Molecular Evidence for Precursors of Sjögren's Foci in Histologically Normal Lacrimal Glands.

Understanding the formation of Sjogren's lymphocytic infiltrates could permit earlier diagnosis and better outcomes. We submitted gene transcript abundances in histologically normal rabbit lacrimal glands to principal component analysis. The analysis identified a cluster of transcripts associated with Sjögren's foci, including messenger RNAs (mRNAs) for C⁻X⁻C motif chemokine ligand 13 (CXCL13) and B-cell activating factor (BAFF), which dominated the major principal component. We interpreted the transcript cluster as the signature of a cluster of integrally functioning cells. Pregnancy and dryness increased the likelihood that the cluster would develop to high levels, but responses were subject to high levels of stochasticity. Analyzing microdissected samples from high- and low-cluster-level glands, we found that certain transcripts, including mRNAs for C⁻C motif chemokine ligand 21 (CCL21), CXCL13, cluster of differentiation 4 (CD4), CD28, CD25, BAFF, and interleukin 18 (IL-18) were significantly more abundant in immune cell clusters (ICs) from the high-cluster-level gland; mRNAs for CCL2, CD25, and IL-1RA were significantly more abundant in acinus-duct axis samples; mRNAs for CCL4, BAFF, IL-6, and IL-10 were more abundant in some acinus-duct samples; cells with high prolactin immunoreactivity were more frequent in interacinar spaces. In conclusion, integrated functional networks comprising Sjögren's infiltrates, such as ICs, acinar cells, ductal cells, and interacinar cells, can form in histologically normal glands, and it is feasible to detect their molecular signatures.

A randomized, controlled study to evaluate the efficacy of intra-articular, autologous adipose tissue injections for the treatment of mild-to-moderate knee osteoarthritis compared to hyaluronic acid: a study protocol.

BACKGROUND: Osteoarthritis (OA) is a highly debilitating joint disease that causes progressive, irreversible damage to articular cartilage. OA takes a massive toll on society that has grown in recent decades, but no therapy has been shown to halt or reverse the progression of the disease. The critical need for better treatments and increased interest cellular therapies has spawned a new generation of "minimally manipulated" cell treatments. Autologous adipose tissue injections are among the most controversial of these new treatments. Despite a lack of clinical evidence, adipose tissue injections are often marketed as "stem cell" injections with wide-ranging regenerative benefits. The purpose of this study is to estimate the effect size of the treatment by comparing the efficacy of autologous fat to hyaluronic acid (HA). As a secondary aim, we will test for preliminary evidence of efficacy of autologous fat vs. HA. METHODS: This is a prospective, single-center, parallel-group, randomized, controlled trial. Participants (n = 54) will receive either a single intra-articular, ultrasound-guided injection of autologous adipose tissue or a single intra-articular, ultrasound-guided injection of HA (1:1 ratio). Outcome data will be obtained at baseline, week-6 and month-6. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain domain (WOMAC-A) will be used as the primary outcome measure. Secondary clinical outcome measures include WOMAC (full), clinical anchors (pain, function, and stiffness), and the 29-point Patient-Reported Outcomes Measurement Information System (PROMIS®) profile. We will also take synovial fluid samples and assess sway velocity using a force plate, as well as analyze excess/discard adipose tissue to gain a better understanding of how intra-articular adipose tissue injections influence the biochemical environment of the joint. DISCUSSION: Given the widespread use of intra-articular fat injections in the United States, it is critical that randomized, controlled human studies evaluating efficacy and biological activity be performed. This study is the first step in addressing this unmet need, but it is not without limitations. The most notable limitations of this study are its small size and lack of blinding, which predisposes the study to both investigator and participant bias. TRIAL REGISTRATION: NCT03242707 // HS-17-00365 // Registration Date (First Posted): August 8, 2018.

Pregnancy probabilistically augments potential precursors to chronic, immune-mediated or autoimmune lacrimal gland infiltrates.

