Human milk components cross-reacting with antibodies against bovine β-lactoglobulin

The human whey components cross-reacting with antibodies raised against bovine and/or equine beta-lactoglobulin were screened systematically. The milk of six women on a normal diet was collected within 72 h of confinement and whey components were fractionated by high-speed size exclusion chromatography and reversed-phase techniques. The fractions which were immunoreactive in double diffusion experiments with antisera anti-bovine and/or equine beta-lactoglobulin were subsequently purified by native PAGE and then electroblotted on Pro-blott membrane (Western blotting). Pro-blot membranes were stained in parallel with Coomassie and by immunostaining using antibodies against bovine and/or equine beta-lactoglobulin as first antibody solution. The immunoreactive bands were cut out from the membrane and N-terminally sequenced; all the immunoreactive components were clearly identified as human beta-casein or its (mainly tryptic) fragments. The strong antigenic similarity between human beta-casein and beta-lactoglobulin (bovine and equine) might be of immunological importance; it could mean that breast-fed neonates risk being sensitized to beta-lactoglobulin irrespective of the presence of cow's milk in the mother's diet.     

BCG vaccine modulates intestinal and systemic response to β-lactoglobulin

Beta-Lactoglobulin (BLG) is a clinically important antigen in cow's milk and one of the major allergens causing cow's milk allergy. Bacillus Calmette-Guerin (BCG) vaccination has been suggested to modify immune response possibly decreasing the risk of allergy to some antigens in both human and experimental animals. In the present study, we have analyzed whether the early BCG vaccination has any effect on the markers of systemic and gastrointestinal (GI) sensitization to BLG. We immunized two groups of Hooded-Lister rat puppets with intraperitoneal injections of native BLG at 43 and 62 days with pertussis vaccine as adjuvant, one group receiving additionally BCG. The animals were then fed native and denatured milk products twice weekly from 73 to 131 days of age, when they were killed. Control group was not vaccinated and received normal rat forage. Total immunoglobulin E (IgE) levels and BLG-specific IgG(1) and IgG(2a) concentrations were determined in serum samples. Spontaneous interleukin (IL)-4 and interferon (IFN)-gamma production from duodenal specimens were measured, and the inflammatory cells were quantitated in specimens from different sections of the GI tract. Administration of BCG simultaneously with BLG resulted in reduced IgE concentration in serum, while the specific IgG(1) and IgG(2a) antibody responses and the spontaneous secretion of IL-4 and IFN-gamma were not affected. Furthermore, BCG-induced eosinophilic infiltration and increase of intraepithelial lymphocytes (IEL) in the GI mucosa, and a trend toward increased number of lamina propria mononuclear inflammatory cells in the colon (BCG compared with BLG, p = 0.09; BCG compared with controls, p = 0.02). Controls showed increment of IgG(1) response in comparison with the BLG group (p = 0.04) and increase of mucosal eosinophilic infiltration. The BCG modified the response to BLG both at the systemic level as shown by decrease of total IgE and at GI mucosa where increase of eosinophilic infiltration and increased number of IEL were seen. Increment of IgG(1) level and eosinophils in the controls might be related with the lack of modulatory effect of pertussis vaccination. A shift of response toward the lower GI tract after BCG immunization as shown by a trend for increase of mononuclear inflammatory cells in colon lamina propria mimics disease development in some cases of clinical food allergy, and emphasizes the need for evaluation of the changes in the whole GI tract in food allergy models.     

