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PubMed1981

The differential localization of IgA, IgM and IgG in the gut of suckled neonatal piglets.

The localization of immunoglobulin IgG, IgM and IgA in tissue sections prepared from the ileum of neonatal and adult swine were compared. Eighty percent of the immunoglobulin-containing lymphoid cells in the lamina propria of conventional adult German Landrasse swine were IgA-positive with lower numbers of IgM cells and occasionally an IgG cell. Anti-mu and alpha-chain reagents also stained the cytoplasm of the crypt epithelial cells. By comparison to these adult control tissues, the ileum of unsuckled neonates contained no immunoglobulins although after the ingestion of colostrum, the entire cytoplasm of the villus epithelial cells stained intensely when tested for IgG with only faint staining for IgM and IgA. On the other hand, IgA and IgM were readily localized on what appears to be only the apical border of the crypt epithelial cells but in contrast to the adult, the cytoplasm of these cells was unlabelled. IgG was absent from the crypt region. We interprete these findings to indicate an important, selective role for the villus epithelium in the absorption into the neonatal circulation of colostral IgG and probably IgA and IgM, and a specialized role for the crypt epithelium in adsorbing colostral IgA and IgM; possibly by complexing with mucin-bound secretory component.

Aging↗

PubMed1988

Up-regulation of receptors for IgA on activated human B lymphocytes.

Expression of receptors for the Fc part of IgA (Fc alpha R) by T lymphocytes was recently shown to be up-regulated after activation by T cell mitogens in the absence of IgA. We describe a similar increase on activated human B lymphocytes. Fc alpha R were determined by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-IgA or goat anti-secretory component F(ab')2 fragments. B-enriched cell suspensions were prepared from peripheral blood or tonsils and activated by Staphylococcus aureus Cowan I, anti-IgM antibodies or E. coli lipopolysaccharide. All three activators increased the percentage of Fc alpha R positive cells although only the former induced significant DNA synthesis. Finally recombinant interleukin 1 (10 nM) and interleukin 2 (10 IU/ml) but not interleukin 4 (300 units/ml) nor low-molecular-weight B cell growth factor induced an increase of Fc alpha R expression. The data show that Fc alpha R can be up-regulated on human B cells in the absence of exposure to IgA.

Antibodies, Anti-Idiotypic↗

PubMed1989

Specificity and association constants of 33 monoclonal antibodies to human IgA epitopes.

We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants (Ka) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. Ka ranged from 0.37 to 690 x 10(7) liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.

Animals↗

PubMed1995

Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA.

Trans-epithelial transport of polymeric immunoglobulins (pIg) into mucosal and glandular secretions is carried out by the pIg receptor (pIgR). Therefore, expression of the pIgR gene in epithelial cells of mucosal and glandular tissues is an absolute requirement for achieving mucosal immunity. We report the cloning and characterization of the bovine pIgR cDNA. Three overlapping cDNA clones with a total length of 3608 bp yielded an open reading frame encoding a 757-amino-acid (aa) transmembrane (TM) glycoprotein. Although polymorphism was found in two separate clones, Northern blot analysis showed a single pIgR mRNA (approx. 3.8 kb) to be present in the mammary gland, liver, lung, kidney and intestine of a lactating cow. There was no detectable expression of pIgR in the spleen of the same animal. Comparison of the deduced bovine pIgR as sequence with those of rat, mouse, man and rabbit shows that this receptor is highly conserved both in aa sequence and structural organization. The degree of conservation in the TM sequence and the C-terminal cytoplasmic tail, which contains the various signals for intracellular trafficking of the receptor, is 65-73%. We also find a high degree of conservation (61-66%) in the ectoplasmic part of the receptor, known as the secretory component (SC), with an exception for that of the rabbit SC, which is much lower (47%). Among the five Ig-like domains in the SC, the N-terminal domain I, where the primary pIg-binding site is located, showed the highest (72-83%) aa sequence conservation.

Amino Acid Sequence↗

PubMed1995

Histological and immunohistochemical analysis of lung development, with particular reference to lung hypoplasia.

