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Possibilities of immunohistochemical investigation on human temporal bone.

After preliminary studies on human tonsillar tissue, lymph node tissue, or tissue from the appendix, it could be shown that after a fixation in Bouin solution or in sublimate-formaldehyde solution and subsequent embedding of the tissue in paraffinwax, the structure of the immunoglobulins present in the tissue remains intact. Even after long-term decalcification of the fixed tissue in EDTA, its structure does not alter and the immunoglobulins can be detected in high dilution either by the direct immunofluorescence method or with peroxidase-antiperoxidase. Human temporal bones from healthy individuals were fixed in Bouin solution or in sublimate-formaldehyde solution and incubated for sufficient time in EDTA decalcification solution until they were radiologically free of calcium. After embedding in paraffinwax or Paraplast, the immunoglobulins of the middle ear mucosa could be identified with the immunohistochemical methods described above. For the first time, localized immunoglobulins including the secretory component in the endolymphatic sac could also be demonstrated. The method is so sensitive that the binding of antibodies from the serum of patients with possibly immunopathological inner ear conditions can be detected by means of the indirect immunofluorescence test. Furthermore, other immunohistochemical assay methods can also be carried out with these methods (e.g. neuropeptide detection.

Decalcification Technique↗

The effects of vaccination with inactivated uropathogenic bacteria in recurrent urinary tract infections of children.

Secretory IgA (sIgA) is an important parameter in the predisposition to recurrent urinary tract infection (UTI). We investigated whether sIgA and frequency of UTI could be positively influenced by intramuscular vaccination with inactivated uropathogenic bacteria (Solco-Urovac). Ten otherwise healthy girls aged from 5 to 11 years (mean 9 1.7 years) with recurrent UTI were immunized with Solco-Urovac by i.m. injections three times at weekly intervals. A booster injection was given after 6 months. Urinary sIgA secretory component (SC) concentration was determined by radial immunodiffusion assay. Ten other age-matched girls with UTI were not immunized. Immunization therapy caused a significant reduction in the frequency of infection and an increase in urinary sIgA SC, while in the nonvaccinated group the values remained constant.

Adjuvants, Immunologic↗

[Secretory immunoglobulin A in amniotic fluid].

Secretory immunoglobulin A (S-IgA) was estimated in amniotic fluid samples by means of the single radial immunodiffusion according to Mancini. A monospecific antiserum against human secretory component was used. 163 amniotic fluid samples from normal pregnancies and risk pregnancies respectively were investigated. Within the 3rd trimenon the S-IgA content in amniotic fluid increased significantly. With respect to literature and examinations performed previously a connection between S-IgA content in amniotic fluid and fetal lung maturity seems to be possible.

Amniotic Fluid↗

The effect of recombinant human interleukin-5 on eosinophil accumulation and degranulation in human nasal mucosa.

Recombinant human interleukin-5 (rhIL-5) was administered repeatedly onto the nasal mucosa of individuals with Japanese cedar pollinosis outside the pollen season. The numbers of eosinophils and epithelial cells and the amount of eosinophil cationic protein (ECP), secretory IgA (S-IgA), and IgA in the nasal lavage fluid increased significantly after the application of rhIL-5. Responsiveness to histamine was also enhanced after the application. When S-IgA was administered onto the nasal mucosa after application of rhIL-5, the amount of ECP in the nasal lavage fluid was significantly more increased. The above findings together with the facts that IL-5 promotes production of IgA, that IgA receptors are present on eosinophils, and that rhIL-5 does not increase release of ECP from eosinophils isolated from the peripheral blood suggest that a series of possible reactions consisting of (1) IL-5--induced production of IgA from the immune-mediating cells, (2) binding of secretory components released from either serous glandular cells or epithelial cells of the nasal mucosa with IgA, (3) release of ECP from eosinophils induced by S-IgA and/or IgA, (4) epithelial damage to the nasal mucosa, and (5) development of nasal hyperreactivity to histamine.

