Langerhans cell expression of neuropeptide Y and peptide YY.

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides with a variety of known functions. The role of these peptides in the skin is largely unknown, although NPY-like immunoreactivity has been reported in the epidermis. The recent report that these peptides have antimicrobial properties suggests that NPY and PYY may contribute to the skin's defense mechanisms against invading microorganisms. We have demonstrated that Langerhans cells (LC) and a certain BALB/c epidermis-derived dendritic cell line contain mRNA for NPY and PYY using RT-PCR. Furthermore, this dendritic cell line as well as an epidermis-derived dendritic cell line from A/J mice were found to produce NPY and PYY and LC produced PYY, as assessed by radioimmunoassay. These data suggest that the protective function of LC include not only antigen presentation, but also production of antimicrobial peptides.

Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice.

PURPOSE: To examine the pathologic changes in the retina of apolipoprotein E (apoE)-deficient mice fed a high-cholesterol diet. METHODS: ApoE-deficient mice (ApoE) were maintained on either regular mouse chow (ApoE-R) or a high-cholesterol diet (ApoE-C) for 25 weeks. Age-matched control C57BL/6J mice (C57) were also maintained on either regular mouse chow (C57-R) or a cholesterol-containing diet (C57-C). Retinal function was assessed by dark-adapted electroretinography (ERG). The eyes were embedded, sectioned, and analyzed by histologic and immunohistochemical methods, as well as by light and transmission electron microscopy. RESULTS: After the 25-week feeding period, ERG tracings of ApoE-C mice revealed significant increases of a- and b-wave implicit times when compared with the C57-R group of mice. In addition, there were reductions in oscillatory potential (OP) amplitudes in the ApoE-C group. However, a- and b-wave amplitudes appeared to be unchanged among the four groups of mice. Light microscopic examination of the retinas showed that compared with control C57-R mice, ApoE-C mice had significantly lower cell numbers in the inner and outer nuclear layers (85.1% +/- 4.6%, P < 0.05 and 81.4% +/- 3.7%, P < 0.01 of C57-R controls, respectively). Transmission electron microscopy of apoE-deficient mice revealed cells of the inner nuclear layer with condensation of nuclear chromatin and perinuclear vacuolization in focal areas. Bruch's membrane was also found to be thicker, and its elastic lamina appeared disorganized and discontinuous. Immunohistochemistry demonstrated diminished or no immunoreactivity for carbonic anhydrase II and calretinin in the retinal layers of apoE-deficient mice. CONCLUSIONS: Overall, there were increasing abnormalities of retinal function and cellular morphology among the four groups of mice in the order of C57-R < C57-C < ApoE-R < ApoE-C. These findings suggest that apoE and/or cholesterol play an important role in retinal function.

Nucleoside analogues as highly potent and selective inhibitors of herpes simplex virus thymidine kinase.

A series of carboxamide derivatives of 5'-amino-2',5'-dideoxy-5-ethyluridine has been prepared as inhibitors of HSV-TK (herpes simplex virus thymidine kinase). The most potent compounds were derived from xanthene, thioxanthene and dihydroanthracene carboxylic acids. The lead compounds show subnanomolar IC(50) values against HSV TKs.

Androgens and dry eye in Sjögren's syndrome.

Sjögren's syndrome is an extremely complex and currently incurable autoimmune disorder, which occurs primarily in females, and is associated with lacrimal gland inflammation, meibomian gland dysfunction, and severe dry eye. We hypothesize that androgen deficiency, which reportedly occurs in primary and secondary Sjögren's syndrome (e.g., systemic lupus erythematosus, rheumatoid arthritis), is a critical etiologic factor in the pathogenesis of dry eye syndromes. We further hypothesize that androgen treatment to the ocular surface will promote both lacrimal and meibomian gland function and alleviate both "aqueous-deficient" and "evaporative" dry eye. Our results demonstrate that androgens regulate both lacrimal and meibomian gland function, and suggest that topical androgen administration may serve as a safe and effective therapy for the treatment of dry eye in Sjögren's syndrome.

