Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix.

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.

Histamine transport and metabolism are deranged in salivary glands in Sjogren's syndrome.

OBJECTIVE: To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS: Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS: mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION: Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to ∼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 µM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.

Exosomal secretion of death bullets: a new way of apoptotic escape?

Ovariectomy/estrogen deficiency causes selective apoptosis of the serous epithelial cells of the submandibular glands (SMG) in female mice. Because such apoptosis does not occur in healthy, estrogen-deficient male mice, it was hypothesized that dihydrotestosterone (DHT) protects epithelial SMG cells against apoptosis. The antiapoptotic effect of DHT on human epithelial HSG cells exposed to tumor necrosis factor-α and cycloheximide was studied. Correspondingly, the proapoptotic effect of androgen deficiency was studied in orchiectomized (ORX) androgen-knockout (ARKO) and wild-type (WT) mice. The health state of the SMG cells was studied with Alcian blue-periodic acid Schiff (AB-PAS) and amylase staining and transmission electron microscopy (TEM). The eventual protective antiapoptotic effect of dehydroepiandrosterone (DHEA) treatment was tested in this model. Apoptosis was assessed using immunohistochemisty of cleaved effector caspase-3 and its activator caspase-8 and the TUNEL assay. To test for the bioavailability, intracrine metabolism and sex steroid effects of DHEA, cystein-rich secretory protein-3 (CRISP-3), and leucine-isoleucine-valine transport system 1 (LIV-1) were used as androgen- and estrogen-regulated biomarkers, respectively. DHT protected HSG cells against induced apoptosis. In mice, androgen deficiency resulted in extensive activation of apoptotic caspase-8/3 cascade in serous epithelial cells. However, in salivary glands, active caspases were not translocated to nuclei but secreted to salivary ducts in exosome-like particles, which are associated with weak AB-PAS and amylase staining of the androgen-deprived cells and reduced number of intracellular secretory granules. DHEA treatment suppressed induction of proapoptotic caspases and almost normalized mucins and amylase and ultramophology of the serous epithelial cells in WT ORX but not ARKO ORX mice. According to the CRISP-3 and LIV-1 markers, DHEA probably exerted its effects via intracrine conversion to DHT.

Sex steroids in Sjögren's syndrome.

The purpose of the review is to consider pathomechanisms of Sjögren's syndrome (SS), which could explain the female dominance (9:1), the most common age of onset (40-50 years) and targeting of the exocrine glands. Estrogens seem to specifically protect secretory glandular acinar cells against apoptosis whereas lack of estrogens during menopause and climacterium specifically leads to increased apoptosis of the exocrine secretory cells. Male gonads produce testosterone and convert it in exocrine glands to dihydrotesterosterone (DHT), which is anti-apoptotic and protects against acinar cell apoptosis. Estrogen-deficient women need to produce dehydroepiandrosterone (DHEA) in the adrenal glands and convert it to DHT in exocrine glands in a complex and branching reaction network in which individual enzymatic reactions are catalyzed in forward and backward directions by a myriad of different isoforms of steroidogenic enzymes. Tailoring DHT in peripheral tissues is much more complex and vulnerable in women than in men. In SS the intracrine steroidogenic enzyme machinery is deranged. These endo-/intracrine changes impair acinar remodeling due to impaired integrin α1ß1 and integrin α2ß1 expression so that the intercalated duct progenitor cells are unable to migrate to the acinar space, to differentiate to secretory acinar cells upon contact with laminin-111 and laminin-211 specifically found in the acinar basement membrane. The disarranged endo-/intracrine estrogen/androgen balance induces acinar cells to release microparticles and apoptotic bodies and to undergo apoptotis and/or anoikis. Membrane particles contain potential autoantigens recognized by T- (TCRs) and B-cell receptors (BCRs) and danger-associated molecular patterns (DAMPs) recognized by Toll-like receptors (TLRs). In membrane particles (or carrier-complexes) antigen/adjuvant complexes could stimulate professional antigen capturing, processing and presenting cells, which can initiate auto-inflammatory and autoimmune cascades, break the self-tolerance and finally lead to SS.

Healthy human salivary glands contain a DHEA-sulphate processing intracrine machinery, which is deranged in primary Sjögren's syndrome.