Adult↗

Elevated platelet-derived growth factor-BB concentrations in premature neonates who develop chronic lung disease.

BACKGROUND: Chronic lung disease (CLD) in the preterm newborn is associated with inflammation and fibrosis. Platelet-derived growth factor-BB (PDGF-BB), a potent chemotactic growth factor, may mediate the fibrotic component of CLD. The objectives of this study were to determine if tracheal aspirate (TA) concentrations of PDGF-BB increase the first 2 weeks of life in premature neonates undergoing mechanical ventilation for respiratory distress syndrome (RDS), its relationship to the development of CLD, pulmonary hemorrhage (PH) and its relationship to airway colonization with Ureaplasma urealyticum (Uu). METHODS: Infants with a birth weight less than 1500 grams who required mechanical ventilation for RDS were enrolled into this study with parental consent. Tracheal aspirates were collected daily during clinically indicated suctioning. Uu cultures were performed on TA collected in the first week of life. TA supernatants were assayed for PDGF-BB and secretory component of IgA concentrations using ELISA techniques. RESULTS: Fifty premature neonates were enrolled into the study. Twenty-eight infants were oxygen dependent at 28 days of life and 16 infants were oxygen dependent at 36 weeks postconceptual age. PDGF-BB concentrations peaked between 4 and 6 days of life. Maximum PDGF-BB concentrations were significantly higher in infants who developed CLD or died from respiratory failure. PH was associated with increased risk of CLD and was associated with higher PDGF-BB concentrations. There was no correlation between maximum PDGF-BB concentrations and Uu isolation from the airway. CONCLUSIONS: PDGF-BB concentrations increase in TAs of infants who undergo mechanical ventilation for RDS during the first 2 weeks of life and maximal concentrations are greater in those infants who subsequently develop CLD. Elevation in lung PDGF-BB may play a role in the development of CLD.

Exudates and Transudates↗

The pleomorphic adenoma of salivary glands transplanted on athmymic mice. A lightmicroscopical and immunohistochemical investigation.

10 pleomorphic adenomas of the human parotid gland were transplanted on several groups of nude mice. For comparative reasons, 10 other pleomorphic adenomas, a neurinoma and a chordoma and transplants of squamous cell carcinomas and of normal salivary gland tissue were also analysed. In the primary tumours and in the transplants, the presence of keratin, carcinoembryonic antigen, tissue polypeptide antigen, lactoferrin, lysozyme, immunoglobulins, secretory component, amylase, fibronectin and of several lectin-receptors (PNA, WGA, HPA, Ulex europaeus) was sought. The immunohistological observations show that many of the features of a pleomorphic adenoma are constant under the conditions of transplantation. In the transplanted tumour, the same heterogeneity as in the primary tumours can be observed. Autoradiographic studies show little labelling with 3-H thymidine, which is in good accordance with the biological behaviour of the tumour. The distribution of fibronectin shows an interesting association with myoepithelial-like cells. Our results support the hypothesis that the histogenetic origin of the pleomorphic adenoma is a cell pool of the terminal ductal segment. A differentiation towards ductal cells (with production of secretory substances) and towards myoepithelial cells (associated with large amounts of basal membrane like substances) is observed.

Adenoma, Pleomorphic↗

Immunoglobulin-containing cells in jejunal mucosa of children with protein-energy malnutrition and gastroenteritis.

Jejunal biopsies from 20 well nourished children (average age 12.8 months) with gastroenteritis, and 20 children (average age 20 months) with protein-energy malnutrition were examined by immunofluorescent technique for immunoglobulins A, G, M, E, and D, and for epithelial glycoprotein secretory component. Compared with previous studies on normal infants, the children with gastroenteritis showed a moderate increase in IgA-containing cells, a large increase in IgM-containing cells, and no change in IgG-containing cells. These findings are similar to previously recorded findings on adults with gastroenteritis. In contrast there was a pronounced and highly significant decrease in IgA-containing cells in the jejunal mucosa of the children with protein-energy malnutrition. No significant differences were noted between the populations of IgG-, IgM-, IgE-, and IgD-containing cells in the two groups. It is suggested that this selective deficiency in mucosal IgA results from a delay in maturation of the secretory IgA system, and the mechanisms of such a deficiency are discussed.