Vascular anatomy of the lacrimal gland.

This study was undertaken to establish the detailed vascular architecture of the lacrimal gland. The common carotid arteries of seven fresh human cadaver heads were injected with a compound consisting of a partially polymerized monomer, to which a catalyst and promoter were added to cause hardening. The soft tissue was then digested, using 40% potassium hydroxide, to obtain detailed casts of the lacrimal artery. The authors describe the anatomy of 14 cadaver lacrimal arteries from their entrance into the lacrimal gland to their terminal conjunctival branches. Consistent vascular patterns within the lacrimal gland were observed. A better understanding of the vascular anatomy of the lacrimal gland should allow modification of surgical techniques to reduce bleeding during biopsy or excision of the lacrimal gland.

Do cytokines have a role in the regulation of lacrimal gland acinar cell ion transport and protein secretion?

The present study explored the potential involvement of cytokines (IL-1 alpha, IL-1 beta, IL-6, and IFN-gamma) in the regulation of basic physiological processes carried out by lacrimal acinar cells. Overall, evidence gathered supports the hypothesis that cytokines may be involved in the regulation of lacrimal secretory processes. When combined with earlier studies, these data suggest that acute treatment (i.e., 20 min) of acinar cells with cytokines does not significantly impact either basal or carbachol-stimulated ion transport or protein secretion. However, chronic treatment of cultured acinar cells with cytokines does appear, in some instances, to influence these cell functions. For example, 24 h treatment of acinar cells with IL-6 or IFN-gamma did not alter basal or carbachol-stimulated protein (beta-hexosaminidase) secretion, whereas a combination of IL-1 alpha and IL-1 beta decreased carbachol-stimulated beta-hexosaminidase secretion by 80%. The mechanism behind this effect is unknown, but it appears to stem from an increase in the basal secretion rather than a decrease in the stimulated secretion. Future studies will attempt to identify the mechanism behind the actions of IL-1 alpha and IL-1 beta, in addition to testing other pertinent cytokines (e.g., TNF-alpha, TGF-beta). The study of cytokine involvement in the regulation of lacrimal acinar cell physiology has received relatively little attention. Thus, substantial gaps remain concerning the identity of cytokines, their source(s) and interplay, receptor distribution, second messenger involvement, and ultimate influence over the secretion of aqueous tears.

Neuropeptides and Langerhans cells.

The immune system and nervous system are intimately related. In addition to neuroendocrine mechanisms, neuropeptides have a variety of effects on immune cells and are responsible at least in part for neurogenic inflammation. The presence of neuropeptides in the skin has been well documented. The influence of neuropeptides on Langerhans cells is the focus of this paper. The physical presence and effects of calcitonin gene-related peptide on Langerhans cells is emphasized. Discussion also includes the putative inflammatory and immunologic roles of vasoactive intestinal peptide, substance P, neurotensin, neuropeptide Y, and somatostatin in the skin.

The design, synthesis and properties of highly potent and selective inhibitors of herpes simplex virus types 1 and 2 thymidine kinase.

The rational design and synthesis of nucleotide analogues as inhibitors of herpes simplex virus (HSV) thymidine kinase is described. Starting from thymidine, product analogues which included phosphates, phosphonates, sulphonates, sulphonamides and carboxamides were prepared. The carboxamide series showed good structure-activity relationships and afforded a lead structure which inhibited the HSV-2 enzyme in the low micromolar range. Replacing the 5-methyl group in thymidine by ethyl enhanced the potency of the lead structure 10-fold. Further optimization of the carboxamide moiety afforded inhibitors active in the sub-nanomolar range and finally the introduction of a 2'-beta-fluoro substituent improved the potency a further twofold. The low water solubility of the most potent inhibitor was overcome by conversion to the 3'-valyl ester, which had good oral bioavailability and showed activity by the oral route in murine models of infection.

Effect of sialodacryoadenitis virus exposure on acinar epithelial cells from the rat lacrimal gland.

