D. M. Samant

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 animals remaining inferior LG. Schirmers 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 Sjögrens patients may benefit from long-term CsA treatment.

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 Sjögrens-like autoimmune dacryoadenitis when injected directly back into the donor animals 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 groups 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.

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.

Visual functional effects of constant blue light in a retinal degenerate rat model

Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line‐3 rat pups (21 days old) were exposed for 5–6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S‐antigen, red‐green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree−1 in 38‐day‐old line 3 rats. With a 0.125 cycles degree−1 stimulus, the head tracking performance of 80‐day‐old BL rats were similar to that of 220‐day‐old no–BL‐treated line‐3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light‐damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.

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 rabbits 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.