Human milk proteins may interfere in ELISA measurements of bovine β-lactoglobulin in human milk

It is widely believed that cow's milk proteins ingested by the mother, in particular β-lactoglobulin ([β-LG), can pass into breast milk and thus sensitize predisposed infants. However, studies to evaluate bovine β-LG in human milk have given conflicting results. The aim of this study was to analyse the correlation between the amount of cow's milk in the mother's diet and the presence of bovine β-LG in breast milk. Human milk samples from 14 healthy non-atopic women on diets with different cow's milk contents were examined. The total concentration of bovine β-LG or β-LG immuno-like proteins (β-LGIP) was determined by enzyme-linked immunosorbent assay (ELISA). Two separation procedures utilizing ELISA plates and an affinity chromatography column were set up to identify the human whey components recognized by the anti-β-LG antibodies. β-LGIP reactivities of milk from three groups on different diets were not significantly different. After splitting the antigen antibody complexes, three main protein components, human lactoferrin, human β-casein and human α-lactalbumin, were identified. This study would suggest that, at least in healthy subjects, false-positive results in ELISA determinations of bovine β-LG in human milk might be due to cross-reactions between polyclonal antibodies and different protein antigens.     

Dietary Bovine β Mactoglobulin is Transferred to Human Milk

ABSTRACT. Human milk from 38 mothers was analysed by radioimmunological method for content of bovine (5-Iactoglobulin. Detectable amounts (5-33 μg/1) of immunoactive β-lactoglobulin were found in 18 human milk samples. Milk from 3 mothers, whose infants suffered from infantile colic contained high amounts of fi-lactoglobulin (32, 18 and 14 μg/1 respectively). With the mothers on a cow's milk free diet the contents fell to non-detectable amounts in two mothers and to 6 μg/1 in the third. All three infants became free from colic,     

Successful pregnancy in β-thalassaemia major

ABSTRACT. Successful pregnancy is described in a patient with β-thalassaemia major, transfusion-dependent from four months of age and treated with desferrioxamine from 13 years of age. The pregnancy was concealed. No antenatal care was given nor any planned alteration in management of her thalassaemic state.     

Coexisting Hereditary Methaemoglobinaemia and Heterozygous β-Tialassaemia

ABSTRACT. Cyanosis was noted within a few weeks after birth in two sisters. On investigation the cause of cyanosis was found to be congenital methaemoglobinaemia due to NADH diaphorase deficiency. Heterozygous β-thaiassaemia was present as an additional incidental finding in one of the sisters, but did not contribute to the symptoms, thus showing that the two diseases, when coexistent, do not pose additional haematological problems. However, it is possible that the β-thalassaemia counteracts the tendency to compensatory erythrocytosis induced by methaemoglobinaemia     

PLASMA AND URINARY β-HEXOSAMINIDASE IN JUVENILE DIABETES MELLITUS

ABSTRACT. The activity of β-hexosaminidase (E C 3.2.1.30) in plasma and urine was determined in 99 patients with juvenile diabetes mellitus and in 40 age-matched controls. Plasma enzyme activities were above normal in plasma from diabetics and showed significant correlations with blood glucose and glycosylated HbA₁ (GHbA₁) but not with the presence of retinopathy. The urinary excretion of β-hexosaminidase, which is a sensitive indicator of renal injury, was also excessive in the diabetics and showed a significant correlation with blood GHbA₁. The results show that plasma and urinary β-hexosaminidase correlate with parameters of diabetic control and that urinary β-hexosaminidase may be more sensitive than urinary albumin as an indicator of early renal damage.     

αβ and γδ T cell subsets in chronic renal failure in children on dialysis treatment

BACKGROUND: Impaired immunity, particularly cell-mediated, is one of the features of chronic renal failure. This also concerns impaired T cell dependent responsiveness. METHODS: The expression of T cell surface antigens (CD3, CD25, TCRalphabeta, TCRgammadelta) was evaluated on peripheral blood (PB) mononuclear cells using two-color flow cytometry in 10 children on continuous ambulatory peritoneal dialysis (CAPD) and in 13 children on maintenance hemodialysis (HD) with polysulfone and cuprophane dialysers. RESULTS: In HD children absolute numbers of leukocytes, lymphocytes, CD3+, alphabeta, gammadelta T cells and a percentage of gammadelta T cells were decreased versus healthy children. Also, we observed a relative increase of CD3+, CD3+/CD25+ and alphabeta T cells after sessions with cuprophane membranes, and an increase of CD3+/CD25+, alphabeta T cell percentages after sessions with the polysulfone membranes. Additionally we found a decrease of both relative and absolute numbers of gammadelta T cells after HD with polysulfone. In CAPD children we found declined absolute numbers of total lymphocytes, CD3+ and alphabeta T cells and higher relative values of CD3+ and alphabeta T cells versus controls. CONCLUSIONS: The T cell depletion in chronic renal failure (CRF) patients primarily results from uremic-related toxicities, rather than from CAPD or HD-related incompatibilities. We showed a significant decrease of gammadelta T cells in CRF patients on HD, that may be partly responsible for impaired T-dependent responsiveness in that group. The intradialytic changes of gammadelta Tcells may result from a different degree of biocompatibility during the application of various dialysis membranes.     

