Comparison of the arthritogenic properties of dietary cow's milk, egg albumin and soya milk in experimental animals

The arthritogenic effect of dietary cow's milk, egg albumin and soya milk were compared in Old English rabbits. The 12-week cow's milk feeding regimen produced the highest incidence of significant joint lesions. Lesions were evident but mild at 5 weeks and the synovitis had resolved by 32 weeks. It is suggested that the transient nature of the synovitis may be due to the development of specific secretory IgA antibodies which were detectable in faecal pellet extracts. Sandy Lop rabbits were less susceptible to the arthritogenic effect than were Old English rabbits. Dietary ovalbumin was less arthritogenic than cow's milk despite high titres of serum and synovial fluid antibodies and immune complexes. The rabbits were 'tolerant' to dietary soya due probably to pre-existing levels of soya protein in their diet. Lewis and Wistar strain rats, CBA, Balb/c and C57/BL6 mice fed on cow's milk for 3 months did not develop serum antibodies or synovial lesions. It is suggested that this allergic synovitis is not a model for early rheumatoid joint disease because of the transience of the lesions and lack of stimulation of rheumatoid factor. It may well, however, be a model for the arthralgia seen in patients with certain food allergies.     

Early rheumatoid-like synovial lesions in rabbits drinking cow's milk. I. Joint pathology

Rheumatoid-like lesions developed in 9 of 25 (36%) Old English rabbits drinking cow's milk for 12 weeks. The incidence of lesions in male and female animals was similar. The majority of rabbits drinking cow's milk developed increased numbers of nucleated cells and raised percentages of T lymphocytes in their synovial fluids, compared to control rabbits. The cell counts and T cell percentages correlated with the severity of the histological lesions. No evidence of glomerulonephritis was observed in any of the experimental animals.     

Effect of cow's milk protein absorption on the anaphylactic and systemic immune responses of young rabbits during bacterial diarrhoea.

In vivo and in vitro studies recently showed that intestinal permeability to cow's milk proteins such as beta-lactoglobulin increased transiently in infant rabbits infected by the enteroadherent Escherichia coli strain RDEC-1 at weaning. The consequences of this enhanced permeability for local anaphylactic and/or systemic immune responses were studied in infected and age-matched control rabbits at weaning, given either water or cow's milk diluted in water in addition to their solid diet. The systemic immune response was determined by measuring IgG, IgA, IgM, and IgE antibodies to cow's milk proteins. A rise in IgG and IgM, but not IgA or IgE, antibody titres to milk protein was observed. Further, infected milk-drinking rabbits had significantly higher beta-lactoglobulin and casein IgG titres than milk-drinking controls. The anaphylactic response was tested by mounting ileal segments in Ussing chambers, adding the milk proteins to the serosal side of the tissue and recording the variation in short-circuit current in order to detect any electrogenic chloride secretion. No change in short-circuit current was observed in the presence of milk proteins in infected or age-matched control milk-drinking rabbits, indicating the absence of any immediate hypersensitivity reaction. These results indicate that at weaning, the rabbit is not prone to developing anaphylactic responses to milk proteins. The rise in milk protein IgG antibodies that followed infection with RDEC-1 might favour the elimination of milk protein-IgG immune complexes from the systemic circulation. The present results emphasize the evidence that genetic background and/or animal species greatly influence the immune response to an antigenic load.     

The Level of Antibodies to the Proteins of Cow's Milk in the Serum of Normal Human Infants

The coated tanned red cell technique has been used to measure the level of serum antibody in normal infants to the proteins of cow's milk.

In 286 sera of infants between the ages of 7 and 97 weeks titres up to 1000 were found with the mode around 64. This distribution was quite different from that found with adult and infant cord sera. Antibodies specific for casein, α-lactalbumin and bovine plasma albumin were shown to be present but none could be shown to β-lactoglobulin.

The object of the study was to get evidence that some normal infants were sufficiently sensitized to cow's milk proteins to support the hypothesis that sudden `cot death' in infants is due to an anaphylactic type of reaction consequent on inhalation of cow's milk.