Sjögren's syndrome (SS) patients have low salivary dehydroepiandrosterone (DHEA) and androgen biomarker levels, but high salivary oestrogen levels. The hypothesis was that the healthy glands contain DHEA-sulphate processing intracrine machinery; the local androgen/oestrogen imbalance suggests that this is disarranged in SS. Indirect immunofluorescence and quantitative real-time PCR (qRT-PCR) of steroid sulphatase, sulfotransferase, 3beta- and 17beta-hydroxysteroid dehydrogenases (3beta- and 17beta-HSD), 5alpha-reductase and aromatase were performed for labial salivary glands of healthy controls and persons with SS. In control acini steroid sulphatase and sulfotransferase immunoreactivities were located in the basolateral cell parts. 3Beta- and 17beta-HSD formed strong, interrupted bands along the basal cell parts. 5alpha-reductase was mainly located in acinar cell nuclei and aromatase in the apical cell membrane. All enzymes were more widespread in ducts. In SS, steroid sulphatase was weak and deranged, 3beta- and 17beta-HSD had lost their strict basal acinar cell localization and 5alpha-reductase was mainly found in the cytoplasm of the acinar cells, whereas aromatase showed similar staining in SS and controls. qRT-PCR of labial salivary glands disclosed all corresponding enzyme mRNAs with the levels of 3beta-HSD in SS being the lowest. Healthy tubuloacinar epithelial cells contain complete intracrine machineries for DHEA(-sulphate) pro-hormone processing. These enzymes have in healthy acini an organized architecture, which corresponds with DHEA uptake from the circulation, nuclear site of production of the active dihydrotestosterone (DHT) end product and production of oestrogens into saliva for export to ductal and oral epithelial cells. SS is characterized by low 3beta-HSD levels, which together with impaired subcellular compartmentalization of HSDs and 5alpha-reductase may explain the low local DHT and androgen biomarker levels in SS.

Intracrine androgenic apparatus in human bone marrow stromal cells.

It was suggested that human mesenchymal stromal cells might contain an intracrine enzyme machinery potentially able to synthesize the cell's own supply of dihydrotestosterone (DHT) from dehydroepiandrosterone (DHEA) pro-hormone produced in the adrenal cortex in the reticular zone, which is unique to primates. Indeed, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 5alpha-reductase enzyme proteins were expressed in resting mesenchymal stromal cells (MSCs) in vitro. However, the 'bridging' enzymes 17beta-HSDs, catalysing interconversion between 17beta-ketosteroids and 17beta-hydroxysteroids, were not found in resting MSCs, but 17beta-HSD enzyme protein was induced in a dose-dependent manner by DHEA. Quantitative real-time polymerase chain reactions disclosed that this was mainly due to induction of the isoform 5 catalysing this reaction in 'forward', androgen-bound direction (P < 0.01). This work demonstrates that the MSCs have an intracrine machinery to convert DHEA to DHT if and when challenged by DHEA. DHEA as substrate exerts a positive, feed-forward up-regulation on the 17beta-hydroxy steroid dehydrogenase-5, which may imply that DHEA-DHT tailor-making in MSCs is subjected to chronobiological regulation.

Human parainfluenza virus type 2 (HPIV2) induced host ADAM8 expression in human salivary adenocarcinoma cell line (HSY) during cell fusion.

BACKGROUND: The aim of the study was to investigate expression of ADAMs (A Disintegrin and A Metalloproteinase) of host cell origin during cell-cell fusion induced by human parainfluenza virus type 2 (HPIV2). RESULTS: Induction of host cell ADAM9 was observed in GMK cells, but the applicability of this model was restricted by lack of cross-reactivity of the anti-human ADAM8 antibodies with the corresponding green monkey antigens. HSG cells were not susceptible to HPIV2 virus infection. In contrast, in human parotid gland HSY cells, a natural host cell for paramyxoviruses, HPIV2 induced ADAM8 expression. ADAM8 staining increased dramatically over time from 7.9 +/- 3% at zero hours to 99.2 +/- 0.8% at 72 hours (p = 0.0001). Without HPIV2 the corresponding percentages were only 7.7% and 8.8%. Moreover, ADAM8 positive cells formed bi- (16.2%) and multinuclear cells (3.5%) on day one and the corresponding percentages on day three were 15.6% for binuclear and 57.2% for multinuclear cells. CONCLUSION: ADAM8, well recognized for participation in cell-to-cell fusion especially in osteoclast formation, is up-regulated upon formation of multinuclear giant cells after HPIV2 induction in HSY cells. The virus-HSY cell system provides a novel experimental model for study of the molecular mechanism of cell fusion events.