Control of β-catenin/Tcf-directed transcription in medulloblastoma

The β-catenin, glycogen synthase kinase 3β (GSK-3β), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome and patients with Turcot's syndrome, who develop medulloblastomas, have been shown to harbor germline APC mutations. While APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the β-catenin and GSK-3β genes has not been evaluated in this tumor. This study shows that 3 of 67 medulloblastomas harbor β-catenin mutations, each of which converts a GSK-3β phosphorylation site from serine to cysteine. The β-catenin mutation seen in the tumors was not present in matched constitutional DNA in the 2 cases where matched normal DNA was available. A loss of heterozygosity (LOH) analysis of 32 medulloblastomas with paired normal DNA samples was performed with 4 microsatellite markers flanking the GSK-3β locus; LOH with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3β allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the β-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3β gene does not appear to be a target for inactivation in this tumor.     

GLYCOSIDASES IN SKIN AND PLASMA IN HUNTER'S SYNDROME Abnormality of a β-galactosidase in Skin

Two patients with Hunter's syndrome (mucopolysaccharidosis type II) were studied. The exact diagnosis could be settled by the finding of clinical symptoms and signs, typical for this form of mucopolysaccharidosis, and of a greatly increased excretion of glycosaminoglycans (acid mucopolysaccharides) in the urine, as well as by the fact that the patients were half-brothers with unrelated fathers. Analyses of glycosidases in skin demonstrated the existence of low activity of β-galactosidase in both patients. β-acetylglucosaminidase was more active in both patients and β-glucuronidase in one of them than in the controls. In plasma increased activities were noted for β-glucuronidase and β-acetylglucosaminidase in both patients and of β-galactosidase and α-mannosidase activities in one of them and of α-fucosidase activity in the other. The results are very similar to those described in patients with Hurler's syndrome (mucopolysaccharidosis type I).     

Characterization of antigens and allergens in hypo-allergenic infant formulae

The antigenicity and allergenicity of so-called hypo-allergenic infant formulae is mainly determined by the degree of hydrolysis and ultrafiltration. Five different formulae were investigated by means of immunoblotting and RAST in order to characterize the antigens and allergens regarding their molecular weights, molecular origin and their ability to bind human IgG and IgE antibodies: A non hydrolysed infant formula (I-F), a mixture of the major cow's milk proteins (PM), a whey-based infant formula (W-H), a whey-based and ultra-filtrated infant formula (U-H), a casein/whey-based infant formula (CW-H). By immunoblotting we demonstrated that all tested formulae still contain antigens with molecular weights from 3 to 67 kD. But when compared with I-F and PM the antigen content of the hydrolysed formulae was considerably lower. The lowest antigen content could be demonstrated in U-H, which contains casein fragments (3–6 kD) and beta-lactoglobulin and its fragments (6–18 kD). W-H and CW-H contain bovine serum albumin, beta-lactoglobulin, casein and their fragments (3–67 kD). All hydrolysed formulae tested showed a reduced IgE-binding capacity. Three out of 12 cow's milk allergic children possessed IgE binding to U-H or W-H, and 5 of them IgE against CW-H.Conclusion The enzymatic hydrolysis plus ultra-filtration seems to be the most efficient method to reduce the antigen content of so-called hypo-allergenic infant formuale.     