     

An enzyme-linked immunosorbent assay for cow's milk protein-specific IgE using biotinylated antigen Avoidance of interference by specific IgG

Detection of specific IgE by the radioallergosorbent test (RAST) which uses labelled antibody can be hampered by the presence of antibodies other than IgE but with the same specificity and may limit usefulness of the RAST for diagnosis of IgE-mediated milk allergy in infancy when high titres of cow's milk protein-specific IgG antibodies are known to be present. This can be avoided by using a system employing labelled antigen, such as the enzyme-linked immunosorbent assay (ELISA) described here, where IgE in the test serum is immunoadsorbed to anti-human IgE coated to microtitre plates. Biotinylated antigen, in this case cow's milk proteins, binds to specific IgE and the reaction is revealed colorimetrically by adding horseradish peroxidase (HRP)-avidin conjugate.     

Occurrence of specific precipitins against bovine whey proteins in serum from children with gastrointestinal disorders

Children with cow's milk intolerance, clinically non-confirmed cow's milk intolerance, coeliac disease, non-confirmed malabsorption, other gastrointestinal disorders and normal children were investigated for the presence of precipitins against 40 individual bovine whey proteins by means of the crossed immunoelectrophoresis with intermediate gel technique. In the various groups of children the amount and specificity of bovine whey precipitins were neither connected with the various gastrointestinal disorders nor with alternating diet and challenge with milk. Bovine whey-specific IgE could not be detected in any of the patients clinically suspected for cow's milk intolerance by cutaneous test, RAST or CRIE. The immunoglobulin level of the patients investigated did not differ significantly from the normal ranges. It is concluded that investigations of serum precipitins against bovine whey proteins do not give any significant information concerning cow's milk intolerance.     

Cow's milk protein-specific IgE concentrations in two age groups of milk-allergic children and in children achieving clinical tolerance

BACKGROUND: IgE-mediated cow's milk protein allergy (CMPA) is usually outgrown in children by the age of 3 years. The immunological responses to cow's milk proteins in children who achieve tolerance, in comparison with those who remain allergic, however, are not well described. OBJECTIVE: To compare the level of cow's milk protein-specific IgE among children with documented CMPA under the age of 3 years, another group over the age of 9 years (persistent allergy), and in another group of children in whom clinical tolerance developed. METHODS: Stored sera from children with CMPA were analysed for IgE antibodies specific for whole cow's milk, casein, whey, alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and bovine serum albumin (BSA) using the Pharmacia CAP System FEIA. RESULTS: Within each group of CMPA children, the concentration of specific IgE antibody to casein proteins was not significantly different from that to whey proteins. However, children in the group with CMPA over 9 years of age had significantly greater concentrations of whole milk (P = 0.02) and casein-specific (P = 0. 04) IgE antibodies compared with the group of children with CMPA under age 3 years. Children under the age of 3 years had a higher median concentration of casein-specific IgE (20.2 vs. 5.5, P = 0.04) than another group of 11 children (mean age 3.5 years), who later lost their milk allergy. Out of 16 children who lost sensitivity to milk, 75% had milk-specific IgE levels below 14.3 kUA/L (median, 1.3 kUA/L). CONCLUSIONS: Although a dominant allergenic milk protein fraction was not identified within either of the two age groups, those with persistent CMPA over age 9 years had significantly elevated levels of milk and casein-specific IgE compared with younger children with CMPA.     

Cow's milk allergy in an adult patient

A 22-year-old female patient is described who was repeatedly hospitalised on account of severe asthmatic attacks presumably due to the ingestion of cow's milk or milk-containing products. There were no signs of gastrointestinal disturbance, but some urticaria and angioedema occurred. Strongly positive RASTs were observed in the blood serum against the proteins in cow's milk, bovine serum, egg white, cod fish, and house dust. The symptoms were successfully controlled by rigorous dietary measures.     

Total IgE, specific antibodies to cow's milk proteins, beta-lactoglobulin and eczema in one month infants

Total IgE, specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin were measured in 32 term and 23 premature infants. 1) The term infants who developed eczema till one month had significantly high specific IgG titers to whole cow's milk and beta-lactoglobulin in cord blood serum. It is concluded that specific IgG antibody to whole cow's milk and beta-lactoglobulin in cord blood serum have predictive value for the development of eczema till one month. 2) Some of mixed feeding premature infants produced specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin till one month. These infants produced various kind of specific antibodies to whole cow's milk and beta-lactoglobulin. 3) The premature infants who developed eczema at one month had significantly high specific IgA.IgG titers to cow's milk and high specific IgA titers to beta-lactoglobulin in serum at one month. These infants had a tendency to show high total IgE value and high specific IgM titers to cow's milk and high specific IgG.IgM titers to beta-lactoglobulin. It is concluded that specific antibodies to whole cow's milk and beta-lactoglobulin are responsible for the development of eczema in one month infants.     