The influence of sex steroids on Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease affecting the exocrine glands, most typically salivary and lacrimal glands. In Sjögren's syndrome, the acinar cells of these glands are damaged and destroyed, leading to diminished secretion of saliva and tear fluid. Accordingly, the current American-European criteria of Sjögren's syndrome include xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). In addition to these sicca symptoms and signs, the diagnostic criteria require autoimmune features in the form of Sjögren's syndrome SS-A and/or SS-B autoantibodies and lymphocyte infiltrates in labial salivary glands. Majority of patients with Sjögren's syndrome are women and the diagnosis is usually done when they are 40-50 years old. The cause of Sjögren's syndrome is unknown, but taking into account the female dominance and the late onset, our hypothesis is that sex steroids play a key role in the etiology of Sjögren's syndrome. More specifically, we believe that the driving factor behind Sjögren's syndrome could be lack of androgens. It has been shown that patients with Sjögren's syndrome have low concentrations of circulating dehydroepiandrosterone sulfate (DHEA-S) compared to age-matched healthy controls. Our hypothesis is that patients with Sjögren's syndrome suffer from an insufficient local androgen effect in the exocrine target tissues of the disease because of low systemic levels and/or ineffective local intracrine handling of DHEA-S prohormone. To further clarify the role of sex steroids and the eventual deficiency of androgens, salivary glands are studied using protein markers regulated by androgens or estrogens.

Immunohistopathology of Sjögren's syndrome.

Sjögren's syndrome (SS) is characterized by keratoconjunctivitis sicca and xerostomia, which occur in an autoimmune lacrimal and salivary gland disease characterized by lymphocyte infiltrates of exocrine glands and/or Sjögren's syndrome autoantibody production. It has been reported that aquaporin-5 distribution is abnormal in SS, perhaps as a result of paracrine effect of TNF-alpha. Also the neurogenic regulation of the salivary gland is impaired in SS. Apart from functional changes, the syndrome is also characterized by structural abnormalities of the secretory acinar apparatus. The acinar basement membrane is abnormal as it lacks laminin alpha1 chain, which may impair its capability to induce the progenitor cells to differentiate to acinar cells. CRISP-3 and TMPRSS-2 can be used as androgen markers and LIV-1 and Cyr61 as estrogen markers to study the sexual dimorphism of the salivary glands. Patients with SS seem to have low concentrations of dehydroepiandrosterone, which may predispose women and the exocrine glands to this syndrome.

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.

Laminin isoform profiles in salivary glands in Sjögren's syndrome.

Five different laminin (LM) alpha, four LM-beta, and three LM-gamma chains form the 15-16 currently known approximately 400-900 kDa heterodimeric LM-monomers, which self-assemble in the lamina lucida of the basement membrane (BM) to a network, connected with nidogens and perlecans with the underlying type IV collagen network. In labial salivary glands (LSG), the structurally organizing/polarizing BM separates the tubuloacinar epithelium from the connective tissue stroma but plays regulatory roles as well. Tissue distribution of LM-alpha, -beta, and -gamma chains is described, and application of the known combinatorial rules allows some conclusions also on the corresponding distribution of the LM-trimers. Currently, known integrin (Int) and non integrin (e.g., dystroglycans and Lutheran blood group antigens) LM-receptors are described. LMs are regulated at transcriptional, translational, and posttranslational levels, together with the regulation of alternative splicing, binding partners (assembly), secretion, and degradation. In LSGs, LM-alpha1, -alpha2, and -alpha4 are only found in the acinar (not ductal) BM, LM-alpha4 also in the periductal/ interstitial stroma. Pattern recognition disclosed irregular expression in the acinar BM, suggesting some dynamic and/or regulatory role. It seems that in a female-dominant autoimmune exocrinopathy, Sjögren's syndrome (SS), LM-alpha1 and -alpha2 are decreased, together with their Int alpha1beta1 and alpha2beta1 receptors. Because LM-111/211-to-Int-alpha1beta1/alpha2beta1 interactions play a crucial role in the transdifferentiation of the intercalated duct progenitors to secretory acinar cells, acinar remodeling is impaired in SS. Disturbed hemidesmosomal Int alpha6beta4/LM-332 interactions in SS may lead to acinar cell anoikis. Interestingly, dehydroepiandrosterone (DHEA) prohormone and its intracrine androgenic dihydrotestosterone (DHT) end product upregulate at least Int alpha1beta1/alpha2beta1, whereas LM-alpha1 is upregulated by outside-in LM-111/211-to-Int-alpha1beta1/alpha2beta1 signaling. It seems that LM alterations precede the lymphocyte infiltration, suggesting that acinar BM-Int pathology, perhaps related to endo- and intracrine sex steroid metabolism, represents an early pathogenic phases in SS.

