.

Secretory antibodies against turkey coronaviral enteritis.

Studies on local immunity to transmissible coronaviral enteritis of turkeys (bluecomb) was made. Intestinal secretions and bile from affected birds contained secretory immunoglobulins against coronaviral antigen throughout the 6 months' duration of the experiment. Attempts to purify and to characterize the globulins in intestinal secretions and bile of the affected birds were made, using the techniques of gel filtration, DEAE chromatography, and immunoelectrophoresis. Class-specific anti-turkey IgA antiserum in the agar gel precipitin test further established the presence of IgA in the intestinal secretions and bile.

Humoral immunity in the pig.

The newborn pig relies on colostrum as its sole source for serum antibody and milk for its intestinal antibody during most of the post natal period. Colostrum and milk are well adapted to perform their very different immune functions--immunoglobulin in colostrum being derived from serum, whereas milk antibodies are locally produced in the mammary gland and mirror the immunoglobulin profile of adult intestinal juice. Intramammary vaccination is far superior to intramuscular vaccination because it produces not only a local but a systemic response. Oral vaccination is similarly effective. Vaccination of one mammary gland results in antibody activity in the secretion of all glands. Irrespective of the route of vaccination, antibody activity is found in all immunoglobulin classes. The main site of immunoglobulin-containing cells is the lamina propria of the intestinal tract, suggesting that the gut is a major site of immunoglobulin formation. In the piglet, immunoglobulin producing cells first appear in the gut at the end of the first week of life and reach a mature profile after a month. During this period the piglet is likely to be capable of responding to orally presented antigens.