Child, Preschool↗

Isolation of secretory IgA from feline bile and bile IgA levels in growing cats.

Secretory IgA was isolated from feline bile, using ammonium sulphate precipitation, gel-filtration chromatography and affinity chromatography. It formed a precipitating line between anti-cat whole serum or anti-cat bile serum by immunoelectrophoresis and double diffusion. It consisted of three subunits that molecular weight were 80 kd, 62 kd and 27 kd by SDS-PAGE analysis. The 80 kd protein was equivalent to secretory component of human and other animals. Immunoglobulin levels were observed on bile collected continuously by gallbladder cannulation in growing cats. Although immunoglobulins were not detected in bile of feline fetus, after birth its levels increased gradually. Biliary IgA levels reached the adult levels earlier than that of their serum.

Aging↗

[Secretory immunoglobulin A (S-IgA) in amniotic fluid and in pharyngeal mucus and urine of newborn infants].

S-IgA was estimated in amniotic fluid and in mucus samples of pharyngeal cavities and urine samples of newborns for assistance of the hypothesis that there is a connection between S-IgA content in amniotic fluid and fetal pulmonary maturity. It was used an antiserum against human secretory component and an S-IgA standard in the single radial immunodiffusion according to Mancini and coworkers. High S-IgA concentrations in the mucus samples of pharyngeal cavities refer to fetal bronchial-pulmonary system as the probable essential part in the synthesis of S-IgA established in amniotic fluid of the 3rd trimenon in pregnancy.

Amniotic Fluid↗

Secretory immunoglobulin A in oral and respiratory passages in man.

Secretory IgA (SIgA) is the predominant immunoglobulin in certain external secretions and may have an important role in immunological mucosal resistance. SIgA differs in chemical and immunological properties from serum IgA. The present study was undertaken to investigate the antigenic relationship between SIgA, free secretory component (FSC) and serum IgA and the localization of SIgA as well as other immunological classes in tissues of oral and respiratory passages by use of immunofluorescence technique. SIgA and FSC were highly purified from human colostrum and rabbit anti-SIgA and anti-SC antisera were prepared. On the basis of antigenic relationships between SIgA, FSC and serum IgA, it was emphasized that individual specific antisera for SC and IgA and/or SIgA should be used in immunochemical or immunohistological investigations for SIgA. The present study failed to detect SC determinants in palatine and lingual tonsils. However, it was evident that cells present in the pharyngeal tonsillar epithelium contain SC determinants. SC molecules may be synthesized in certain secretory cells of mucous membrane and glandular epithelium and the combining of SC with IgA could occur in the cytoplasm of epithelial cells, the intercellular spaces and/or in the lumens of glandular acini and ductules.

Adenoidectomy↗

Gut-liver interactions in the IgA system.

Recent experimental work in rodents has demonstrated that the liver can function as an 'IgA-pump', transporting polymeric IgA from serum to bile via a secretory-component mediated uptake at the surface of the hepatic parenchymal cell. Studies are surveyed which indicate that, although this process occurs in man, it is to a strikingly lesser degree, and interruption of this pathway does not explain the high serum IgA levels often seen in chronic liver disease: the explanation for this lies in enhanced synthesis of IgA, and diminished polymeric IgA catabolism unrelated to hepatobiliary transport.

Animals↗

IgA-associated glomerular deposits in liver disease.

