The in vitro transfer of bovine immune lactoglobulin across the intestine of new-born pigs

1. Everted sacs of pig intestine, used soon after birth, maintained transmural potentials and transferred water and glucose to the serosal surface.2. Immune globulin, fed as bovine colostrum to the new-born pig, appeared in the serosal fluid of everted sacs during incubation in bicarbonate saline. The particular segment showing maximum transferring ability varied between limits and appeared to depend on the amount or concentration of colostrum fed to the pig. Sacs from unfed pigs incubated in bovine colostrum also transferred colostral IgG to the serosal fluid. This transfer was dependent on the concentration of colostral IgG in the incubation medium and became more pronounced in the middle third of the small intestine.3. Human serum albumin inhibited the transfer of colostral IgG and about twenty molecules of albumin were transferred for every molecule of colostral IgG, when both were presented together in equal concentration on the basis of weight, to the middle segment of the small intestine.4. Some of the immune globulin collected in vitro after feeding bovine colostrum was found in a degraded form, but the amounts present could not be estimated. There was no apparent degradation of immune globulin in the purely in vitro experiments.5. The in vitro transfer of bovine colostral IgG showed selectivity between molecules of albumin and colostral IgG, the nature of which warrants further study.     

The absorption of polyvinyl pyrrolidone by the new-born pig intestine

1. The intestinal absorption of [(131)I]polyvinyl pyrrolidone of mean mol. wt. 160,000 (K. 60) and 40,000 (K. 30) after oral administration has been measured in unsuckled conscious pigs less than 20 hr old. Absorption was assessed by the measurement of the concentration of [(131)I]PVP in venous blood during the 6 hr after feeding and also by the distribution at the end of the experiment of [(131)I]PVP between homogenates of the alimentary tract and homogenates of the rest of the animal.2. The concentration of [(131)I]PVP in the peripheral blood after feeding was dependent upon the mol. wt. of the polymer, when comparable amounts had been absorbed from the intestine. PVP K. 60 attained higher blood concentrations than PVP K. 30 and the blood concentrations of PVP K. 60 were close to the values to be expected if all the material which had left the intestine had remained in the blood. The lower blood concentrations found when PVP K. 30 was fed were associated with the disappearance of labelled solute from the gut and were thus the consequence of the relatively rapid escape of labelled solute from the plasma after absorption had taken place.3. The ability of the intestine to absorb [(131)I]PVP K. 60 declined progressively after birth but did not terminate abruptly unless the animal was fed colostrum. In unsuckled animals the rate and extent of absorption at 3 hr was much greater than at 20 hr after birth, but some absorption was still present at least 65 hr after birth.4. The transfer of PVP K. 60 to the peripheral blood was dependent upon factors in sow colostrum, since significant absorption did not occur when PVP was fed in water or simple salt solutions.5. The factors which accelerated absorption were present in colostrum from the goat, cow and ewe as well as that from the sow; they remained in the whey, but, in contrast to the factors which accelerate absorption in the calf, were largely inactivated by boiling. Similarly, neither phosphate, lactate, pyruvate, nor lower volatile fatty acid salts, which were effective in the calf, accelerated absorption in the pig.6. The absorption of [(131)I]PVP K. 30 was found to be much less dependent upon the composition of the solvent than the absorption of [(131)I]PVP K. 60, although absorption was most rapid when PVP K. 30 was fed in colostrum.     

Interdependence of albumin and sodium transport in the foetal and new-born pig intestine

1. Everted sacs of new-born pig intestines incubated in bicarbonate saline at 37 degrees C, transferred bovine plasma albumin across the mucosa into fluid bathing the serosa, the amount transferred increasing as the concentration of albumin in the mucosal fluid was raised from 0.5 to 16 g/100 ml.2. The rate of albumin transfer across the foetal pig intestine showed an apparent maximum, about 400 mug/g intestine/hr, 2 weeks before birth. The transfer at birth, about 200 mug/g intestine/hr, fell sharply during the next 2 days but later returned to that previously found at birth.3. When sacs were prepared from the intestines of 1 to 7-day-old pigs part of the recovered albumin was degraded. No digestion was found when the intestines of new-born or foetal pigs were used.4. The transfer of water and sodium, but not glucose, measured across the foetal and new-born pig intestine, was consistently higher when albumin was present in the mucosal fluid: the transmural potential difference was lowered by the presence of albumin. These differences disappeared during the first 2 days of life.5. Both the total and ouabain-sensitive adenosine triphosphatase (ATPase) activities of the pig intestinal epithelium fell within 24 hr of birth. There was some increase in total ATPase activity in older pigs but the ouabain-sensitive activity remained low.6. The relation between albumin and sodium transport, seen at a time when albumin is not being metabolized, suggests that the transfers are closely coupled. The movement of sodium into a mucosal cell down its own concentration gradient may provide energy for the translocation of albumin.     