Breast milk β-glucuronidase and prolonged jaundice in the neonate

Breast milk samples from mothers of breast-fed, healthy, term newborns with unexplained prolonged jaundice were analyzed for β-glucuronidase activity. Mean enzyme activity was 75.7 ± 34.5 modified Sigma units/ml in the breast milk samples ingested by the study group of jaundiced babies ( n = 25) and 82.2 ± 40.1 modified Sigma units/ml in the samples ingested by the control group of non-jaundiced babies ( n = 20) ( p > 0.05). Enzyme activities at 2, 3 and 4 postnatal weeks were 101.0 ±39.9, 66.0 ± 20.7 and 57.0 ± 22.4 modified Sigma units/ml in the study group and 87.9 ±36.1, 58.5 ± 15.0 and 88.3 ±49.1 modified Sigma units/ml in the controls. The differences were not statistically significant ( p > 0.05). We conclude that breast milk β-glucuronidase activity may be a contributory factor, in the presence of other variables, in hyperbilirubinemia but it is neither the main nor the only cause of prolonged jaundice in neonates.     

Mucolipidosis with β-Galactosidase Deficiency: a Type of Sialidosis

Sialidosis is characterized by the Hurler-like clinical pictures, progressive neurodegenerative course and an increase in sialic acid-rich saccharides in urine. However, it looks a heterogenous category consisted of several clinical disorders. Recently it has been established that acid neuraminidase is deficient primarily in this disease. We reviewed sialidosis and discussed biochemical characteristics including coexisting β-galactosidase abnormality.     

In vivo and in vitro studies on the residual allergenicity of partially hydrolysed infant formulae

AIM: Because allergen-reduced formulae are widely used in the prevention and treatment of cow's milk allergy in children and because anaphylactic reactions have been reported for some hydrolysed formulae, it is of clinical relevance to know about the residual allergenicity of so-called hypoallergenic formulae. METHOD: We therefore studied the reactions of 20 children (mean age 1.6 years) with proven cow's milk allergy to a variety of formulae, using skin prick test, specific IgE in serum, protein content and RAST inhibition. RESULTS: Whereas all but two children with a clinically relevant cow's milk allergy had a positive skin prick test to cow's milk, some children still showed positive responses to the partially hydrolysed formulae. No child had a positive skin test to the amino acid formula. Specific IgE to the partially hydrolysed whey formula (median 0.28 U/ml) was significantly lower (p < 0.003) than to cow's milk. Specific IgE to the partially hydrolysed whey/casein formula, soy/pork collagen hydrolysate and the amino acid formula was in a low range (median values 0.19, 0.23 and 0.21 U/ml, respectively). While determination of the protein content of the formulae gave no valid information, RAST/EAST inhibition was highest for cow's milk, followed by the partially hydrolysed whey formula, partially hydrolysed whey/casein formula, soy/pork collagen formula, and the amino acid formula. CONCLUSION: Skin prick test and RAST inhibition test are suitable methods for determining the residual allergenicity of hydrolysed infant formulae, while determination of protein content using the applied modified Lowry method is not helpful.     

Circulating noradrenaline and β-adrenergic receptors in children with congestive heart failure

This study was designed to investigate changes in plasma catecholamine concentrations and the number of β-adrenoceptors (β-AR) of circulating lymphocyte in 94 noncyanotic congenital heart patients, in 43 patients with congestive heart failure, β-AR density was significantly lower ( p < 0.001) and plasma noradrenergic levels were significantly higher ( p < 0.001) compared with corresponding values in 51 patients without heart failure. A significant negative correlation between lymphocyte β-AR density and plasma noradrenergic levels was observed ( r =−0.61, p < 0.001). The degree of left-to-right shunt and pulmonary pressure was correlated directly with noradrenaline level and inversely with lymphocyte β-AR density. Both plasma noradrenaline level and lymphocyte β-AR density return to normal in children with heart failure after surgical repair. Our results support the idea that changes in noradrenaline level and lymphocyte β-adrenoceptor density occur concurrently with the presence and severity of heart failure in children.     