Using immunohistochemical (IH) staining for epithelial membrane antigen (EMA), keratin, Leu-7, Ca 19-9, secretory component (SC) and surfactant protein A (SPA) on formalin-fixed, paraffin-embedded autopsy cases of stillbirth and early neonatal death, the normal profile of IH-positive cells in the airway of the developing fetal lung was clarified. IH-positive cells first appeared in the proximal region of the lung, and then in the distal region. Keratin-positive cells and EMA-positive cells were abundant at the 10th gestational week (10 GW), the earliest stage observed in this study. Cells positive for Leu-7 and Ca 19-9 appeared during the time when bronchial branches developed, and were abundant at around 16 GW, the time of completion of bronchial branching. On the other hand, SPA- and SC-positive cells appeared after the completion of bronchial branching, and were abundant around 29 GW and 34 GW, respectively. Acinous structural development was observed in terms of radial alveolar count (RAC). Cases of lung hypoplasia defined as a lung weight to body weight ratio of under 0.012 at > or = 28 GW or under 0.015 at < 28 GW showed a rather high tendency of abnormalities of cellular differentiation revealed by IH staining.

Bronchi↗

PubMed1989

The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line.

The human colonic cancer cell line HT29, when grown in DMEM, forms a morphologically unpolarized cell culture in which the cells are covered with irregular microvilli and devoid of belt zonula occludens type tight junctions. However, by modifying the culture medium and growing the cells in RPMI, a different morphology was obtained. A large number of intracellular luminae appeared and at late confluency 90-95% of cells exhibited an apical brush border after four subsequent passages. Junctional complexes and a well developed zonula occludens were revealed under the apical brush border. Immuno-electron microscopical localization of specific markers, sucrase isomaltase (SI), secretory components (SC) and beta 2 microglobulin (beta 2M) showed that SI was limited to the apical surface, whereas 2M and SC were located at the basolateral surfaces. These results indicate that modification of culture conditions affects the ability of HT29 cells to express epithelial cell polarity.

Carcinoma↗

PubMed1989

Induction of phenylethanolamine N-methyltransferase mRNA expression by glucocorticoids in cultured bovine adrenal chromaffin cells.

The effects of glucocorticoids on the expression of phenylethanolamine N-methyltransferase (PNMT) mRNA and proenkephalin A (ProEnk A) mRNA in cultures of bovine adrenal chromaffin cells were examined. The expression of PNMT mRNA (approx. 1.1 kilobases) was induced in the presence of glucocorticoids. This induction was of high potency with an EC50 in the range of 1-10 nM for dexamethasone, and was blocked by high concentrations of the glucocorticoid antagonist RU-38486. Cortisol, prednisolone and Reichstein substance S (11-deoxy-17-hydroxycorticosterone) were all effective in stimulating PNMT mRNA expression while cortisone, progesterone and beta-estradiol were without effect. These results indicate that the effects are mediated by specific glucocorticoid receptor activation and exhibited a strict structural requirement for the ability of glucocorticoids to induce PNMT mRNA expression. By contrast, glucocorticoids had no significant effect on the expression of ProEnk A mRNA. In summary, this study provides evidence that glucocorticoids act to regulate PNMT (but not ProEnk A) at the transcriptional level. This differential effect of glucocorticoids suggests that different mechanisms govern the expression of mRNAs required for synthesis of the co-stored secretory components, the enkephalins and adrenaline within the chromaffin cells of the adrenal medulla.

Adrenal Glands↗

PubMed2005

Human placental explants in culture: approaches and assessments.

Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

Biocompatible Materials↗

PubMed2006

Human reporter gene assays: transcriptional activity of the androgen receptor is modulated by the cellular environment and promoter context.