PURPOSE: This study asked whether pregnancy, a risk factor for dry eye disease associated with both chronic, immune-mediated- and autoimmune etiologies, augments development of clusters of coordinately functioning cells (CCFC) that may be precursors to pathological lacrimal gland infiltrates. METHODS: Lacrimal glands were from six virgin- and six term-pregnant rabbits of the same age and environmental exposure history. Seventy-two immune response-related gene transcripts were assayed by real time RT-PCR. Principal component (PC) analysis identified transcript signatures of CCFC contributing negative (⊖) or positive (⊕) PC loadings and determined gland PC projections, which reflect levels of CCFC development. RESULTS: Three CCFC were of interest as potential precursors to pathological infiltrates. CCFC 1⊖ was suggestive of an ectopic lymphoid structure with resting T cells and B cells. CCFC 1⊕ was suggestive of an immune-mediated infiltrate with TH1 cells and mature, cytotoxic B cells. CCFC 2⊖ was suggestive of an ectopic lymphoid structure with activated T cells, mature B cells, germinal center, and plasmacytes. CCFC 4⊖ and CCFC 5⊖ also included plasmacytes. Pregnancy augmented CCFC 1⊖ in some glands; augmented CCFC 1⊕ in others; and augmented CCFC 2⊖, CCFC 4⊖, and CCFC 5⊖ different combinations. CONCLUSIONS: Potential precursors of pathological infiltrates form in the lacrimal glands by the time of sexual maturity. Pregnancy augments lacrimal gland plasmacyte populations, and it can augment development of potential precursors to either chronic, immune-mediated infiltrates or autoimmune infiltrates of various phenotypes. Systemic and strictly local, probabilistic phenomena interact with pregnancy to determine which combinatorial phenotypes are favored.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes.

PURPOSE: To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. METHODS: Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1°ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). RESULTS: Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1°ATD-eyes and exacerbated 1°ATD-LG atrophy. IMIP were evident in 2°ATD-LG as well as 1°ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. CONCLUSIONS: The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms.

Dry eye in postmenopausal women: a hormonal disorder.

OBJECTIVE: This review examines the etiology and pathophysiology of dry eye disease in postmenopausal women, and describes the steroid reproductive hormone influences that may contribute to its development. METHODS: We have reviewed the relevant studies on dry eye disease related to hormonal status and hormone therapy (HT) in both animal models and humans. RESULTS: Although both low and high estrogen levels have been associated with symptoms of dry eye disease, low androgen levels are a more consistent factor in its etiology. Postmenopausal HT with estrogen or estrogen plus progestogen has shown a limited benefit for dry eye symptoms and may even result in progression of meibomian gland dysfunction, decreased tear film break up time, and tear flow reduction. However, systemic or local androgen treatment has shown promising results in improving dry eye symptoms. CONCLUSIONS: Because of the high incidence of dry eye among postmenopausal women that may be related to the hormonal treatment, we propose that a multidisciplinary approach should be considered between gynecologists and ophthalmologists in management of this disorder.

Potentially pathogenic immune cells and networks in apparently healthy lacrimal glands.

Lacrimal glands of people over 40 years old frequently contain lymphocytic infiltrates. Relationships between histopathological presentation and physiological dysfunction are not straightforward. Data from rabbit studies have suggested that at least two immune cell networks form in healthy lacrimal glands, one responding to environmental dryness, the other to high temperatures. New findings indicate that mRNAs for several chemokines and cytokines are expressed primarily in epithelial cells; certain others are expressed in both epithelial cells and immune cells. Transcript abundances vary substantially across glands from animals that have experienced the same conditions, allowing for correlation analyses, which detect clusters that map to various cell types and to networks of coordinately functioning cells. A core network--expressing mRNAs including IL-1α, IL-6, IL-17A, and IL-10--expands adaptively with exposure to dryness, suppressing IFN-γ, but potentially causing physiological dysfunction. High temperature elicits concurrent increases of mRNAs for prolactin (PRL), CCL21, and IL-18. PRL is associated with crosstalk to IFN-γ, BAFF, and IL-4. The core network reacts to the resulting PRL-BAFF-IL-4 network, creating a profile reminiscent of Sjögren's disease. In a warmer, moderately dry setting, PRL-associated increases of IFN-γ are associated with suppression of IL-10 and augmentations of IL-1α and IL-17, creating a profile reminiscent of severe chronic inflammation.

