Characterization of the Pelodiscus sinensis polymeric immunoglobulin receptor (P. sinensis pIgR) and its response to LPS and Aeromonas sobria.

The polymeric immunoglobulin receptor (pIgR) is one of the most vital components of mucosal immunity that plays a pivotal role in mediating transcytosis of polymeric immunoglobulin (pIg) on epithelial surfaces for protection against invading pathogens. Herein, we cloned the full-length cDNA of Pelodiscus sinensis pIgR, designated as P. sinensis pIgR, made of an open reading frame (ORF) of 1848 bp, molecular weight of 68.2 kDa and estimated isoelectric point of 7.00. The deduced P. sinensis pIgR sequence had a leader peptide, extracellular region containing four immunoglobulin-like domains (Ig like domains), transmembrane and intracellular regions comparable with other vertebrates. P. sinensis pIgR contained four Ig like domains that corresponded with mammalian D1, D3, D4 and D5 similar with reptile and avian Ig like domains. It had 40 potential phosphorylation sites, four putative N-glycosylation sites and several motifs resembling mammalian pIgR motifs. Phylogenetic analysis showed a close relationship between P. sinensis pIgR with avian and reptile pIgRs. P. sinensis pIgR basal levels were higher in the esophagus, small intestine and intestinnum crissum than in other organs of health turtles. Intragastric delivery of LPS and Aeromonassobria led to significant upregulation of P. sinensis pIgR in tissues of the gastrointestinal tract. A polyclonal anti- P. sinensis pIgR antibody produced in rabbit reacted with the recombinant P. sinensis pIgR protein expressed in Escherichia coli in Western blot. These studies demonstrate the existence and immune response of P. sinensis pIgR to stimulation in mucosal organs in Chinese soft-shelled turtles.

Identification of proteomic signatures associated with COPD frequent exacerbators.

AIMS: Acute exacerbation is a major event that alters the natural course of chronic obstructive pulmonary disease (COPD), and recurrent exacerbation results in worse clinical outcomes and greater economic consequences. While some patients suffer frequent exacerbations, others experience no exacerbations; this study was designed to detect proteins that were differentially abundant in COPD frequent exacerbators and assess whether those expression profiles are unique among COPD patients. MAIN METHODS: Tandem mass tag labeled quantitative proteomics combined with two-dimensional liquid chromatography-tandem mass spectrometry was used to detect the changes in the lung proteome in COPD frequent exacerbators and infrequent exacerbators. A series of bioinformatics analyses were performed to screen potential signatures of COPD frequent exacerbations. The accuracy of proteomic results was further verified by western blot studies. KEY FINDINGS: Compared with infrequent exacerbators, 23 proteins in the lung tissues from frequent exacerbators showed significant degrees of differential expression; combined bioinformatics analyses of proteome indicated that the immune network for IgA production and the phenylalanine metabolism pathway were associated with frequent exacerbations. The Western blot analysis confirmed the expression pattern of three significantly regulated proteins (HLA-DQA1, pIgR and biglycan). SIGNIFICANCE: These findings indicate that immune response might play a key role in the pathophysiological mechanisms of COPD frequent exacerbations. Our results make a crucial contribution to the search for a comprehensive understanding of potential pathophysiological mechanisms associated with the frequent exacerbations of COPD, and might provide guidance for treating frequent exacerbations.

Expression and prognostic significance of the polymeric immunoglobulin receptor in epithelial ovarian cancer.

BACKGROUND: High expression of the polymeric immunoglobulin receptor (PIGR) has previously been associated with a favourable prognosis in a few cancer forms, but its expression and relationship with clinical outcome in epithelial ovarian cancer (EOC) has not yet been reported. The aim of this study was therefore to examine the clinicopathological correlates and prognostic significance of PIGR expression in EOC. METHODS: After an initial screening in the Human Protein Atlas portal, a validated antibody was selected for extended analysis of immunohistochemical PIGR expression in tissue microarrays with tumours from 154 incident cases of EOC from two pooled prospective population-based cohorts. Subsets of corresponding benign-appearing fallopian tubes (n = 38) and omental metastases (n = 33) were also analysed. Kaplan-Meier analysis and Cox regression analysis were applied to examine the impact of PIGR expression on overall survival (OS) and ovarian cancer-specific survival (OCSS). RESULTS: PIGR expression was significantly higher in fallopian tubes compared to primary tumours and metastases (p < 0.001) and lower in carcinoma of the serous subtype compared to other carcinomas (p < 0.001). PIGR expression was significantly associated with lower grade (p = 0.001), mucinous histological subtype (p = 0.002), positive progesterone receptor expression (p = 0.009) and negative or low Ki-67 expression (p = 0.003). Kaplan-Meier analysis revealed a significantly improved OS (p = 0.013) and OCSS (p = 0.009) for patients with tumours displaying high expression of PIGR. These associations were confirmed in unadjusted Cox regression analysis (HR = 0.48; 95% CI 0.26-0.87; p = 0.015 for OS and HR = 0.43, 95% CI 0.22-0.82; p = 0.011 for OCSS) but did not remain significant after adjustment for age, grade and clinical stage. CONCLUSIONS: This study provides a first demonstration of PIGR expression in human fallopian tubes, primary EOC tumours and metastases. High tumour-specific expression of PIGR was found to be associated with a favourable prognosis in unadjusted, but not in adjusted, analysis. These findings are novel and merit further investigation.