The androgen receptor (AR) is a member of the nuclear receptor superfamily and mediates the physiological effects of androgens. Androgens are essential for male development and disruption of androgen signaling may cause androgen-dependent developmental defects and/or tumors. Here we present a comparative analysis of various model systems for the investigation of endocrine active compounds in human cell lines. We generated reporter plasmids containing androgen response elements derived from the human secretory component or the rat probasin genes as well as the glucocorticoid consensus response element and compared their activities to that of the mouse mammary tumor virus promotor. Additionally, we generated an expression plasmid containing the AR cDNA derived from LNCaP cells. In 22Rv1 cells transiently transfected with human AR, all reporters displayed a dose-dependent, high activity when treated with androgens. Interestingly, the potency of testosterone and its metabolite dihydrotestosterone was very low in HepG2 but not in 22Rv1 cells, independent of the reporter used. The efficacies of the androgens tested were comparable in both cell lines but highly dependent on the reporter used. Based on these results, 22Rv1 cells provide a highly sensitive in vitro test system to analyze endocrine activities of xenobiotics. Furthermore, this study highlights the need to investigate the (anti-) androgenic activity of compounds in dependence of the cellular and promoter context.

Androgen Antagonists↗

PubMed1998

Molecular maturation and functional expression of mouse polymeric immunoglobulin receptor.

Mouse polymeric immunoglobulin receptor (pIgR) cDNA was stably introduced into a hamster-derived fibroblastic cell line, Chinese hamster ovary (CHO) cell, by the calcium phosphate method. Surface expression of pIgR was detected by immunostaining and FACS analysis. The immunoprecipitated products of cell lysates revealed that the molecular mass of the most mature form of pIgR was approximately 120 kDa. Western blotting and metabolic labeling experiments followed by immunoprecipitation with an anti-mouse secretory component (SC) Ab demonstrated the existence of a 110 kDa immature form of pIgR. The reason for the existence of two forms of pIgR molecule was examined by conducting pulse-chase experiments which revealed the pIgR underwent molecular maturation. During this process, the 110 kDa form of pIgR was converted into a 120 kDa form by glycosylation. Moreover, tunicamycin treatment revealed the core form of pIgR had a molecular mass of approximately 100 kDa. The pIgR expressed on the surface of the transfectant could specifically bind and take up mouse polymeric IgA (MOPC 315), suggesting that, at least in this mouse system, cell type-specific molecules are not necessary for surface pIgR expression and polymeric immunoglobulin (pIg) binding and uptake.

Animals↗

PubMed2000

Detection by ELISA of low transferrin levels in bronchoalveolar secretions of preterm infants.

Transferrin levels in bronchoalveolar secretions (BAS) are very low compared to serum levels in humans. For the exact measurement of transferrin concentrations in BAS a very sensitive assay was developed as a double sandwich enzyme immunoassay using the combination of a polyclonal and a monoclonal antibody against human transferrin. The measurable range of the assay was 1.5 to 100 ng/ml of human transferrin. The lowest measurable value was 0.84 ng/ml and the sensitivity of the assay was 0.88 ng/ml. The coefficient of variation was 14.1% for 25 ng/ml (intra-assay) and 11-20% (inter-assay). The levels measured in 123 samples of BAS of preterm infants ranged between 0.03 and 8.93 (microgram/microgram secretory component (SC)). The determination of transferrin in BAS of preterm infants is helpful in determining oxidative damage, e.g. the availability of free iron, in the neonatal lung. The transferrin concentration in BAS of neonates who recovered from respiratory distress syndrome (RDS) in the first six days of life was 0.48 compared to 0.52 ((microgram/microgram SC), median range) for infants who developed chronic lung disease.

Antibodies, Monoclonal↗

PubMed1992

Glomerular alterations in children with biliary atresia.

To investigate the association of glomerular involvement with biliary atresia, we carried out the following studies: (1) review of the clinical records of 120 patients, (2) histologic study of the kidneys obtained at autopsy from 28 patients, and (3) measurements of circulating immune complexes. Of 90 patients with adequate follow-up information, 40 (44.4%) had hematuria, proteinuria, or both. All the kidney specimens showed a wide variety of mesangial proliferation, and immunoglobulins were present in 23 of 26 cases. There was a good correlation between the glomerular alterations and the period of reduced hepatic function. When IgA was present in the mesangium, IgA2 and secretory components were detected. Elevated serum IgA and circulating IgA-containing immune complex levels were found in patients with prolonged obstructive jaundice. These findings indicate that glomerular alterations may occur subsequently in patients with biliary atresia, and that IgA of intestinal mucosal origin plays some role in the development of these lesions.

