Growth hormone increases connexin-43 expression in the cerebral cortex and hypothalamus.

Several studies indicate that systemic GH influences various brain functions. Connexin-43 forms gap junctions that mediate intercellular communication and establish the astroglial syncytium. We investigated the effects of peripheral administration of bovine GH (bGH) and recombinant human insulin-like growth factor I (rhIGF-I) on the expression of connexin-43 in the rat brain. Hypophysectomized female Sprague Dawley rats were substituted with cortisol (400 microg/kg x day) and L-T4 (10 microg/kg x day) and treated with either bGH (1 mg/kg x day) or rhIGF-I (0.85 mg/kg x day) for 19 days. The abundance of connexin-43 messenger RNA (mRNA) and protein in the brainstem, cerebral cortex, hippocampus, and hypothalamus was quantified by means of ribonuclease protection assays and Western blots. Treatment with bGH increased the amounts of connexin-43 mRNA and protein in the cerebral cortex and hypothalamus. No changes were found in the brainstem or hippocampus. Infusion of rhIGF-I did not affect connexin-43 mRNA or protein levels in any of the brain regions studied. These results show that administration of bGH increases the abundance of cx43 in specific brain regions, suggesting that GH may influence gap junction formation and thereby intercellular communication in the brain.

Cytochrome P450 2D6 (CYP2D6) genotyping on postmortem blood as a supplementary tool for interpretation of forensic toxicological results.

Debrisoquine hydroxylase (CYP2D6) is involved in the metabolism of many toxicologically important drugs. The gene encoding for this enzyme displays a polymorphic distribution in all populations examined. We report a study on 46 cases, where analyses of the CYP2D6 gene were conducted on postmortem femoral blood in order to investigate the occurrence of poor metabolizers (PM). A polymerase chain reaction (PCR) method, designed and routinely used for therapeutic drug monitoring, was employed, only slightly modified. Samples from 22 cases, where the parent drug to metabolite ratio was unexpectedly high were analyzed as well as samples from 24 control cases. Genotyping could be carried out in all but one case. Previous freezing or addition of potassium fluoride as preservative did not prevent analysis. Only one PM (from the control group) was discovered, implying an occurrence of only 2.2% as compared to the reported frequency of approx. 7% in Sweden. Among the extensive metabolizers (EM), however, a number of individuals with mutated genes were identified. Although it seems reasonable to suspect a PM genotype in cases with a high concentration of a drug metabolized by CYP2D6, but without suspicion of acute overdose, our study does not support the opinion that this interpretation pitfall is particularly common. This study rather indicates that drug interactions in EMs constitute a more frequent and important problem.

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Obese (ob) gene defects are rare in human obesity.

Our knowledge of the role of the recently cloned ob-protein (leptin) in the regulation of body fat stores is largely derived from experiments performed in mice. Different mouse models exhibit abnormalities in ob-gene expression, with extreme overexpression in mice which lack bioactive ob-protein, have nonfunctional ob-receptors or hypothalamic lesions, and undetectable expression in mice with suggested defects in regulatory elements. The aim of this study is to examine if defects, corresponding to those in mice, exist in human obesity. Adipose tissue was obtained from 94 adult obese subjects and from six children who had developed obesity after surgery in the hypothalamic region. Total RNA was isolated and ob-gene expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot. The coding region of the ob-gene was sequenced in both directions in the 94 obese adults. No mutations were detected in the coding region of the ob-gene and ob-gene expression was detectable in all subjects and none of the subjects had an extreme overexpression. There was no systematic increase in ob-expression in obese children with hypothalamic disease compared to their healthy brothers and sisters. These results show that severe abnormalities involving the ob-gene, analogous to those described in mouse models, are rare in human obesity. We therefore conclude that the cloning and subsequent analysis of the ob-gene has not provided information that can, by itself, explain the genetic component in the development of human obesity.

Specific antibodies to poliovirus type I in breastmilk of unvaccinated mothers before and seven years after start of community-wide vaccination of their infants with live, oral poliovirus vaccine.

Secretory IgA (SIgA) antibodies against poliovirus type 1 were determined using the ELISA method in breastmilk samples obtained each month from 100 young, healthy, unvaccinated mothers living in urban slum areas of Lahore, Pakistan. The study covered two different groups, one in 1980-1981 and the other in 1987, before and seven years after a nation-wide expanded programme of childhood immunization (EPI) had started. The SIgA titres did not change neither with duration of lactation nor with time after vaccination in the infants of the mothers studied. The seasonal breastmilk IgA antibody titres to poliovirus type 1 corresponded to the epidemiological conditions existing both before (1980-81) and after general vaccination coverage with live, oral poliovirus vaccine (OPV) had reached 80% of the infant population (1987). Neutralization titres did not seem to correlate well with ELISA titres although colostrum samples had high levels of neutralizing antibodies. The wide variation between high (greater than 10,000) and low (less than 500) individual breastmilk IgA antibody titres observed during various seasons could be of consequence for the breast-fed baby. Colostrum, which was also found to have significant neutralization capacity, might interfere with the OPV now often given on the day of birth.