CYP11β1 (11-beta-hydroxylase) deficiency in congenital adrenal hyperplasia

Objective : To review experience of CYP11β1 deficiency (previously known as 11β-hydroxylase) at the Royal Children's Hospital, Melbourne, Victoria.
Methodology : A retrospective case review was conducted from 1974 to 1995 with five cases identified.
Results : Age of presentation ranged from 1 day to 7 years. Presentation was with ambiguous genitatia at birth (two females), simple virilization (two males) and suspected early puberty in mid childhood (one female). Associated clinical features were hypertension (three cases) and tall stature with markedly advanced bone age (four cases). Biochemical abnormalities consistent with CYP11β1-deficiency were elevated urinary tetrahydro-11-deoxycortisol ( n = 5) and elevated serum 11-deoxycortisol ( n = 3). Additional abnormalities were elevated 17-hydroxyprogesterone ( n = 3), elevated androstenedione ( n = 4) and elevated dehydroepiandrosterone sulphate ( n = 4). The clinical features and investigations suggested CYP11β1-classical deficiency in four patients and CYP11β1-non-classical deficiency in one patient.
Conclusions : The five cases of CYP11β1-deficiency demonstrate a spectrum of clinical abnormalities, with diagnostic difficulties in two cases and delayed presentation in three cases. Prompt diagnosis of CYP11β1-deficiency is facilitated greatly by the availability of a gas chromatography-mass spectrometry instrument and is essential to avoid the long-term effects of hypertension and hyperandrogenism.     

The Epidemiology of Lost Residual β-cell Function in Short Term Diabetic Children

ABSTRACT. Using the country-wide Swedish childhood diabetes register 526 children, who had had diabetes for 6–30 months were traced for measurements of 24-hour urinary C-peptide. Lost ft-cell function was defined as a 24-hour urinary C-peptide excretion per kg body weight less than 10% of the mean for healthy children (<0.025 nmol/kg). The estimated cumulative incidence of lost β-cell function was 0.64 at 30 months. The incidence of lost β-cell function did not differ by sex. Neither was there any significant variation in season at onset for cases with lost β-cell function. A significant age dependency was shown for the cumulative incidence of lost β-cell function with the highest incidence in the young age groups, i.e. a reversed age dependency compared to that of clinical onset. In contrast to the clinical onset of diabetes no significant geographical variation was found for lost β-cell function when comparing standardized morbidity ratios. The urinary C-peptide excretion was significantly correlated to age at onset but not to degree or duration of ketonuria at onset. It is concluded that there are striking differences when comparing the epidemiology of lost β-cell function to that of clinical onset in terms of age, sex, seasonal and geographical variations. The timing of clinical onset may thus partly be determined by factors different from those determining the rate of fall in β-cell function.     