Sialodacryoadenitis virus (SDAV), a RNA coronavirus, induces degenerative, necrotic and atrophic alterations in acinar epithelial cells of the rat lacrimal gland. To begin to explore the underlying mechanism(s) of this viral effect, we sought in the present study to: (1) determine whether SDAV invades and replicates in lacrimal gland acinar cells in vitro and (2) assess whether short-term SDAV challenge interferes with the viability or function of acinar cells in vitro. For comparison we also evaluated the relative infectivity of SDAV in acinar epithelial cells from lacrimal, submandibular and parotid glands, given that salivary tissues are known to be highly susceptible to SDAV infection in vivo. Acinar epithelial cells from lacrimal, submandibular or parotid glands were isolated from male rats, exposed briefly to SDAV or control cell antigen and then cultured for four, eight or twelve days. At experimental termination, SDAV titers in both media and sonicated cell extracts were evaluated by plaque assay titration on mouse L2 cell monolayers. To evaluate functional aspects of lacrimal gland acinar cells, SDAV-infected cells were incubated in the presence or absence of dihydrotestosterone and culture media were analyzed by RIA to measure the extent of the androgen-induced increase in secretory component (SC) production. Our results showed that: (1) SDAV invades and replicates in lacrimal gland acinar cells, Viral challenge resulted in a significant, time-dependent increase in SDAV titers, that were primarily cell-associated and greatly exceeded amounts contained in the original inoculum; (2) SDAV infection did not compromise lacrimal acinar cell viability or prevent the cellular SC response to androgens. Viral presence, though, did often attenuate the magnitude of this hormone action; and (3) SDAV infects salivary acinar cells, but the kinetics and magnitude or viral replication in lacrimal, submandibular and parotid cells showed considerable variations. These findings demonstrate that SDAV invades and replicates in acinar epithelial cells from lacrimal and salivary glands. The resulting release of infectious progeny may play a role in the SDAV-induced pathology of exocrine tissues in vivo.

Comparison of two microkeratome systems.

BACKGROUND: Microkeratomes are currently used for keratomiluesis in situ (automated lamellar keratoplasty) for myopia and hyperopia and for laser in situ keratomileusis (LASIK). Visual and refractive complications have been reported with these refractive surgical procedures. We compared two microkeratomes in their ability to resect corneal lamellae to gain insight into possible mechanism(s) of refractive and visual complications following lamellar refractive procedures. METHODS: Using an eyebank eye model, we performed automated lamellar keratoplasty to theoretically correct 10.00 diopters (D) of myopia using the Automated Corneal Shaper, manufactured by Chiron, Inc. and the MicroPrecision microkeratome, manufactured by Eye Technology, Inc. Diameters before (wet) and after fixation, thicknesses of excised tissue, and scanning electron microscopy were measured in a masked evaluation to compare instruments. Ultrasonic corneal pachymetry and a mechanical tissue compression gauge were also used to assess thickness of excised tissue. RESULTS: The Chiron automated corneal shaper created blade chatter marks at the edges of all excisions, smaller than anticipated excision diameters, and a wide range of tissue thicknesses. In contrast, the MicroPrecision microkeratome created smoother resections of all tissues without creating blade marks; tissue diameters and thicknesses were closer to the intended dimensions compared to the Chiron automated corneal shaper. CONCLUSION: Different microkeratomes create different morphologic features as they excise corneal tissue. Differences in instrument design, mechanics of the tissue excision and blade oscillation, and instrument traverse combined with surgical skill influence the configuration of lamellar keratotomies.

Excimer laser effects on human corneal endothelium. Modulation by serum factor(s).

OBJECTIVE: To determine the possibility of endothelial cell damage after excimer laser ablation. METHODS: Endothelial cell densities and morphology of human corneas after photoablations or mechanical keratectomy were compared with those of the untreated mates after 1 week of culture with or without serum. RESULTS: Corneas cultured in serum-free medium after ablation to a depth of 150 microns showed endothelial cell densities reduced to 60% of untreated, mate corneas; ultrastructural analysis showed endothelial cell damage not seen in untreated mates. Corneas ablated to the same depth and cultured in serum-enriched medium showed no endothelial cell density loss, nor did corneas cultured in serum-free medium after an ablation to a depth of 50 microns or mechanical keratectomies averaging 95 microns. CONCLUSIONS: Endothelial cell loss in deep laser resections may be prevented by factor(s) in fetal bovine serum. The apparent lack of cell loss in clinical studies may be related to the protective action of similar factors in aqueous humor.