Androgens and integrins in salivary glands in Sjogren's syndrome.

OBJECTIVE: Laminin alpha1-chain normally induces intercalated duct progenitors to differentiate to acinar cells through integrin (INT) alpha1ss1 and alpha2ss1 receptors. Maintenance of acinar cells is impaired in Sjögren's syndrome (SS), which is also characterized by low levels of serum and salivary androgens. We hypothesized that androgens normally support salivary gland remodeling by upregulating either laminin alpha1 chain or its cellular alpha1 or alpha2 INT subunit-containing receptors. METHODS: Intercalated duct and acinar human salivary gland (HSG) cells and labial salivary gland (LSG) biopsies from healthy controls and patients with SS were cultured without or with sex steroids. Laminin alpha1 chain and INT alpha1 and alpha2 subunits were studied using quantitative reverse-transcription real-time polymerase chain reaction and INT alpha1 and alpha2 subunits using immunofluorescence staining. RESULTS: INT alpha1-subunit and alpha2-subunit messenger RNA (mRNA) levels were increased in intercalated duct and acinar cells by DHEA and testosterone. In contrast, laminin alpha1-chain mRNA levels were not affected. The upregulating effect of DHEA on INT subunits was also seen at the protein level. DHEA also increased mRNA levels of both INT subunits in healthy but not SS LSG. CONCLUSION: Androgens increased INT alpha1 and alpha2 subunits in tubuloepithelial cells and in healthy LSG, but in SS salivary glands this androgen regulation was defective, which is likely to contribute to defective outside-in signaling, acinar atrophy, and ductal cell hyperplasia.

Dehydroepiandrosterone (DHEA) substitution treatment for severe fatigue in DHEA-deficient patients with primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by fatigue and low levels of serum dehydroepiandrosterone/dehydroepiandrosterone sulfate (DHEA/DHEAS). Our aim was to study whether SS patients with severe fatigue and low serum DHEAS values benefit from DHEA substitution (50 mg/day). METHODS: A multicenter, investigator-based, powered, randomized controlled clinical trial (crossover, washout design) using fatigue as the primary outcome measure was performed on patients with primary SS (n = 107) who had a general fatigue score > or =14 on the 20-item Multiple Fatigue Inventory (MFI-20), combined with age- and sex-adjusted serum DHEAS values below the mean. Fatigue was assessed using MFI-20 subscales, i.e., general fatigue, physical fatigue, mental fatigue, reduced motivation, and activity (scale 4-20), and with a visual analog scale (VAS; scale 0-100). RESULTS: In an intent-to-treat analysis, a 50-mg DHEA substitution dose and placebo similarly improved fatigue. All of the MFI-20 subscales and the fatigue VAS improved from the baseline levels as a result of treatment (P < 0.001), but with negligible differences between these 2 treatments. The mean between-treatment difference was -0.1 for general fatigue (the primary outcome measure), 0.0 for physical fatigue, 0.0 for mental fatigue, 0.0 for reduced motivation, 0.3 for reduced activity, and 2.2 for the fatigue VAS. None of these differences was statistically significant. CONCLUSION: Similar to earlier results using pharmacologic doses, substitution treatment with 50 mg of DHEA in DHEA-deficient and severely tired primary SS patients does not help against fatigue better than placebo. This may relate to the prohormone nature of DHEA and its recently described defective intracrine tissue-specific conversion to active sex steroids in SS.

Androgen deficiency and defective intracrine processing of dehydroepiandrosterone in salivary glands in Sjögren's syndrome.