Antigen-Antibody Complex↗

PubMed1986

Glomerular lesions associated with liver cirrhosis: an immunohistochemical and clinicopathologic analysis.

Clinicopathologic and immunohistochemical analyses of the kidneys in 30 autopsy cases of liver cirrhosis of different types revealed glomerular lesions in 27 cases. Clinical nephritis was present in three cases. Characteristic diffuse mesangial proliferation with focal mesangial interposition and/or subendothelial deposits were present. Glomerular immunoglobulin deposition was observed in several cases, IgM in 23 cases, IgG in five, and IgA in 17; the latter were predominantly IgA1. Secretory component binding was found in 11 of the 17 cases with IgA deposits (65 per cent). Liver weight was related to IgA deposition (P approximately 0.05), and the volume of ascitic fluid was significantly correlated (P less than 0.05).

Adult↗

PubMed1995

Protein content in bronchoalveolar lavage fluid of patients with asthma and control subjects.

BACKGROUND: Secretory component (SC), Clara cell protein (CC10) and to some extent, IgM are proteins locally synthetized in airways. Albumin, alpha 2-macroglobulin and alpha 1-antitrypsin are mainly plasma proteins. In patients with asthma, blood proteins may occur in greater amounts in bronchoalveolar lavage fluid (BALF) than in control subjects because of plasma extravasation. These proteins were measured in BALF to define markers of local synthesis and plasma exudation. METHODS: Twenty-four patients with asthma (mean age, 40 +/- 3.1 years) and 24 control subjects (mean age, 29 +/- 11 years) were tested. Five aliquots of saline solution (50 ml) were instilled, and the recovered BALF was stored. Clara cell protein was measured by a sensitive immunoassay technique based on the agglutination of latex particles. SC, immunoglobulins, alpha 2-macroglobulin, alpha 1-antitrypsin, and albumin were measured by an immunoradiometric assay. Protein concentrations were normalized to albumin. RESULTS: In BALF from patients with asthma there was a significant increase in alpha 2-macroglobulin and IgM and a significant decrease of SC, alpha 1-antitrypsin, and Clara cell protein compared with control subjects. Also, the ratio to albumin was significantly increased for alpha 2-macroglobulin and IgM and decreased for SC. There was no correlation between the severity of asthma and the amount of proteins. CONCLUSIONS: BALF protein content is significantly different in patients with asthma and control subjects, for proteins produced locally and derived from plasma.

Adolescent↗

PubMed2002

Helicobacter pylori-specific immunoglobulin synthesis in gnotobiotic piglets: evidence for the induction of mucosal immunity in the stomach.

Short term tissue biopsy cultures and paired, sera, bile and gastric and intestinal contents from Helicobacter pylori-infected gnotobiotic piglets were tested for the synthesis of H. pylori-specific immunoglobulin (Ig) isotype production by antigen-specific ELISA from post-infection days (PIDs) 2-28. Serum antibody levels in all three Ig isotypes were elevated from baseline values by PID 14, serum IgM levels reached peak levels on PID 14 and by PID 28 bile was strongly positive for IgA and IgG.Intestinal, but not gastric contents from infected piglets, contained IgA-specific antibody from PID 14 onward. Gastric mucosal epithelia adjacent to areas of inflammation in infected but not uninfected control piglets produced readily detectable amounts of porcine secretory component (SC); IgA-positive plasma cells were identified in gastric submucosa and lamina propria in these areas. Culture fluid supernatants, collected from explanted gastric cardia and antra and intestinal ilea of H. pylori-infected piglets had trace amounts of IgA as early as PID 2 in some animals, and strong IgA reactivity in all by PID 28. Supernatants also contained H. pylori-specific IgG by PID 14. A strong gastric lymph node IgA response contrasted with moderate IgA production in mesenteric lymph nodes and spleen. Mucosal biopsy production of H. pylori-specific IgG was more evenly distributed throughout the lymphoid system. These data support the contention that the Ig response to H. pylori is initiated within the gastric compartment and matures over time to a generalized IgA-dominated mucosal and IgG-dominated nonmucosal humoral immune response.