Diverse mediators modulate the chloride ion fluxes that drive lacrimal fluid production.

PURPOSE: To learn whether locally expressed and systemic mediators might modulate the cholinergically induced transepithelial Cl(-) fluxes that underlie lacrimal fluid production. METHODS: Reconstituted epithelial monolayers were exposed to a submaximal dose of the muscarinic agonist, carbachol (CCh), or to one of several paracrine mediators for 18 hours, then acutely stimulated with an optimal dose of CCh. Secretory Cl(-) fluxes were assessed as negative short-circuit currents (ISC). RESULTS: Exposure to IL-6 at concentrations of 1 and 10 ng/mL and IL-1ß at 10 ng/mL significantly decreased CCh-induced Cl(-) secretion. Prolactin decreased CCh-induced Cl(-) secretion, but the extent of the decrease diminished as the prolactin concentration increased from 20 to 200 ng/mL. CCh, 10 µM, prevented CCh, 100 µM, from eliciting Cl(-) secretion. Exposure to histamine, 10 mM, prevented formation of confluent monolayers. Exposure to histamine, 1 mM, decreased CCh-induced Cl(-) secretion, whereas exposure to 5-HT, 1 mM, potentiated CCh-induced Cl(-) secretion. CONCLUSIONS: Chronic exposure to inflammatory cytokines may significantly impair cholinergically induced lacrimal fluid production. Concentrations of prolactin within the high range of normal values also may impair fluid production, but this effect is reversed at levels associated with pregnancy. Autonomic neurotransmitters and paracrine mediators that signal through different G protein-coupled receptors appear to exert varying influences, which range from complete suppression to potentiation of cholinergically induced fluid production. Thus, some hormones and paracrine mediators may impair secretion in apparently homeostatic glands as well as diseased glands, whereas mediators produced by certain immune cell infiltrates may actually enhance fluid formation.

Autoimmune dacryoadenitis and sialadenitis induced in rabbits by intravenous injection of autologous lymphocytes activated ex vivo against lacrimal antigens.

PURPOSE: Autologous peripheral blood lymphocytes, activated in a mixed cell reaction when cocultured with purified rabbit lacrimal epithelial cells, are known to induce a severe autoimmune dacryoadenitis when injected directly into the donor animal's remaining inferior lacrimal gland (LG) or subcutaneously at a site remote from the LG. The purpose of the present study was to determine the ability of intravenously (IV) injected autologous stimulated lymphocytes to home to the LG and salivary gland (SG) and induce disease. METHODS: One inferior LG was surgically excised from each rabbit. Acinar epithelial cells were purified, cultured for 2 days, gamma-irradiated, and cocultured for 5 days with purified autologous peripheral blood lymphocytes. The activated lymphocytes were used for autoadoptive transfer. RESULTS: Tear production was reduced 50% by 4 weeks and tear breakup time was 70% less than normal. Ocular surface defects assessed by rose bengal staining were present but not as pronounced as after direct injection. Four weeks after IV injection, as after direct injection, glands contained large infiltrates composed of predominantly CD4(+) T cells close to interlobular and intralobular ducts; however, they also contained unique areas of streaming lymphocytes. Histopathology at 8 weeks was more severe than at 4 weeks, and SG also showed clusters of abnormal epithelial cells and streaming lymphocytes. CONCLUSIONS: Lymphocytes activated against lacrimal antigens and injected IV can home to the LG and SG and initiate autoimmune processes, suggesting that these sites constitutively contain not only antigen-presenting cells displaying potentially pathogenic autoantigen epitopes but also chemokines and homing molecules that recruit CD4(+) T cells. This new rabbit model more closely mimics Sjögren syndrome, in that SG manifestations accompany the LG disease. It should be well suited to elucidating Sjögren pathogenesis and pathophysiology and to evaluating experimental therapies.