OBJECTIVE: .We hypothesized that in addition to dehydroepiandrosterone (DHEA) depletion, Sjögren's syndrome (SS) is characterized by local androgen deficiency in salivary glands and defects in local processing of DHEA. METHODS: Sex steroid levels in serum and saliva were measured using enzyme immunoassays. Androgen effects on salivary gland cells were analyzed using the cysteine-rich secretory protein-3 (CRISP-3) androgen biomarker. RESULTS: Serum and salivary concentrations of androgens were low in SS. Substrate to end-product ratios and correlations suggest that in SS salivary glands DHEA is effectively converted to testosterone, but that there are defects in converting testosterone further to dihydrotestosterone (DHT). In healthy controls no such phenomenon was seen, but testosterone is effectively converted to DHT. Salivary glands contained type I 5-alpha-reductase, and its inhibition with dutasteride completely blocked the upregulating effect of DHEA, but not of DHT, on CRISP-3 in human salivary gland acinar cells. CONCLUSION: DHEA and DHT upregulate CRISP-3, which is reportedly low in SS. The effect of DHEA on CRISP-3 is indirect and is inhibited by dutasteride, showing that there is intracrine processing of DHEA in salivary glands. In healthy glands, but not in SS, DHEA is effectively taken up and converted to DHT. Sex steroid concentrations in saliva in part reflect glandular uptake of DHEA-sulfate and local intracrine DHEA metabolism, which seem to be defective in SS. Our study demonstrates a prominent androgen deficiency and a defect in intracrine production of active androgens in SS salivary glands, also suggesting that salivary DHT cannot be maintained at a normal level in this female-dominant autoimmune exocrinopathy.

Low salivary dehydroepiandrosterone and androgen-regulated cysteine-rich secretory protein 3 levels in Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS), an autoimmune disease of exocrine glands, typically starts at the time of adrenopause. We undertook this study to test the hypothesis that SS is characterized by an insufficient androgen effect at the target tissue level. METHODS: We searched for androgen response elements (AREs) in the cysteine-rich secretory protein 3 (crisp-3) gene. Dehydroepiandrosterone (DHEA) responsiveness was experimentally studied using quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence staining of human submandibular gland-derived acinar cells and labial salivary gland explants with or without DHEA. Finally, glandular and salivary CRISP-3 in healthy controls and SS patients was analyzed using immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assay. Serum DHEA sulfate (DHEAS) and salivary DHEA levels were measured using a radioimmunometric method. RESULTS: Literature analysis and a search for AREs in gene banks suggested androgen dependency of human CRISP-3, and this was verified by studies of human submandibular gland acinar cells cultured with or without DHEA, in which DHEA increased CRISP-3 messenger RNA (mRNA) levels (P = 0.018). This finding was confirmed by the results of DHEA stimulation of labial salivary gland explants. Glandular CRISP-3 mRNA and protein labeling was weak and diffuse, coupled with low secretion in saliva (mean +/- SEM 21.1 +/- 2.7 mug CRISP-3/15 minutes in SS patients versus 97.6 +/- 12.0 mug CRISP-3/15 minutes in healthy controls; P < 0.0001). Compared with healthy controls, SS patients had low serum levels of DHEAS (P = 0.008) and also low salivary levels of DHEA (mean +/- SEM 224 +/- 33 pmoles versus 419 +/- 98 pmoles; P = 0.005). CONCLUSION: CRISP-3 pathology was seen in acini remote from lymphocyte foci and is apparently not secondary to local inflammation, but may represent some systemic effect in SS. Indeed, androgen deprivation in the salivary glands of SS patients is evidenced both by low salivary levels of DHEA and by low levels of DHEA-regulated CRISP-3. This may explain some of the characteristic features of SS.

Abnormal distribution of aquaporin-5 in salivary glands in the NOD mouse model for Sjögren's syndrome.

OBJECTIVE: To localize aquaporin-5 in healthy salivary gland acinar cells and to check if it is abnormally translocated in an experimental NOD mouse model for Sjögren's syndrome (SS). METHODS: Healthy BALB/c control mice and autoimmune focal adenitis NOD mice were studied. Aquaporin-5 was stained using avidin-biotin-peroxidase complex and indirect immunofluorescence staining methods, and visualized using light and laser scanning confocal microscopy. RESULTS: Aquaporin-5 was found in the apical domain of the acinar cell plasma membrane in healthy BALB/c mice. In contrast, aquaporin-5 was found in the apical and basolateral acinar plasma cell membrane in parotid, submandibular, and sublingual glands in NOD mice. This was confirmed using laser scanning confocal microscopy for optical sectioning and image reconstruction. CONCLUSION: Our findings reveal an abnormal translocation of aquaporin-5 in an experimental SS animal model and support observations that implied a similar loss of the ordered and polarized expression of aquaporin-5 in human SS labial and lacrimal glands.