Animals↗

PubMed2000

Sympathetic decentralization abolishes increased secretion of immunoglobulin A evoked by parasympathetic stimulation of rat submandibular glands.

Salivary secretion of immunoglobulin A (IgA) in response to electrical stimulation of the parasympathetic nerve supply was assessed bilaterally in the submandibular glands of anaesthetized rats 1 week following unilateral pre-ganglionic sympathectomy (decentralization). Nerve-mediated stimulation on the non-denervated side increased IgA secretion several fold above an unstimulated rate of secretion whereas sympathetic decentralization reduced the parasympathetically stimulated secretion of IgA without affecting the basal rate. Glandular levels of IgA were increased following decentralization compared to the control glands. Salivary levels of free secretory component (FSC), the cleaved polymeric immunoglobulin receptor (plgR), were increased by parasympathetic stimulation and reduced by sympathectomy, though not as much as IgA. The decreased secretion of FSC suggests a reduced production of plgR and may account in part, for reduced IgA secretion following long-term removal of sympathetic nerve impulses.

Animals↗

PubMed2003

Antisecretory factor counteracts secretory diarrhoea of endocrine origin.

BACKGROUND: Dietary induction of antisecretory factor (AF) can reduce diarrhoea in patients with inflammatory bowel disease. Patients with neuroendocrine tumours may suffer from diarrhoea with a prominent secretory component. We studied if AF-therapy could affect this type of diarrhoea. METHODS: Six patients with the midgut carcinoid syndrome and two with metastasizing medullary thyroid carcinoma (MTC) participated. Effects of intake of AF, in the form of AF-rich egg powder (AF-egg), and induction of endogenous AF-activity by intake of specially processed cereals (SPCs) were studied. In an initial open part of the study all patients received AF-egg for 4 weeks, followed by a double-blind crossover period with SPC and control cereals (CCs) for 6 weeks each. Daily number of bowel movements at the end of each treatment period was registered. RESULTS: Treatment with AF-egg resulted in a decrease of bowel movements in seven patients (P<0.01). Registrations of bowel movements from both SPC and CC diet periods were obtained from five patients. The daily number of bowel movements was lower during the SPC-period compared to the period with CC (P<0.05). All patients had low levels of AF-activity in serum at baseline. During treatment with AF-egg, the mean level increased slightly. AF-activity was higher (P<0.05) after SPC compared to the CC diet. CONCLUSIONS: In a group of patients with endocrine diarrhoea, AF-activity could be induced, and AF-therapy reduced the number of bowel movements.

Antidiarrheals↗

PubMed1986

Clinical significance of polymeric and monomeric IgA complexes in patients with IgA nephropathy.

IgA nephropathy is currently considered an immune complex (IC) disease. However, though several groups have demonstrated the presence of IgA-IC in the sera of patients by various techniques, a correlation with clinical activity of the nephropathy has not always been found. Since these assays detect (simultaneously) polymeric and monomeric IgA-IC, the pathogenicity of these two classes of complexes could not be established. In this work, we have studied in 66 patients with IgA nephropathy the existence and significance of such IC, by means of a technique described in our laboratory, based on the specific binding of secretory component for polymeric IgA. Furthermore, IgG-ICs were also determined by the standard Raji cell assay in ELISA. The prevalence of these complexes was as follows: Multimeric (polymeric and monomeric) IgA-ICs were detected in 55% of 66 patients studied, polymeric IgA-ICs in 30%, monomeric IgA-ICs in 39%, and IgG-ICs in 46%. The intermittency of all these complexes was clearly noted in sequential examinations. A significant correlation (P less than .025) with hematuria was only found with polymeric IgA-IC, but not with multimeric IgA-IC, monomeric IgA-IC, or IgG-IC. Polymeric IgA-ICs were more frequently observed at the initial phases of the disease. Analytical ultracentrifugation showed that polymeric IgA-IC was of larger size than monomeric IgA-IC. The major pathogenicity of polymeric IgA-IC is in agreement with the finding of this immunoglobulin at the mesangial level in patients and animals with IgA nephropathy.

Adolescent↗