Maternal dietary antigens and the immune response in the offspring of the guinea-pig.

Guinea-pig dams and their litters were raised on either a cow's milk protein-containing diet (MCD) or a milk-free diet (MFD). At 8 weeks of age all litters were challenged i.p. with 50 micrograms milk whey-protein concentrate (V67) and 100 mg A1(OH)3 in saline. The immune response was estimated 2 weeks later as the serum IgG antibody titres against V67, beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) using enzyme-linked immunosorbent assay (ELISA) and the tracheal Schulze-Dale response to these antigens. Feeding milk protein antigen to dams from birth and during pregnancy induces antigen-specific hyporesponsiveness (tolerance) in their offspring, despite no direct contact between the offspring and the milk proteins. Tolerance seems to be induced by the antigen itself since withdrawal of the MCD 10 days before delivery reduced tolerance in the offspring. No tolerance was produced in the offspring of dams fed the antigen from 3 months of age (adult). beta-LG appears to be a major antigen in milk whey while alpha-LA is a minor one since there was almost no antibody or tracheal response to alpha-LA in any of the animals tested. The results indicate that maternal antigen experience and antigens present during pregnancy are important for the subsequent immune response to these antigens in offspring.     

Effect of maternal diet during lactation on development of bovine insulin-binding antibodies in children at risk for allergy

BACKGROUND: The role of exposure to dietary antigens through maternal milk is intriguing, because it may result either in immunization or in tolerance. Exposure to cow's milk proteins results in antibody formation against bovine insulin in infants at risk for type 1 diabetes. OBJECTIVE: To study the appearance of IgG antibodies to bovine and human insulin in infants with an atopic family history whose mothers followed a cow's milk-free diet during the first 3 months of lactation. METHODS: In a prospective cohort study on prevention of food allergies, 123 infants were exclusively breast-fed or received supplementation with a hydrolyzed casein-based formula (Nutramigen) until the age of 6 months. The mothers either avoided cow's milk during the first 3 months of lactation (diet group) or had an unrestricted diet (nondiet group). The levels of IgG antibodies to bovine and human insulin were determined by enzyme immunoassay at 3, 6, 12, and 18 months and at 4 years. In addition, cord blood was obtained at birth and a maternal sample at delivery. RESULTS: At 3 months, IgG antibodies to bovine insulin were low in both dietary groups (median levels 0.150 and 0. 114 optical density units in the diet and nondiet groups). After exposure to dietary insulin, IgG antibodies to bovine insulin increased in both groups, reaching a peak at 12 months in the nondiet group and at 18 months in the diet group. At 18 months, IgG antibodies to bovine insulin were lower in infants in the nondiet group than in infants in the diet group (0.287 vs 0.500, P<.0001). At 4 years, the antibodies no longer differed between the groups. CONCLUSION: The exposure to cow's milk proteins through breast milk during the first 3 months of life resulted in decreased levels of antibodies to dietary bovine insulin at 18 months of age, suggesting a role for breast milk antigens in early tolerance induction.     

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in seropositive and seronegative rheumatic disease

An ELISA has been developed for serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-GSP). A significantly increased prevalence of serum anti-PG-GSP antibody was found in juvenile chronic arthritis and both seropositive and seronegative rheumatoid arthritis (RA), compared with ankylosing spondylitis, systemic lupus erythematosus, myeloma and healthy controls. Anti-PG-GSP antibody was always of the IgG class and there was no correlation of anti-PG-GSP levels with C reactive protein, rheumatoid factor (RF) or anti-streptolysin O titres. There was no direct cross-reaction of RF with PG-GSP, nor did the presence of IgM-RF significantly interfere with the assay. Examination of paired serum and synovial fluid samples offered no evidence for local production of anti-PG-GSP antibody in synovial tissue. These data are compatible with an increased systemic immunization by bacterial fragments in RA.     