Immunochemistry of serum albumin. X. Five major antigenic sites of human serum albumin are extrapolated from bovine albumin and confirmed by synthetic peptides

In a recent report from this laboratory, we have predicted and confirmed by synthesis the locations of five major antigenic sites of bovine serum albumin. In view of the high structural similarity between bovine and human serum albumins, analogous regions of human serum albumin are predicted here to comprise antigenic sites in this protein. Inimunochemlcal studies with antlsera to human albumin and the synthetic antigenic sites of bovine albumin verified this prediction and also identified the major structural locations responsible for the immunochemical cross-reaction between these two albumins.     

Delayed type hypersensitivity to bovine serum albumin and to lipid-conjugated bovine serum albumin in mice.

Delayed type hypersensitivity (DTH) to bovine serum albumin (BSA) and to lipid-conjugated BSA were studied comparatively. Unlike the case of BSA with which no DTH can be detected with native antigen, injection of butyric-conjugated BSA (Bu-BSA) in sensitized mice provokes a typical DTH for an early and limited period. Alum-precipitated Bu-BSA (Al-Bu-BSA) provokes from the beginning a stronger DTH which persists a much longer period.     

Bicarbonate and bovine serum albumin reversibly 'switch' capacitation-induced events in human spermatozoa

We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle's balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its 'uncapacitated' size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.     

Tissue deposition of immune complexes in mice receiving daily injections of bovine serum albumin

The daily intraperitoneal injection of 0.3 mg of 0.5 mg BSA into preimmunized mice produced chronic serum sickness (CSS) within several weeks. The glomerulonephritis which developed was characterized in most cases (74%) by the deposition of immune complexes in the glomerular capillary wall. Associated pathological changes included crescent formation, hypercellularity, capillary occlusion and exudative and degenerative lesions in the glomeruli. In other animals (26%) a less severe renal disease developed in which immune complex deposition and histological abnormalities were limited to the glomerular mesangium. Mice with membranoproliferative immune complex glomerulonephritis had deposits of immune complexes in many other organs besides the kidney. A model of CSS in mice opens the possibility of studying the cellular basis of the immune response and genetic determinants in experimentally induced systemic immune complex disease.     

Immune recognition of serum albumin—XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin

Recently, this laboratory reported an autoreactivity of myoglobin (Mb)3 antisera with the Mb of the species in which the antisera are raised. Also, animals injected with autologous Mb mounted an autoimmune antibody and T-lymphocyte proliferative response against this protein. This posed the possibility that autoimmune recognition might be a general phenomenon not confined only to sequestered proteins such as Mb. Using RSA, we have demonstrated unambiguously that RSA cross-reacted with rabbit antisera to bovine (BSA) or to human (HSA) albumin. In exchange experiments, ¹²⁵I-labelled RSA was bound by the IgG in the immune complexes isolated from rabbit antisera to BSA or HSA. Also, ¹²⁵I-antibodies were bound by RSA-adsorbents. Both binding activities were inhibited specifically by RSA. RSA was isolated from three rabbits and each rabbit was immunized with its own RSA. In each rabbit autoantibodies were found by exchange between immune complexes and the rabbit's own [¹²⁵I]-RSA. Also, ¹²⁵I-antibodies from each rabbit were bound by adsorbents of the rabbit's own RSA. Inhibition studies, data on preimmune sera and on antisera against other proteins confirmed the specificity of the binding. The findings confirm the universality of autoimmune recognition and lend support to our previous suggestion that antigenic sites are ‘structurally-inherent’ in the protein.     