β-lactam antibiotic resistance in aerobic commensal fecal flora of newborns

BACKGROUND: The purpose of the present paper was to prospectively determine the rate of beta-lactam antibiotic resistance in commensal fecal flora of newborns and the risk factors leading to this colonization. METHODS: One hundred and eighteen newborns in the neonatal intensive care unit (NICU) group (n = 38), the neonatal ward group (n = 36), and the control group (n = 44) were enrolled. Three or four stool samples were obtained from each infant, 15 days apart. Bacterial growth in Eozin Methylene Blue (EMB) agar + 10 microg ampicillin/mL was considered to be ampicillin-resistant bacteria, and antibiotic susceptibility and extended spectrum beta-lactamases (ESBL) production was investigated in those bacteria. RESULTS: Colonization with ampicillin-resistant commensal fecal flora microorganisms was determined in 75.2% of 367 stool samples. Klebsiella spp. and Escherichia coli were identified in 59% and 41% of the samples, respectively. The lowest rate of ampicillin-resistant bacterial colonization was determined in the NICU group. Microorganisms producing ESBL were identified in 33.7% of 367 stool samples. Fifty-one and 73 of ampicillin-resistant E. coli and Klebsiella spp. isolates were determined to produce ESBL, respectively. There was no difference with respect to colonization with ESBL-producing microorganisms between the three groups. When risk factors related to colonization with ESBL-producing microorganisms in stool samples were evaluated through the whole study period, very low birthweight, vaginal delivery, infant antibiotic usage, maternal antibiotic usage, male sex and premature rupture of membranes were determined as risk factors, while feeding with nasogastric tube was identified as a protective factor. When the risk factors related to colonization with ESBL-producing bacteria in stool samples after discharge from the hospital were evaluated, failure to feed breast milk was determined as the only risk factor. CONCLUSIONS: To decrease mortality and morbidity due to infection caused by resistant microorganisms colonized in the intestine flora of the infant, protection of normal non-pathogenic bacterial colonization is important. This can be provided by feeding neonates with breast milk, application of infection control measures efficiently, and limitation of antibiotic usage only to strict clinical indications.     

Neuroendocrine and circadian aspects (melatonin and β-endorphin) of atopic dermatitis in the child

Atopic dermatitis (AD) is a disease of increasing incidence among paediatric patients. Among the factors involved in its pathogenesis is the alteration of the immune response, and so the objective of this study was to evaluate the involvement of certain neuroendocrine factors with immune properties in the development of the disease. Fifty-five subjects were selected and divided into the following three groups: healthy subjects, those diagnosed with symptomatic AD and those with asymptomatic AD. Plasma levels of melatonin and beta-endorphins were measured by radioimmunoassay, in serum samples obtained at 9 am and 9 pm, with two samples being obtained from each of the patients and controls. In the phases of AD outbreaks, there is a reduction in the serum levels of both melatonin and beta-endorphin. In the case of melatonin, the difference is statistically significant only during the day, although nocturnal levels are greater for both hormones. In AD, a central neuroendocrine dysfunction may be a primary pathogenic event. Our hypothesis is that the physiological nocturnal peak of melatonin due to pineal gland production may mask the decline of melatonin of possibly extrapineal (immunological) origin during episodes of dermatitis outbreaks. Further studies are required, particularly of neurovegetative and hormonal aspects, to better define this process. Such a definition would also be of therapeutic interest.     

Abnormal β-D-glucosidase and Different Growth Characteristics of Cultured Skin Fibroblasts from Infantile Gaucher's Disease

Enzyme kinetics and the isoelectric point of S-D-glucosidase (EC 3.2.1.21) and growth characteristics of cultured skin fibroblasts from infantile type Gaucher's disease were examined. Growth characteristics of fibroblasts from the patient were different from those of normals; the population doubling times of the patient and the normal fibroblasts were 9.4 and 18.4 hours in the logarithmic phase, and the mean values of cell density in the stationary phase were 6.68 times 10! and 1.67 times 10! cells per square centimeter, respectively. S-D-glucosidase activity of patient's fibroblasts decreased to 22% of normal. No differences in the activities of several hydrolases such as S-D-galactosidase (EC 3.2.1.23) S-L-fucosidase (EC 3.3.1.51) and acid phosphatase (EC 3.1.3.2) in fibroblasts were observed between the patient and the normal. The apparent Km and pH profile of the enzyme in a crude preparation from the patient fibroblasts were the same as those from the normal one. The isoelectric focusing profile of S-D-glucosidase in cultured skin fibroblasts from infantile Gaucher's disease was different from that of the enzyme in fibroblasts from normal individuals; the predominant isoelectric point from the patient was at pH 5.5 and that from the normal at pH 4.5. These results indicate that the β-D-glucosidase of fibroblasts from infantile Gaucher's disease is a mutant enzyme. (Acta Paediatr Jpn 23(2): 208–213 1981)