Immune response patterns in coeliac disease. Serum antibodies to dietary antigens measured by an enzyme linked immunosorbent assay (ELISA)

Serum IgG, IgA and IgM activities to wheat, egg and cow's milk antigens were measured by an ELISA method in children and adults with coeliac disease (CD). In untreated patients, the IgA activity was characteristically raised to gluten antigens but often also to proteins from egg or cow's milk. Setting the upper reference range for gluten antibodies as the highest IgA reading obtained in healthy controls and patients with other intestinal disorders, IgA measurements afforded virtually 100% diagnostic sensitivity and specificity and detected 94% of children and 80% of adults with untreated CD. Such measurements, therefore, represent a valuable adjunct in the diagnosis of this disease. IgA activity to beta-lactoglobulin, casein or ovalbumin higher than the normal 95 percentile was found in 44-89% of untreated patients. Reduction of these antibody titres seemed to reflect relatively well the response to treatment with a gluten free diet, particularly the activity to beta-lactoglobulin. Monitoring of IgA antibodies to dietary antigens other than gluten may therefore be of particular importance in the follow-up of CD patients.     

Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats.

Six months after inoculation with caprine arthritis-encephalitis virus, the serum and synovial fluid of virus-infected goats had antibodies to [35S]methionine-labeled viral proteins with apparent molecular weights of 125,000, 90,000, 28,000, and 15,000. The 125,000-, 90,000-, and 15,000-molecular-weight methionine-labeled proteins were identified as virion surface glycoproteins by lactoperoxidase iodination and galactose oxidase-boro[3H]hydride reduction labeling techniques. Radioimmunoassay antibody titers to purified p28, the most abundant viral structural protein, averaged 1:182 in synovial fluid and 1:67 in serum 6 months after inoculation. High dilutions of serum and synovial fluid reacted with gp90 and gp125 electroblotted onto nitrocellulose paper from polyacrylamide gels. Anti-gp90 activity was detected at dilutions with an immunoglobulin G content of 0.02 to 11 micrograms, whereas antibody to p28, when detectable on Western blots, was present in samples with an immunoglobulin G content of 0.1 to 2 mg, representing 100- to 1,000-fold-greater titers of antibody to the surface glycoprotein. Synovial fluids often contained more anti-gp90 antibody than did sera. Immunoprecipitation of lactoperoxidase-iodinated virus confirmed the presence of high antibody titers to the two virion surface glycoproteins. Because antiviral gp90 and gp125 antibody is abundant in the synovial fluid of infected goats, it probably contributes to the high immunoglobulin G1 concentrations seen at this site 6 months after caprine arthritis-encephalitis virus infection.     

Studies on the Pathogenesis of Experimental Chronic Active Hepatitis in Rabbits: II. Demonstration of Immunoglobulin on Isolated Hepatocytes

Isolated hepatocytes of rabbits with experimentally induced chronic active hepatitis (CAH) in different stages and liver cirrhosis, respectively, and of animals without liver lesions were studied by immunofluorescence with regard to surface bound immunoglobulin. In 8 of 9 animals with CAH immunoglobulin could be demonstrated on the membranes of the hepatocytes. One animal with liver cirrhosis did not show fixed gammaglobulin; 8 animals without liver lesions which had been immunized with xenogeneic (human) together with allogeneic liver specific proteins (RLP) had no immunoglobulin bound to their hepatocytes, with one exception. The antibody titres in the serum of these animals against liver specific proteins (passive haemagglutination and gel diffusion) were not different from those in animals with liver lesions. The skin test reactivity with RLP as antigen corresponded largely to the development of liver lesions. The pathogenic importance of an antibody mediated lymphocytic cytotoxicity for the induction of CAH is discussed.     

A hydrolysed rice-based formula is tolerated by children with cow''s milk allergy: a multi-centre study