COLORECTAL Polymeric Immunoglobulin Receptor Expression is Correlated with Hepatic Metastasis and Poor Prognosis in Colon Carcinoma Patients with Hepatic Metastasis.

BACKGROUND/AIMS: Increased expression of polymeric immunoglobulin receptor (pIgR) in tumor tissue has been detected in various cancer forms. However, the clinical relevance of pIgR in colon cancer hepatic metastasis remains uncertain. The aim of this study was to assess the prognostic value of pIgR in patients with colon carcinoma hepatic metastasis after hepatic resection. METHODOLOGY: Genome-wide gene expression analysis was used to evaluate the expression of pIgR in cryopreserved tissue from liver metastases of colon cancer and in the corresponding primary colon cancer tissues from one patient with hepatic metastatic colon cancer. pIgR expression was further confirmed by quantitative real-time PCR in cryopreserved primary colon carcinoma and paired hepatic metastasis tissues from 32 patients with hepatic metastatic colon cancer and by immunohistochemistry in paraffin-embedded primary colon carcinoma and paired hepatic metastasis tissues from 136 patients with liver metastasis from colon carcinoma who underwent hepatic resection. The relation between pIgR expression and clinicopathologic factors and long-term prognosis in these 136 patients was retrospectively examined. The prognostic significance of negative or positive pIgR exspression in colon carcinoma hepatic metastasis was assessed using Kaplan-Meier survival analysis and log-rank tests. RESULTS: Positive expression of pIgR was correlated with liver metastasis of colon cancer. Univariate analysis indicated significantly worse overall survival (OS) for patients with a positive pIgR expression in colon carcinoma hepatic metastasis than for patients with a negative pIgR expression. Multivariate analysis showed positive-pIgR in colon carcinoma hepatic metastasis to be an independent prognostic factor for OS after hepatic resection (P = 0.021). CONCLUSIONS: Positive expression of pIgR was statistically significantly associated with poor prognosis of patients with colon carcinoma hepatic metastasis. pIgR could be a novel predictor for poor prognosis of patients with colon carcinoma hepatic metastasis after hepatic resection.

iTRAQ reveals candidate pancreatic cancer serum biomarkers: influence of obstructive jaundice on their performance.

BACKGROUND: The aims of our study were to identify serum biomarkers that distinguish pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) patients from benign pancreatic disease patients and healthy subjects, and to assess the effects of jaundice on biomarker performance. METHODS: Isobaric tags for relative and absolute quantification were used to compare pooled serum and pancreatic juice samples from a test set of 59 and 25 subjects, respectively. Validation was undertaken in 113 independent subjects. RESULTS: Candidate proteins Complement C5, inter-α-trypsin inhibitor heavy chain H3, α1-ß glycoprotein and polymeric immunoglobulin receptor were elevated in cancer, as were the reference markers CA19-9 and Reg3A. Biliary obstruction had a significant effect on the performance of the markers, in particular within the PDAC group where the presence of jaundice was associated with a significant increase in the levels of all six proteins (P<0.01). Consequently, in the absence of jaundice, proteins showed reduced sensitivity for PDAC patients over benign subjects and healthy controls (HCs). Similarly, in the presence of jaundice, markers showed reduced specificity for PDAC patients over benign subjects with jaundice. Combining markers enabled improved sensitivity for non-jaundiced PDAC patients over HCs and improved specificity for jaundiced PDAC patients over jaundiced benign disease subjects. CONCLUSIONS: The presence-absence of jaundice in the clinical scenario severely impacts the performance of biomarkers for PDAC diagnosis and has implications for their clinical translation.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

Sputum proteomics identifies elevated PIGR levels in smokers and mild-to-moderate COPD.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood.

mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC-multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK-M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK-M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK-M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.