IgA-associated immunopathologic renal injury has been reported in patients with cirrhosis. In an effort to elucidate the pathogenesis of this phenomenon, the livers and kidneys obtained at autopsy from a group of patients with cystic fibrosis were studied; these patients were selected because of their broad spectrum of liver abnormalities. On the basis of histologic examination of sections of liver, the patients were divided into two groups: Group I (20 patients) included patients with focal biliary cirrhosis and multilobular biliary cirrhosis; all had anatomic distortion of the biliary system, and many had cholestasis. Group II (28 patients) showed no bile duct anomalies. Immunofluorescence studies of the corresponding kidneys for immunoglobulin, complement, and free secretory component (FSC) revealed significantly more numerous IgA-containing glomerular deposits in group I (P less than 0.02). Although FSC was virtually absent in these deposits, significant in vitro binding of this protein revealed the polymeric nature of the glomerular IgA. This is consistent with previous observations of elevated serum levels of polymeric IgA, which forms the dominant component of glomerular deposits in cirrhotic patients. Since IgA glomerular deposition occurred in patients with focal biliary and no hepatocellular dysfunction, it seems that the source of this polymeric IgA is related to its impaired serum clearance by a distorted and stagnant bile duct system. However, the mechanism that leads to the deposition of this immunoglobulin in the glomeruli and other tissues remains conjectural.

Bile Ducts↗

Amelioration of habu venom-induced glomerular lesions: potential role for platelet secretory proteins.

Platelets have been implicated as mediators of mesangial cell proliferation. Of interest is a potential role for platelet secretory proteins (some of which are known to be growth factors) in proliferative glomerular disease. This study examines the effect of sulindac, an inhibitor of platelet thromboxane A2 generation and platelet activation, on the development of glomerular cystic and proliferative lesions after injection of habu snake venom (HSV). To examine the association of platelet secretory proteins with glomerular lesions after HSV, antiserum against a pool of platelet secretory cationic proteins (PSCPs) was used, by immunofluorescence, as a marker of the secretory component of platelet activation in platelet-compromised and normal rats. Uninephrectomized rats received sulindac (60 mg/kg body weight) or vehicle daily before and after HSV (2 mg/kg body weight, IV). Glomerular cysts, proliferative nodules, and mixed lesions (cystic plus proliferative) were quantitated and PSCP localization was examined 48 hours after HSV. Sulindac substantially reduced the total number of glomerular lesions and preferentially reduced proliferative lesions when compared with controls. PSCPs localized in glomerular lesions in both groups and paralleled the severity of disease, but overall intensity of PSCP staining was less in sulindac-treated rats. Sulindac did not alter renal function before HSV, ruling out hemodynamic factors. The concomitant localization of PSCPs in glomerular lesions and amelioration by antiplatelet therapy supports a role for platelet secretory proteins in this model of proliferative glomerular disease.

Animals↗

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition.

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.

Animals↗

Secretion of immunoglobulins and plasma proteins from the colonic mucosa: an in vivo study in man.

There are no available data on immunoglobulins and albumin outputs into the normal human colon. We thus measured the intracolonic secretion rates of IgA, IgG, IgM, secretory component (SC) and plasma proteins (albumin (Alb), orosomucoid (Oro), transferrin (Transf) and alpha 2-macroglobulin (alpha 2-M)). Using a pancolonic perfusion technique in 10 healthy volunteers (six females, four males, mean age 24 years), concentrations and outputs of Alb, immunoglobulins, SC, Oro, Transf and alpha 2-M were measured in the rectal effluents by immunoradiometric assay. Monomeric (m) and polymeric (p) IgA distribution was analysed by sucrose density ultracentrifugation. The secretion of polymeric IgA (p-IgA) was 153 micrograms/min, i.e. 220 mg/day, exceeding that of other immunoglobulins (m-IgA 8.5 micrograms/min; IgG 33.5 micrograms/min; IgM 17 micrograms/min) and of non-immunoglobulin proteins (Alb 104 micrograms/min; Oro 9 micrograms/min; Transf 7 micrograms/min; alpha 2-M 4.5 micrograms/min). p-IgA was entirely linked to SC (secretory IgA) and 12% of SC was in free form. About 62% of total IgA was IgA2. For each protein, a relative coefficient of excretion (RCE) was calculated (colon to serum concentration ratio expressed relative to that of Alb). The p-IgA, IgM and m-IgA RCE were 277, 6 and 2.2 times higher than the values predicted from their molecular weight. RCE of non-immunoglobulin proteins also exceeded the values expected from a passive seepage from the vascular compartment. The intracolonic clearance of Alb extrapolated to 24 h was only 3.7 ml/day. These results show the high local production and/or the facilitated transport to the colonic lumen of p-IgA, and are in very good agreement with the distribution of plasma cells in the colonic mucosa.