Differences in level and avidity of secretory IgA antibodies in breast milk of Swedish, Indian and Japanese mothers to soybean protein.

Colostrum was collected from Swedish, Indian and Japanese mothers. The samples were as a mean, collected 4.00-4.25 days after delivery of term infants. The level of specific IgA antibody to 2S, 7S and crude soybean antigen were measured by the enzyme-linked immunosorbent assay (ELISA). The avidity of the IgA antibodies to 7S soybean antigen was also measured with an ELISA system using different molarities of potassium thiocyanate for elution of the specific IgA antibody from solid phase-bound antigen. The level of specific IgA antibody to 7S and crude soybean antigen in the milk of the Indian mothers was significantly higher than in the milk of the Japanese mothers (p less than or equal to 0.01). In contrast, the avidity expressed as the molarity of KSCN for 50% elution of IgA antibody to 7S soybean antigen in the milk of the Japanese mothers was significantly higher than in the milk of the Indian mothers (p less than 0.01).

Protective factors in milk and the development of the immune system.

The neonate is immature in certain immunologic functions. The slow development of secretory immunoglobin A (IgA) seems to be compensated by selective transfer of secretory IgM into exocrine secretions on mucous membranes during the first few months of life. Secretory IgA and secretory IgM antibodies against Escherichia coli and poliovirus are already found in the neonate, possibly in response to the maternal anti-idiotypic IgG antibodies transplacentally exposing the fetus. Via such a mechanism, food antibodies could occur before direct food exposure in the infant. Human milk provides large amounts of antibodies (as a crude comparison, about 50 times the amount of antibodies given to a patient with hypogammaglobulinemia). The milk antibodies, dominated by secretory IgA, protect especially against intestinal infections. The milk also contains oligosaccharide analogues to epithelial receptors for bacteria. They, as well as a number of milk components such as lactoferrin and lysozyme, may contribute to host defense. The food antibodies in human milk may influence the infant's immune response to foreign food proteins introduced during weaning.

The protein tolerance of very low birth weight infants fed human milk protein enriched mother's milk.

Pooled breast milk was ultra-filtrated and freeze-dried to give a product with a protein content of 60 g/100 g powder. More than half of the secretory IgA activity against E. coli O antigen was preserved. This concentrated protein was added to the mother's fresh milk providing a protein supply of 3.0-3.5 g/kg/24 hours in four VLBW infants, at a calorie supply of 110 kcal/kg/24 hours. Growth followed the intrauterine rate. Free amino acid levels, acid-base balance and urea concentrations of peripheral whole blood indicated tolerance of the increased supply of human milk protein.

Studies on human milk III. Secretory IgA quantity and antibody levels against Escherichia coli in colostrum and milk from underprivileged and privileged mothers.

We studied the milk content of secretory IgA (SIgA) and of specific IgA antibodies to E. coli in relation to volume, in 24 h samples from mothers belonging to different socio-economic groups and living under different ecologic conditions in Ethiopia, Guatemala and Sweden. There were no statistically significant differences in the daily output of milk SIgA among the population groups investigated at different times after onset of lactation. There was, however, a certain trend towards lower SIgA levels among the Guatemalan poor women, compared to the corresponding privileged one (Tables 2 and 3). Three days after delivery the underprivileged Ethiopian mothers showed significantly lower antibody levels than the privileged Ethiopian. These differences were no longer seen when the values were corrected for differences in volume (Table 5). One month after delivery, the levels of SIgA antibodies in milk from Swedish women and Guatemalan privileged women, against a pool of eight E. coli somatic antigens were comparable; these two groups of mothers had significantly higher antibody levels than the Guatemalan rural and urban ones (Table 4). The same pattern was observed after correction for differences in 24 h volumes (Table 5). At 3 months after delivery, the Guatemalan urban privileged women, again showed higher levels and daily output of antibodies against the E. coli antigens than the urban poor and rural mothers (Tables 4 and 5). The milk samples taken from a population where malnutrition is evident, i.e., mothers from Santa María Cauqué, did not show any changes in the levels of SIgA and the anti-E. coli antibodies 3, 6, 9 months after initiation of lactation. The data presented here provide evidence that chronically malnourished mothers are able to produce SIgA and transfer it to their offspring via breast milk. Furthermore, they do so in quantities that are comparable to those observed in well-nourished populations. There was a wide range of concentrations and daily output of SIgA and of specific antibodies in all groups, suggesting that some of the infants get less than others. The observed differences in levels of antibodies against E. coli may be explained by differences in exposure to E. coli strains of the eight serogroups studied here. The possibility of a deficiency in the SIgA antibody response in the undernourished mothers still remains unanswered.

