B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

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 (M-P). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1 degrees ATD-LG) of two rabbit cohorts, one naive, one immunized with a LG lysate membrane fraction (P-i). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [H-3]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-I-A) 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 degrees ATD-eyes and exacerbated 1 degrees ATD-LG atrophy. IMIP were evident in 2 degrees ATD-LG as well as 1 degrees 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 I-A 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.

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.

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

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.

Autologous peripheral blood lymphocytes (PBL), activated in a mixed cell reaction when co-cultured with purified rabbit lacrimal epithelial cells, are known to induce a Sjögren's-like autoimmune dacryoadenitis and keratoconjunctivitis when injected directly back into the donor animal's inferior lacrimal gland (LG). This study shows that autoreactive lymphocytes injected subcutaneously in a site away from the LG is capable of inducing an autoimmune disease in a rabbit. Induced disease (ID) develops more slowly, taking 4weeks as compared to 2weeks in the direct injection model. Initially, both clinical symptoms and histopathology are less pronounced than in the direct injection ID model, but later the immunocytochemistry shows the same CD4+/CD8+ ratio of 4:1 for both injection methods. The finding that lymphocytes activated against lacrimal antigens can travel or home from the injection site back to the inferior and superior LG, as well as the conjunctiva, suggests that these anatomical sites may have common epitopes that induce pathogenic CD4+ T cells that produce a Sjögren's-like syndrome.

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.

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.

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.

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.

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.