The kinetics of immune tolerance to human serum albumin induced in sublethally X-irradiated rabbits

Kinetic studies of immune tolerance induction to HSA in sublethally X-rayed rabbits revealed that tolerance can be induced in this system with small amounts of antigen which in non-irradiated animals would constitute small immunizing doses. This, however, depends on a proper schedule of antigen administration which has to be spread over the 8 weeks of post-radiation recovery, although tolerance can be induced by this method even when this antigen treatment is postponed as long as 4 weeks after X-irradiation.

Since the lymphoid system recovers rapidly from sublethal radiation injury, and since it was found that by the end of 4 weeks the lymphoid organs demonstrate good cellular repopulation, the conclusion was arrived at that the two processes, namely that of lymphoid recuperation and that of transition from susceptibility to tolerance into the state of immunocompetence, progress independently. These observations raise some questions relevant to theories of the cellular basis of immune tolerance.

     

The interaction of anti-glomerular basement membrane antibody deposition with immune elimination of bovine serum albumin in the rabbit.

We studied the interaction of two different forms of immune glomerular damage occurring simultaneously: anti-glomerular basement membrane (GBM) antibody fixation and immune elimination of bovine serum albumin (BSA). 125I-radiolabelled BSA anti-BSA immune complexes, formed in response to a single small intravenous dose (150 mg/kg) of 125I BSA, did not cause proteinuria in control animals within 15 days, despite evidence of immune elimination of the antigen. Similarly, a small dose of nephrotoxic globulin (NTG)(3.0 mg/kg) did not cause immediate proteinuria in controls. Test animals received the BSA injection followed by the NTG injection 5, 7 or 9 days later. In this way, antibody fixed to glomerular basement membrane antigens at various times after BSA anti-BSA complexes first appeared in the circulation. Animals were killed on day 15. Fifteen of the eighteen test animals developed moderate to severe clinical nephritis. The onset of the nephritis coincided with BSA elimination irrespective of when the NTG was given. Greatly increased amounts of nonlinear immunofluorescent deposits were demonstrated in the glomeruli of test animals. We concluded that there was a marked synergistic effect between two forms of immune glomerular damage (i.e. that mediated by anti-GBM antibody and immune complexes), which appeared to be due to the increased deposition of complex material in the presence of active fixation of anti-GBM antibody. The relevance of this finding to human glomerulonephritis is discussed.     

Detection of bovine serum albumin in the circulating IgA immune complexes of patients with IgA nephropathy

Alimentary antigenic challenge has been postulated to have a role in the genesis of IgA circulating immune complexes (CIC), resulting in mesangial IgA disease. In this study, we examined the relationship between bovine serum albumin (BSA) and IgA CIC in patients with IgA nephropathy. Of the 47 patients studied, elevated IgA CIC levels were found in 32% by the F(ab')2 anti-C3 and Raji cell enzyme immunoassays (EIA). Elevated IgA anti-BSA antibody levels were found in 9 patients, and there was a positive correlation between these levels and IgA CIC as measured in the Raji cell EIA (R = 0.60, P less than 0.001). In 4 patients with elevation of both IgA CIC and IgA anti-BSA antibody levels, solubilization experiments were done to demonstrate the presence of BSA antigen in the IgA CIC. Using the Raji cell EIA, the IgA CIC levels decreased significantly after preincubating the sera with serial concentrations of excess BSA. No corresponding effect was seen with human serum albumin used as control. Hence, BSA may be the antigenic stimulus in the formation of IgA CIC in selected patients with IgA nephropathy. The pathogenic capacity of these IgA-BSA CIC remains to be determined.     

Occupational asthma caused by inhalation of bovine serum albumin powder

Bovine serum albumin (BSA), which is present in bovine plasma, is one of the major allergens affecting patients with food allergies induced by milk and meat. It is also commonly used in research laboratories. Although some reports have documented food allergies associated with BSA, BSA-induced occupational asthma has not been reported. We report a case of occupational asthma and rhinitis in a laboratory worker caused by the inhalation of BSA powder, in which an IgE-mediated response was suggested as the pathogenic mechanism.