Comparison of the UniversalKeratome and the Automated Corneal Shaper.

PURPOSE: To compare the morphologic appearance and measurements of in situ keratomileusis performed with the UniversalKeratome (UK) with those done with the Automated Corneal Shaper (ACS). SETTING: Surgical suite within private practice. METHODS: Procedures were performed the same day on mate eye-bank eyes. In situ keratomileusis was done using existing nomograms for each instrument to resect a cap thickness of 160 microns and a myopic resection of 100 microns. Intraocular pressures were increased by inflating the globes with balanced salt solution and were measured with the suction fixation rings in place. The excised caps and stromal resections were measured twice independently after surgery, again after tissue fixation, and then evaluated with light and scanning electron microscopy. RESULTS: No complications were encountered. Compared with the ACS, the UK was easy to set up, use, clean, and take down. Its excised tissue dimensions were greater and more predictable, it resected a concave shaped lenticule (edges imperceptibly blending with the host stroma), and it created a smoother power resection surface and primary resection base. CONCLUSIONS: Smoother, predictable tissue resection, and simple assembly/disassembly and use give the UK an apparent advantage over the ACS. The UK corrects astigmatism and hyperopia by changing the shape of the poly(methyl methacrylate) optical insert.

Analysis of cytomegalovirus infection and replication in acinar epithelial cells of the rat lacrimal gland.

PURPOSE: The objectives of this investigation were threefold: to advance understanding of the nature and impact of herpesvirus infection in the lacrimal gland; to determine the influence of gender and sex hormones on viral infectivity and replication capacity in lacrimal tissue; and to compare the susceptibilities of lacrimal, submandibular, and parotid cells to viral invasion. METHODS: Acinar epithelial cells were isolated from lacrimal or salivary glands from intact, orchiectomized, ovariectomized, or sham-operated rats, cultured on Matrigel in serum-free media, and briefly exposed to rat cytomegalovirus (RCMV). Cells were then incubated for varying time intervals, and RCMV titers and secretory component (SC) levels in media or cell extracts were measured by plaque assay or radioimmunoassay. Exocrine glands also were obtained from rats after RCMV inoculation in vivo and were analyzed for viral infection. RESULTS: These findings demonstrated that RCMV invades the rat lacrimal gland after intravenous or intraperitoneal viral inoculation; RCMV infects and undergoes a time-, dose-, strain-, and gender-dependent replication in acinar epithelial cells from rat lacrimal tissue; the magnitude of RCMV replication in acinar epithelial cells in vitro may, in fact, induce an acute increase in the cellular production of SC; and the extent of viral replication in lacrimal and salivary gland epithelial cells displays distinct, tissue-specific variations. CONCLUSIONS: The herpesvirus RCMV invades and replicates in acinar epithelial cells from the rat lacrimal gland. The magnitude of the viral infection may be significantly influenced by gender and alterations in the hormonal environment.

Cornea1 organ culture.

Long-term organ culture of human corneas offers a tool for conducting a variety of biochemical, histological, and wound-healing studies on human tissue in vitro. A requirement of any organ culture system is that cell differentiation and structural integrity of the tissue be maintained or even improved during culture The latter is particularly relevant in cornea1 organ culture, since available donor corneas are sometimes of poor quality These corneas are usually unsuitable for transplantation and are from older donors (>65 yr), from donors with known bacterial or viral infections at the time of death (e g., septicemia, hepatitis), or are rejected because of a low endothelial cell count, such as might occur following ocular surgery.

Keratocyte cell culture.