Absolute quantification of potential cancer markers in clinical tissue homogenates using multiple reaction monitoring on a hybrid triple quadrupole/linear ion trap tandem mass spectrometer.

Multidimensional liquid chromatography with tandem mass spectrometry with iTRAQ-labeling typically used for differential expression analysis in biomarker discovery does not always detect peptides from these biomarkers in all samples analyzed. Herein we describe the results of targeted analyses using multiple reaction monitoring (MRM) on a hybrid triple quadrupole/linear ion-trap tandem mass spectrometer. The MRM approach when combined with the newly released mTRAQ reagent, a non-isobaric variant of the iTRAQ tag available in two versions, enables absolute quantification of peptides and proteins via isotope-dilution mass spectrometry. This approach was applied to clinical endometrial tissue homogenates in an effort to quantify two endometrial cancer biomarkers, pyruvate kinase (PK) and polymeric immunoglobulin receptor (PIGR). We successfully demonstrated the feasibility of this approach on 20 individual samples and further verified the differential expressions of these two biomarkers in endometrial carcinoma. PK was determined to be present at an average concentration of 58.33 pmol/mg of total proteins and in the range of 9.13-87.66 pmol/mg in the soluble fraction of the normal proliferative endometrium homogenates. By contrast, the average concentration of PK in the cancer sample homogenates was 237.2 pmol/mg of total proteins and in the range of 66.10-570.9 pmol/mg. PIGR was found to be expressed at an average concentration of 8.85 pmol/mg of total proteins with a range of 1.02-49.61 pmol/mg in the normal proliferative control samples, and an average concentration of 200.2 pmol/mg with a range of 7.63-810.4 pmol/mg in the cancer samples. This study confirmed qualitatively the differential expressions previously observed but also showed that the actual relative differential expressions in these samples were much higher than those reported in the discovery study. These results validated earlier observations of dynamic-range compression in iTRAQ-labeling with hybrid quadrupole/time-of-flight mass spectrometry (DeSouza, L.V. et al. J. Proteome Res. 2008, 7, 3525-3534).

Different proteomic protein patterns in saliva of Sjögren's syndrome patients.

OBJECTIVE: To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects. MATERIALS AND METHODS: Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization--tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software. RESULTS: Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects. CONCLUSION: In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.

Polymeric immunoglobulin receptor-negative tumors represent a more aggressive type of adenocarcinomas of distal esophagus and gastroesophageal junction.

CONTEXT: Polymeric immunoglobulin receptor (PIgR) expression has been found in gastric mucosa and gastric cancers, but it is not known whether PIgR expression is related to background intestinal metaplasia nor the patterns of PIgR expression in tumors arising in the distal esophagus and gastroesophageal (GE) junction. OBJECTIVES: To identify clinicopathologic features of tumors associated with PIgR expression and to determine whether PIgR expression is associated with intestinal differentiation of tumors and intestinal metaplasia in background mucosa in 3 groups of upper gastrointestinal adenocarcinomas. These groups are (1) gastric adenocarcinomas, (2) adenocarcinomas of the distal esophagus and GE junction with background intestinal metaplasia, and (3) adenocarcinomas of the distal esophagus and GE junction without background intestinal metaplasia. DESIGN: Expression of PIgR and CDX2 in nonneoplastic mucosa, intestinal metaplasia, and adenocarcinomas was examined by immunohistochemistry in 42 cases: 14 gastric and 28 from the distal esophagus and GE junction, including 13 with esophageal or GE junction intestinal metaplasia. RESULTS: PIgR and CDX2 were expressed in all cases of intestinal metaplasia. PIgR expression was positive in 40% of group 3 versus 77% of group 2 and 71% of gastric adenocarcinomas (P = .06), and the expression of CDX2 was similar in all tumor groups (80%-83%). Metastatic-positive lymph nodes were more frequent in PIgR-negative tumors (94% vs 58%, P = .01). CONCLUSIONS: PIgR is uniformly expressed in intestinal metaplasia and in a subgroup of adenocarcinomas of the distal esophagus, GE junction, and stomach. Esophageal and GE junction adenocarcinomas that do not express PIgR show more frequent lymph node metastasis, suggesting that lack of expression of PIgR identifies a subgroup of more aggressive adenocarcinomas.

Effects of autonomic agonists and immunomodulatory cytokines on polymeric immunoglobulin receptor expression by cultured rat and human salivary and colonic cell lines.