BACKGROUND: Children allergic to cow's milk are fed a soy- or a hydrolysed cow's milk-based substitute. Neither can rule out a sensitization risk. Previous studies have shown that hydrolysed rice is tolerated by animals and children with multiple food hypersensitivities. OBJECTIVE: A prospective clinical assessment of tolerance to a rice-based hydrolysed formula was carried out in children allergic to cow's milk. Patients and methods One hundred children (42 girls and 58 boys, mean age 3.17+/-2.93 years, median 2.20, range 0.18-14.6 years) with a history of immediate reactions to cow's milk and confirmed at double-blind, placebo-controlled food challenge (DBPCFC) when not contraindicated were assessed for clinical tolerance to cow's milk proteins. Their allergy work-up included skin prick tests with whole milk, alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and total caseins, and specific IgE determinations using CAP technology were performed against whole milk, ALA, BLG and casein. Sensitization to rice and rice-based hydrolysed formula was similarly investigated. Patients' sera were evaluated at immunoblotting for specific IgE to cow's milk proteins, rice and rice-based hydrolysed formula. DBPCFC was carried out with increasing doses of a rice-based hydrolysed formula. RESULTS: All patients were sensitized to cow's milk and/or at least one cow's milk protein fraction. Eighty-seven out of 99 were positive to cow's milk and/or a cow's milk protein fraction at skin prick test. Positive (>0.35 kUA/L) specific IgE determinations were found for cow's milk and/or milk fractions (92/95), rice (21/91) and hydrolysed rice infant formula (4/91). At immunoblotting, sera from 96 children were positive to alpha-casein (n=54), beta-casein (n=38), ALA (n=57), BLG (n=37) and bovine serum albumin (n=61). Similarly, although patients' sera often contained specific IgE against rice proteins at CAP (21/91) and immunoblotting (70/96), only six very weakly positive responses were observed against rice-based hydrolysed formula. All DBPCFC with rice-based hydrolysed formula were negative. CONCLUSIONS: Rice-based hydrolysed formula is a possible alternative not only for children with multiple allergies, but also for children with cow's milk allergy.     

Specific antibodies in infants with gastrointestinal intolerance to cow's milk protein

Antibodies of various immunoglobulin classes against cow's milk proteins were studied in infants and children with cow's milk protein intolerance, gluten-sensitive enteropathy and acute gastroenteritis. Their IgE, IgG, IgM and IgA antibody levels determined with the enzyme-linked immunosorbent assay (ELISA) and the IgE antibodies also determined with RAST, were compared with reference groups of children and adults. IgE, IgT or IgA antibodies against unseparated cow's milk proteins, alpha-lactalbumin, beta-lactoglobulin, alpha-casein and beta-casein were present in many of the studied samples, but did not discriminate between the individuals with and without intolerance symptoms. As a group, the infants with late reactions to cow's milk showed increased levels of IgE and IgG antibodies detected with the ELISA, while patients with gluten-sensitive enteropathy had significantly increased levels of IgG and IgA antibodies of cow's milk proteins compared to the reference group. By combining the findings of antibody increases in various immunoglobulin classes, an individual discrimination could be reached. Thus, 8 of 9 of the patients with late reactions to cow's milk had increased levels of IgE or IgG + IgA antibodies as compared to 3 of 22 in the reference group. Serodiagnosis with the ELISA may, therefore, be of some use in patients with a suspicion of cow's milk protein intolerance.     

Two-site enzyme immunometric assays for determination of native and denatured β-lactoglobulin

Two enzyme immunometric assays suitable for measuring native and denatured β-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cow's milk and cow's milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cow's milk in goat's milk.     

Local production of specific antibodies in the aqueous humor in experimental Candida endophthalmia in rabbits

An increasing frequency of ocular lesions due to Candida albicans has been reported, but clinical detection of lesions, which may cause permanent blindness, remains difficult. Hematogenous endophthalmitis caused by Candida albicans in the rabbit is a useful model to study the pathogenesis of the disease, and perhaps to find new methods of biological diagnosis. A suspension of Candida albicans was injected in 15 rabbits, according to the method described by Edwards et al. In 1975, causing uni or bilateral chorioretinitis in 12 of them. Unaffected eyes were chosen as controls. After aspiration of the anterior chamber fluid, serum and aqueous humour titres of specific antibodies were determined. A local synthesis of IgG antibodies was detected, after comparison of the titres of specific antibodies in the aqueous humour and in the serum, related to the serum and aqueous humour concentrations of IgG antibodies. According to a formula proposed by Witmer and Desmonts in ocular lesions due to different pathological agents, we calculated the "C" coefficient: C = antibody titres aqueous humor/serum x IgG serum/aqueous humor. A local synthesis of specific antibodies in cases of experimental endophthalmitis leads us to believe that aqueous humour aspiration could be used in the diagnosis of endophthalmitis in humans.