Keratocytes, or cornea1 fibroblasts, are the primary cell type of the cornea1 stroma. They lie between and are oriented parallel to the orthogonally arranged collagen lamellae, forming a continuous interconnecting cellular network that has been hypothesized to transmit information throughout the cornea concerning the status of the tissue (1). Under normal conditions, the keratocyte in the adult cornea is a relatively quiescent cell. However, in the event of a cornea1 injury or trauma, the keratocytes near the injured area differentiate into active, synthesizing cells and rapidly replace damaged stromal matrix (3, 3).

Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells.

It is well known that lacrimal gland acinar cells retrieve secretory vesicle membrane constituents from their apical plasma membranes after stimulated exocytosis of secretory proteins. There have also been indications of a recycling traffic involving the basal-lateral plasma membranes. In an effort to document this traffic, determine how it is regulated, and discern whether it involves more than one intracellular compartment, we studied internalization of the fluid phase marker, Lucifer Yellow, and its relationship to protein release in acinar cells isolated from rabbit lacrimal glands. Loading of intracellular vesicles was apparent with fluoresence microscopy. Stimulation with carbachol increased both the rate of internalization and the intracellular volume equilibrating with extracellular fluid, suggesting the loading of two compartments. A carbachol concentration of 10 microM increased uptake by 80% during 20-min incubations at 37 degrees C. Increasing the carbachol concentration to 1 mM reduced the response by 50%, and it appeared to do so by decreasing the intracellular volume accessible to extracellular fluid, rather than the rate of endocytosis. Carbachol affected protein release differently, increasing it by 50% at 10 microM and 80% at 1 mM. Acceleration of endocytosis by 10 microM carbachol was transient, becoming negligible after 60 min. Vasoactive intestinal peptide (VIP) and isoproterenol increased internalization 35% and 25% respectively; neither reduced uptake at the highest concentrations tested; and only isoproterenol significantly affected protein secretion. Combinations of VIP and carbachol exerted synergistic effects on both fluid phase internalization and protein release. Steady-state uptake at 18 degrees C in the presence of 10 microM carbachol was equal to uptake at 37 degrees C in the absence of carbachol, suggesting a temperature block in the pathway to at least one endocytic compartment. Decreasing the temperature to 18 degrees C eliminated the inhibitory action of excessive carbachol, suggesting that the compartment whose loading was impaired by excessive carbachol was positioned distal to the temperature block. Carbachol accelerated release of marker from preloaded cells, indicating that it stimulated recycling between the plasma membranes and endocytic compartments. This effect was maximal at a concentration of 10 microM and unchanged with increasing concentrations. In accord with the hypothesis that traffic into and out of a certain compartment was particularly dependent on stimulation, a fraction of the marker taken up by optimally stimulated cells at 37 degrees C was retained unless carbachol or VIP was present in the efflux medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen control of secretory component mRNA levels in the rat lacrimal gland.

The purpose of this investigation was to determine whether the known gender-related differences in, and the endocrine control of, the production of secretory component (SC) by the rat lacrimal gland are associated with alterations in SC mRNA content. Levels of SC mRNA were measured in lacrimal tissues of intact, sham-operated, castrated, hypophysectomized, and testosterone-treated male and female adult rats by Northern blot procedures, which utilized a specific, [alpha-32P]-labelled rat SC cDNA probe. For control purposes, SC mRNA amounts were standardized to the beta-actin content in experimental blots. The location of SC mRNA in lacrimal glands was evaluated by in situ hybridization techniques, which involved exposure of tissue sections to sense or anti-sense [35S]-labelled SC RNA probes. Our results demonstrate that: (1) lacrimal glands of male rats contain a significantly greater amount of SC mRNA than those of female rats, and that this difference co-exists with distinct, gender-associated variations in the distribution of SC mRNA in lacrimal tissue; (2) orchiectomy or hypophysectomy, but not ovariectomy or sham surgery, leads to a marked decline in the lacrimal SC mRNA content; and (3) testosterone, but not placebo, administration to castrated male and female rats induces a significant increase in the SC mRNA levels in lacrimal tissue. Overall, these findings show that gender, androgens and the hypothalamic-pituitary axis exert a considerable influence on the SC mRNA content in the rat lacrimal gland.