OBJECTIVE: Immunoglobulin A (IgA) is transported across glandular epithelial cells by polymeric immunoglobulin receptor (plgR), with each receptor molecule participating in only one round of transcytosis. Nerve-related stimuli rapidly increase salivary secretion of IgA, while concentrations are increased in the autoimmune disease Sjögren's syndrome. Our aim here was to determine whether autonomic agonists and cytokines present in Sjögren's-affected glands can up-regulate salivary cell plgR expression. METHODS: Cultures of rat parotid acinar cells (PAR C5) and human submandibular gland ductal cells (HSG) were exposed to carbachol or adrenaline for 24 h and to interleukin-4 and/or interferon-gamma for 48 h. The human colonic cell line HT-29 served as a positive control for cytokine response. plgR mRNA was quantified by reverse transcription and real-time PCR and protein expression was examined by immunoblotting. RESULTS: Carbachol increased plgR mRNA levels significantly in all cells but adrenaline did so only with PAR cells (P<0.05). HSG and HT-29 cells both up-regulated plgR gene transcription on exposure to interleukin-4 and interferon-gamma either alone or in combination (P<0.05). By contrast, production of plgR mRNA in PAR cells tended to decrease in response to all cytokine treatments. plgR protein levels rose in line with mRNA expression in cytokine-treated HT-29 cultures (P<0.05). CONCLUSIONS: Autonomimetics can up-regulate plgR transcription in transformed and neoplastic salivary and colonic cells, although intracellular coupling mechanisms require further investigation. Immunomodulatory cytokines increased plgR expression in one of the salivary cell lines, but additional work is needed to establish whether this occurs in Sjögren's patients.

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes.

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.

Identification of parotid salivary biomarkers in Sjogren's syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis.

OBJECTIVES: To identify the most significant salivary biomarkers in Sjögren's syndrome (SS) using proteomic methods. METHODS: Parotid saliva from 20 non-SS subjects and 41 primary SS patients was analysed. Protein expression profiles for each sample were generated by surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry (SELDI-TOF-MS). Mean peak intensities of SS patients and non-SS subjects were compared by univariate analyses. Samples pooled by diagnosis (SS and non-SS) and labelled with different Cy dyes were compared by two-dimensional difference gel electrophoresis (2D-DIGE). Two protein levels that were most significantly different by SELDI-TOF-MS and 2D-DIGE were validated by enzyme-linked immunosorbent assay in individual samples. RESULTS: SELDI-TOF-MS of 10-200 kDa peaks revealed eight peaks with >2-fold changes in the SS group that differed from non-SS at P < 0.005. Peaks of 11.8, 12.0, 14.3, 80.6 and 83.7 kDa were increased, while 17.3, 25.4, and 35.4 kDa peaks were decreased in SS samples. 2D-DIGE identified significant increases of beta-2-microglobulin, lactoferrin, immunoglobulin (Ig) kappa light chain, polymeric Ig receptor, lysozyme C and cystatin C in all stages of SS. Two presumed proline-rich proteins, amylase and carbonic anhydrase VI, were reduced in the patient group. Three of these ten biomarkers have not been associated previously with SS. CONCLUSIONS: The salivary proteomic profile of SS is a mixture of increased inflammatory proteins and decreased acinar proteins when compared with non-SS. Future studies will test the ability of these biomarker levels, alone and in combination, to diagnose the salivary component of SS.

Dietary fructooligosaccharides up-regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice.

We examined whether or not dietary fructooligosaccharides (FOS) in infancy can have a beneficial effect on the mucosal immune system. Newborn BALB/c mice, accompanied by their dams until 21 days of age, were fed either a control diet based on casein [FOS- diet group] or a FOS- diet supplemented with 5% (w/w) FOS [FOS+ diet group]. Total IgA levels in tissue extracts from the intestines of mice in the FOS+ diet group at 38 days of age were about twofold higher (P < 0.05) than those in the FOS- diet group in the jejunum, ileum and colon. Ileal and colonic polymeric immunoglobulin receptor (pIgR) expression in the FOS+ diet group at 36 days of age was 1.5-fold higher than in the FOS- diet group (P < 0.05). Consistent with these results, the ileal IgA secretion rate of the FOS+ diet group at 37 days of age was twofold higher than that of the FOS- diet group (P < 0.05). Moreover, the percentage of B220(+)IgA+ cells in Peyer's patches (PP) was significantly higher in the FOS+ diet group than in the FOS- diet group (6.2%versus 4.3%, P < 0.05), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in the small intestine as well as the colon in infant mice.

Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist.

Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 microM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca2+ and secrete beta-hexosaminidase in response to acute stimulation with 100 microM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 microM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of beta-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, Gq and G11, were decreased. Additional biochemical changes included decreased contents of 47 kDa Gs and Gi3, protein kinase Calpha and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of beta-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

Use of proteomic methodology for the characterization of human milk fat globular membrane proteins.