Food antibodies in milk from Guatemalan women.

PIP: This study investigates the presence of antibodies against food products in milk samples from 30 Guatemalan women of 3 different socioeconomic status: 10 rural poor, 10 urban poor, and 10 urban privileged. The 3 groups had varied dietary habits. Both the rural and urban poor mothers do not consume cow's milk. Black beans are consumed more often by the urban groups while soy bean products are consumed by both rural and urban poor regularly. Milk samples were collected from the mothers. The volume of milk produced in a 24-hour period was estimated by weighing the children before and after every meal. The enzyme-linked immunosorbent assay (ELISA) described by Sohe-Akerlund et.al. was used in quantitating milk secretory immunoglobulin A (SIgA). A modification of the ELISA method was used to determine levels of specific IgA antibodies directed against cow's milk, black beans and soybeans. The Wilcoxon rank sum test was used in statistical analysis. Comparable amounts of SIgA were produced by the 3 groups of mothers in a 24-hour period (p0.1). The urban privileged mothers exhibited significantly higher antibody levels against cow's milk (p0.01) and black beans (p0.05) compared to the other 2 groups. There were no differences in the level of anti-soybean antibodies among the 3 groups. The notion that antibodies found in the breast milk reflect the mother's intestinal antigenic experience is supported by this study. It has been suggested that anti-food protein antibodies contribute in the prevention of allergies. If so, cow's milk fed among the rural and urban poor children may be expected to produce negative reactions. This negative reaction can be prevented by feeding the mother, before delivery, cow's milk products to induce them to produce milk antibodies against cow's milk.^ieng

A sensitive method for specific quantitation of secretory IgA.

A method to quantitate specifically secretory IgA (SIgA) has been developed using the enzyme-linked immunosorbent assay. The IgA in the test sample was adsorbed to anti-alpha antibodies attached to plastic tubes via a cost of IgA myeloma protein. The reacted SIgA was determined using anti-secretory component antiserum conjugated with alkaline phosphatase. The technique permitted quantitation of secretory IgA in biological fluids like milk, urine, and saliva with a reproducibility of +/-7%, down to 0.03 mg/l. In contrast to earlier techniques, the presence of up to 157% of serum IgA without secretory component (SC) and free SC did not disturb the measurements of SIgA. Furthermore, variations in pH and osmolarity, within biological ranges in secretions, did not influence the estimations.

Influence of maternal gut flora and colostral and cord serum antibodies on presence of Escherichia coli in faeces of the newborn infant.

From 29 healthy newborn infants and their mothers faecal, serum and milk specimens were obtained on several occasions from one to nine weeks after delivery. Predominant faecal E. coli were serotyped with regard to the O antigen and milk and serum were analysed for their content of E. coli O antibodies by the enzyme-linked immunosorbent assay. In five cases the babies acquired the same O serotype as was found in the stools of their mothers but in 12 out of 29 cases infant and mother never had any dominating faecal E. coli O type in common. There was no apparent correlation between the patterns of feeding and interchange of bacteria. Klebsiella/Enterobacter was the dominating facultative organism on at least one occasion in half the infants. The newborns received colostral IgA and transplacental circulating IgG antibodies against a great number of E. coli O serotypes. These antibodies did not prevent intestinal colonization, as judged from cultures of faeces.

Antibody production by human colostral cells. I. Immunoglobulin class, specificity, and quantity.

The production of antibody by human colostral cells was assayed by the hemolysis in-gel technique. When sheep erythrocytes coated with O antigens from frequently encountered Escherichia coli bacteria were used as detector cells and anti-IgA serum was added for development, numerous plaque-forming cells (PFC) were demonstrated in all samples tested. In contrast, plaques were rarely seen in the presence of anti-IgG developing serum. The direct (IgM) plaques occasionally noted with both antigen-coated and uncoated sheep erythrocytes were mainly due to the production of heterophil antibodies, since they were not formed when human erythrocytes were used as O-antigen carriers. A strikingly high number of the colostral lymphocytes formed antibodies to the E. coli antigens, up to 8%. This suggests that these cells represent a rather selective population--possibly cells from the gastrointestinal tract exposed to enteric bacteria. The large number of plaques observed, the predominance of the cells forming IgA antibodies, and the marked changes in PFC number in relationship to parturition pose a number of questions relevant to the antibody-producing colostrum cells and their relationship to the secretory immune system.