A lacrimal gland is a lacrimal gland, but rodent's and rabbit's are not human.

Research into the physiological processes governing both normal and abnormal functions of the lacrimal gland has used animal models to provide insights that might be applied to improving our understanding of human disease and designing of beneficial therapeutic interventions. Animal models most frequently used are mice, rats, and rabbits. As participants in research into normal and abnormal lacrimal gland function, the authors have observed significant differences between the various animal models, and these differences must be considered in investigational studies. This review summarizes a wide range of topics, including structural organization of the lacrimal gland and the immunological, secretomotor and hormonal processes regulating lacrimal gland function in all three animal models. In addition, comparisons with relevant aspects of the human lacrimal gland are included where permitted by available data.

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

PURPOSE: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease. METHODS: Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS: CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group. CONCLUSIONS: Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

PURPOSE: To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID). METHODS: Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression. The ID group serving as control also received doxycycline. All LGs were removed 16 weeks after disease induction. RESULTS: Clinical symptoms showed overall improvement in the ID/Rx group compared with the ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci and areas of altered or distorted acini, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18(+) cells was almost fivefold lower in the ID/Rx group than in the ID group. Although the total number of RTLA(+) cells did not differ between the groups, the CD4/CD8 ratio was 16-fold smaller in the ID/Rx group. CONCLUSIONS: Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. Quantitative immunohistochemical analysis suggested that the therapy might not have been simply immunosuppressive but rather supported the induction of CD8(+) regulatory cells.

Long-term topical cyclosporine treatment improves tear production and reduces keratoconjunctivitis in rabbits with induced autoimmune dacryoadenitis.

PURPOSE: To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA). METHODS: Autoimmune dacryoadenitis was induced by injecting autologous peripheral blood lymphocytes, which had been activated in a mixed cell reaction with acinar cells isolated from one inferior lacrimal gland (LG), back into the donor animal's remaining inferior LG. Schirmer's test, tear breakup time, and rose Bengal staining were assessed. Animals with established disease were treated topically with either CsA or Endura twice daily for 5 months. RESULTS: Without treatment tear production and tear stability were abnormal for 6 months, and clear signs of ocular surface defects were evident. Severe immune cell infiltration was observed in the LG. Long-term CsA treatment increased tear production only slightly, but the severity of LG histopathology decreased noticeably. CD4(+) T-cell infiltration of the LG was decreased and infiltration by MHC class II-expressing cells was also decreased. For the Endura-treated group tear production did not improve, rose Bengal scores remained high, and histopathology showed infiltration comparable to the untreated group, but by the end of the study the tear breakup time did improve. CONCLUSIONS: The rabbit model of autoimmune dacryoadenitis had signs of chronic dry eye disease 6 months after induction of disease. Tear production improved slightly with CsA treatment and CD4(+) T-cell infiltration decreased significantly in the LG. This suggests that some Sjögren's patients may benefit from long-term CsA treatment.

Microporous poly(L-lactic acid) membranes fabricated by polyethylene glycol solvent-cast/particulate leaching technique.

With the eventual goal of developing a tissue-engineered tear secretory system, we found that primary lacrimal gland acinar cells grown on solid poly(L-lactic acid) (PLLA) supports expressed the best histiotypic morphology. However, to be able to perform vectorial transport functions, epithelia must be supported by a permeable substratum. In the present study, we describe the use of a solvent-cast/particulate leaching technique to fabricate microporous PLLA membranes (mpPLLAm) from PLLA/polyethylene glycol blends. Scanning electron microscopy revealed pores on both the air-cured ( approximately 4 microm) and glass-cured sides (<2 microm) of the mpPLLAm. Diffusion studies were performed with mpPLLAm fabricated from 57.1% PLLA/42.9% polyethylene glycol blends to confirm the presence of channelized pores. The data reveal that glucose, L-tryptophan, and dextran (a high molecular weight glucose polymer) readily permeate mpPLLAm. Diffusion of the immunoglobulin G through the mpPLLAm decreased with time, suggesting the possible adsorption and occlusion of the pores. Cells cultured on the mpPLLAm (57.1/42.9 wt%) grew to subconfluent monolayers but retained histiotypic morphological and physiological characteristics of lacrimal acinar cells in vivo. Our results suggest that mpPLLAm fabricated using this technique may be useful as a scaffold for a bioartificial lacrimal gland device.