Characterization of the major human milk fat globular membrane proteins was carried out using proteomic techniques comprising two-dimensional polyacrylamide gel electrophoresis, followed by in situ PNGase F and trypsin digestion. Matrix-assisted laser desorption/ionization quadrupole time-of-flight and electrospray ionization mass spectrometry identified seven major protein components: alpha-lactalbumin, lysozyme precursor, beta-casein, clusterin, lactotransferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein. Sequence information on the protein-associated glycans was determined by matrix-assisted laser desorption-ionization quadrupole time-of-flight hybrid mass spectrometry. This glycan analysis revealed interesting fucosylation branching patterns which may be influential in maternal protection of the newborn against bacterial and viral pathogenic attack.

Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney.

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to approximately 90% of control levels and urinary FSC and S-IgA concentrations returned to approximately 55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.

Vitamin A is required for regulation of polymeric immunoglobulin receptor (pIgR) expression by interleukin-4 and interferon-gamma in a human intestinal epithelial cell line.

The secretory immunoglobulin A (IgA) antibody response to infections of mucosal surfaces requires transport of IgA from the basal to apical surface of mucosal epithelial cells by a specific transport protein, the polymeric immunoglobulin receptor (pIgR). We have tested the hypothesis that the vitamin A metabolite all-trans retinoic acid (RA) is required for the regulation of pIgR expression by the cytokines interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in HT-29 cells, a well-differentiated human epithelial cell line derived from a colonic carcinoma. pIgR expression is upregulated by IFN-gamma and IL-4 when HT-29 cells are grown in normal media, but this upregulation was significantly lower when cells were grown in vitamin A-depleted media. Treatment with RA at concentrations from 10(-9) to 10(-5) mol/L restored normal levels of pIgR expression. The percentages of cells expressing cell-surface pIgR after 24, 48 and 72 h of treatment with RA, IL-4 and IFN-gamma were 66 +/- 10, 90 +/- 5 and 92 +/- 1, respectively, significantly higher than the percentages seen without RA treatment, which were 32 +/- 2.3, 72 +/- 1.2 and 30 +/- 7, respectively. In addition, the intensity of fluorescence of pIgR-positive cells was significantly higher in the RA-treated cultures than in the cultures without RA treatment. Similarly, pIgR mRNA levels (adjusted for beta-actin mRNA levels) in RA-supplemented cultures were 404, 105 and 949% higher at 24, 48 and 72 h, respectively, than were pIgR mRNA levels in identical cultures grown in the absence of RA. These data indicate that RA strongly interacts with IL-4 and IFN-gamma to regulate pIgR expression in HT-29 cells, suggesting that vitamin A may be required for proper in vivo regulation of IgA transport in response to mucosal infections.

Reduction in the quantity of the polymeric immunoglobulin receptor is sufficient to account for the low concentration of intestinal secretory immunoglobulin A in a weanling mouse model of wasting protein-energy malnutrition.

The main objective of this investigation was to determine the influence of protein-energy malnutrition (PEM) in weanling mice on the expression of the hepatic and intestinal polymeric immunoglobulin receptor (pigR), a molecule that transports mucosal immunoglobulin A (IgA) into the intestinal lumen. An experimental system was used that produces systemic wasting (loss of approximately 1.9% of initial body weight per day) and that exhibits fidelity to human PEM in its influence on the concentration of IgA in critical biological fluids as well as in its influence on lymphoid involution and thymus-dependent immunocompetence. Male C57BL/6J mice were allocated to a zero-time control group (19 d of age) or to groups fed for 14 d as follows: free access to a complete purified diet (19% crude protein, 17 kJ/g gross energy) or free access to a low protein diet (0.5% crude protein). The concentration and total quantity per organ of the pIgR were assessed in the liver and intestine by Western immunoblotting using an antiserum raised against the secretory component portion of rat pIgR. Malnourished mice exhibited low quantities of hepatic and intestinal pIgR relative to well-nourished controls (0.4% and 36% of control, respectively) and also exhibited a low concentration (soluble-protein basis) of hepatic pIgR (2% of control). The concentration of biliary secretory component also was low in the malnourished mice (4% of the value for well-nourished controls). Finally, Western blotting revealed an eightfold increase in serum concentration of dimeric IgA in the malnourished group relative to well-nourished mice, whereas the levels of the monomeric form and of the higher order polymers of IgA were elevated by factors of three and two, respectively. In this experimental system, decreased expression of the pIgR is sufficient to account for the low concentration of IgA that is maintained in the mucous secretions of the intestine.