Adult↗

Immunobiology of the reproductive tract in a female baboon.

The aim of this study was to assess the suitability of various antihuman antibodies directed against immunocomponent cells to identify components involved in cellular and humoral immune responses in the immune organs of a female baboon, and to use these reagents to analyze the immunobiology of its reproductive tract. A female baboon of reproductive age was euthanized in the luteal phase of the menstrual cycle, and samples of spleen, intestines, tonsil, lymph nodes, Fallopian tube, uterus, cervix, and vagina were removed. Tissues were either fixed in 10% unbuffered formaldehyde, Bouin's fluid, or 95% ethanol containing 5% glacial acetic acid, and embedded in paraffin, or frozen unfixed. Frozen sections were then fixed in 100% acetone. Subsequently, tissue sections were reacted with the following antihuman antibodies directed against CD3, CD45RA, CD45RO, CD4, CD8, CD20, CD68, HLA-DR, CD57, CD103, CD15, and TIA-1: IgA, IgG, IgM, J-chain, secretory component, and neutrophil elastase, using routine immunohistology techniques. Human tissues (spleen, small intestine, lymph node, and tonsil) were used as positive controls. All antihuman antibodies crossreacted with baboon tissues, except neutrophil elastase, CD15, CD45RO, CD57, and CD1A. The distribution of immune cells in the reproductive tract of the female baboon was comparable to that in the human and offers the potential for this primate to be used as a model for the study of human reproductive immunology.

Animals↗

Production of secretory IgA antibodies in plants.

Functional antibodies produced in tobacco plants were first reported over a decade ago (1989). The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross. In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants. In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences. Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation. A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed. Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component. Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa [(Ann. NY Acad. Sci.)721(1994)235; (Biotechnol. Bioeng.)20(1999)135]. Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth. The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans. N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose. No adverse effects or human anti-mouse antibodies (HAMA) have been observed in >40 patients receiving topical oral application of a plant produced secretory IgA specific to Streptococcus mutans, for the control of caries [(Nat. Med.)4(1998)601]. The progressive improvement of expression vectors for plantibodies, and purification strategies, as well as the increase in transformable crop species, is expected to lead to almost limitless availability of inexpensive (even edible forms of) recombinant immunoglobulins free of human pathogens for human and animal therapy, and for novel industrial applications (e.g. catalytic antibodies).

Antibody Formation↗

Clustering of IgA-producing immunocytes related to HLA-DR-positive ducts in normal and inflamed salivary glands.

Ig-producing immunocytes and epithelial expression of secretory component (SC) and HLA-DR were evaluated by two-colour immunofluorescence staining in 10 normal and 20 inflamed salivary glands; the latter included specimens from 10 patients with obstructive sialadenitis and 10 with Sjögren's syndrome (SS). Epithelium adjacent to T-cell infiltrates showed extensive co-expression of SC and HLA-DR, suggesting that leucocyte-derived cytokines were responsible for this concurrent up-regulation. Clusters (greater than 2 positive cells) of IgA-producing cells were spatially related to DR-positive ducts. The possibility is discussed that DR-expressing epithelium contributes to local terminal differentiation of IgA-producing plasma cells. A cytokine-mediated up-regulation of SC that simultaneously increases the transport capacity for polymeric IgA would constitute an efficient enhancement of secretory immunity in diseased glandular tissue.

Adult↗