Cytopathology and exocrine dysfunction induced in ex vivo rabbit lacrimal gland acinar cell models by chronic exposure to histamine or serotonin.

PURPOSE: Lacrimal immunohistopathology has diverse clinical presentations, suggesting that inflammatory mediators exert diverse influences. Chronic exposure to agonistic acetylcholine receptor autoantibodies has been studied previously; the present work addressed mediators that signal through other G protein-coupled receptors. METHODS: Acinus-like structures and reconstituted acinar epithelial monolayers from rabbit lacrimal glands were exposed to varying concentrations of histamine or 5-hydroxytryptamine (5-HT) for 20 hours. Net and vectorial beta-hexosaminidase secretion, cytosolic Ca(2+) (Ca(i)) elevation, apical recruitment of p150(Glued), actin microfilament meshwork organization, and ultrastructure were assessed. RESULTS: Histamine and 5-HT acutely stimulated beta-hexosaminidase secretion at lower, but not higher, concentrations. Neither of them acutely elevated Ca(i) levels. Both recruited p150(Glued) at concentrations that failed to induce secretion. Chronic exposure to 10 mM histamine inhibited carbachol (CCh)-induced beta-hexosaminidase secretion and prevented the formation of continuous monolayers; 1 mM 5-HT partially inhibited secretion at the apical medium. Neither altered secretion to the basal medium. Chronic exposure to histamine or 5-HT partially decreased CCh induced Ca(i) elevations and p150(Glued) recruitment, even at concentrations that did not inhibit secretion. Both expanded acinar lumina and thickened microfilament meshworks, and both caused homotypic fusion of secretory vesicles and formation of aqueous vacuoles in the apical and basal cytoplasm. Chronic exposure to forskolin, which activates adenylyl cyclase, induced similar cytopathologic changes but impaired secretion modestly and only at the highest concentration tested. CONCLUSIONS: Inflammatory mediators that signal through G protein-coupled receptors cause acinar cell cytopathology and dose-dependent reductions of CCh-induced beta-hexosaminidase secretion. Although agonistic acetylcholine receptor autoantibodies may cause pervasive functional quiescence, inflammatory mediators may cause varying degrees of exocrine dysfunction.

Animal models of dry eye.

BACKGROUND: The causes of dry eye include lacrimal gland insufficiency, meibomian gland dysfunction, impairment of the neuronal innervation and environmental stress - all leading to irritation of the ocular surface. Several animal models have been developed to imitate different pathophysiologic mechanisms in the development of dry eye. Understanding the characteristics and limitations of these models will help researchers choose the right models to address specific problems and develop new treatment modalities in dry eye. METHODS: Medline searches were performed to identify English language articles relating to different animal models of dry eye. Manual cross-referencing was also performed and some historical articles were included. RESULTS AND CONCLUSION: A huge variety of animal models exists, mimicking different pathophysiologic mechanisms which can cause dry eye. The mouse is the model most commonly used to study autoimmune mechanisms, because of the diversity of different knockout and transgenic strains and good availability of antibodies. For studying dry eye signs, rabbit or dog models are more suitable, because they present decreased tear secretion and ocular surface changes, have longer lifespans, and offer better accessibility of the ocular surface. For studying special causes of dry eye, such as defects of neuronal reflex loops, environmental changes, or evaporative dry eye, the model of choice should recapitulate the underlying pathophysiologic mechanism.

Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells.

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds.

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib.

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2-inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress-associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA-mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes.

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells.

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of beta-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of beta-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.