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Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4(+) T lymphocytes are critical for host defense against this infection, but in the absence of CD4(+) T lymphocytes, CD8(+) T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis, and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8(+) T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8(+) T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8(+) central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8(+) T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8(+) T lymphocytes.

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OBJECTIVES: This article analyses dose measurement and effective dose estimation of dental CBCT examinations. Challenges to accurate calculation of dose are discussed and the use of dose-height product (DHP) as an alternative to dose-area product (DAP) is explored. METHODS: The English literature on effective dose was reviewed. Data from these studies together with additional data for nine CBCT units were analysed. Descriptive statistics, ANOVA and paired analysis are used to characterize the data. RESULTS: PubMed and EMBASE searches yielded 519 and 743 publications, respectively, which were reduced to 20 following review. Reported adult effective doses for any protocol ranged from 46 to 1073 µSv for large fields of view (FOVs), 9-560 µSv for medium FOVs and 5-652 µSv for small FOVs. Child effective doses from any protocol ranged from 13 to 769 µSv for large or medium FOVs and 7-521 µSv for small FOVs. Effective doses from standard or default exposure protocols were available for 167 adult and 52 child exposures. Mean adult effective doses grouped by FOV size were 212 µSv (large), 177 µSv (medium) and 84 µSv (small). Mean child doses were 175 µSv (combined large and medium) and 103 µSv (small). Large differences were seen between different CBCT units. Additional low-dose and high-definition protocols available for many units extend the range of doses. DHP was found to reduce average absolute error for calculation of dose by 45% in comparison with DAP. CONCLUSIONS: Large exposure ranges make CBCT doses difficult to generalize. Use of DHP as a metric for estimating effective dose warrants further investigation.

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OBJECTIVE: The presence of myocilin was investigated in a colony of Beagles, a canine model for inherited primary open-angle glaucoma (POAG). The myocilin protein was localized in the normal and glaucomatous canine eyes by immunohistochemistry and immunocytochemistry. METHODS: Paraffin- and plastic-embedded specimens from the anterior uveas of 10 Beagles with inherited glaucoma (3 months to 13 years old) and 6 age-matched normal dogs were sectioned, and were then incubated with primary antibody, rabbit polyclonal antihuman MYOC IgG, overnight at 4 degrees C. Specimens were incubated with secondary antibody with one of the following: biotinylated link followed by peroxidase-labeled streptavidin and then by substrate-chromogen for light microscopy; fluorescent marker Texas red; or 18 nm colloidal gold-labeled goat antirabbit IgG for transmission electron microscopy. RESULTS: With normal, pre- and early glaucomatous canine specimens, cell membranes of smooth muscle cells of the iris and ciliary body stained positively, as well as most resident stromal and vascular endothelial cells. The cytoplasm of cells within the nonpigmented ciliary epithelium of the ciliary body processes stained intensely, being weaker along the pars plana. Trabecular meshwork (TM) cells and surrounding extracellular matrix labeled, as well as the sclera adjacent to the angular aqueous plexus. In specimens with moderate and advanced glaucoma, greater intensity of staining was observed within TM cells and adjacent sclera, and portions of the nonpigmented epithelium of the ciliary processes. Fibrinous material labeled intensely within the posterior chamber. CONCLUSIONS: Myocilin in the normal and glaucomatous canine eye was successfully immunolocalized. These findings with regard to the normal eye are nearly identical to those previously reported in humans, and support the original hypothesis that there is an increase in both accumulation and localization of myocilin in glaucomatous canine eyes. It also supports the possibility that changes in the activity of myocilin within the aqueous humor outflow pathway of individuals with spontaneous glaucoma are associated with the rise of intraocular pressure and subsequent development of this disease, but may not be the primary event in the initial raise in intraocular pressure in POAG in the Beagle.

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We have shown previously that phenytoin impairs learning in rats in several different behavioral paradigms (Churchill et al., 1998, 2003; Banks et al., 1999). The present study has examined this drug's effects on performance in a delayed match-to-place water maze paradigm developed by Steele and Morris (1999). We find that phenytoin retards performance, but only when the inter-trial interval (ITI) is short (i.e., 15-sec). With longer ITIs (i.e., 20-min, 2-hr), the performance of the phenytoin-treated rats was quite comparable to the controls. We suggest that this pattern of results stems from a disruption of spatial working memory, perhaps due to the effects of the drug on hippocampal function (cf., Churchill et al., 1998, 2003). This disruption is, however, not so profound that consolidation is prevented.

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PURPOSE: The most common reason for long-term failure of glaucoma filtering surgery (GFS) is scarring of the external filtering "bleb" tissues. The identification of the factors that mediate this process, as well as the development and initial testing of new therapies to limit scarring is enhanced by the use of appropriate animal models. The standard animal model for studying GFS is the rabbit but newer investigative tools that examine changes induced in biologic systems at a genetic level have made development of a rat model desirable. METHODS: Glaucoma filtering surgery was performed on 20 Sprague-Dawley rats by introducing a 30-gauge silicone cannula through a penetrating scleral tunnel, under a limbal-based conjunctival flap and suturing the conjunctiva closed. Identical GFS was performed on 3 additional rats, which underwent histologic evaluation at days 2, 5, and 11, following surgery.Fistulizing surgery was also performed on 6 Sprague-Dawley rats, for comparison, by creating a full-thickness needle sclerostomy under a limbal-based conjunctival flap and suturing the conjunctiva closed. RESULTS: Following the cannula GFS, well-elevated filtering blebs formed and these gradually failed over the course of 8 to 13 days. Needle tract sclerostomy filtering blebs formed at the site of the fistulizing surgery but rapidly failed over the course of 2 to 3 days. CONCLUSION: Cannulated filtering surgery in the rat provides a longer lasting and more predictable model than needle tract sclerostomy for studying wound healing following GFS and may facilitate the study of induced changes at the gene level.

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OBJECTIVES: The phenomenon of 'eye-shine' is seen in a variety of animal species, and is generally thought to be related to the presence of an intraocular reflecting structure, the tapetum lucidum. The tapetum lucidum is a biologic reflector system that is a common feature in the eyes of vertebrates. It normally functions to provide the light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation, thereby enhancing visual sensitivity at low light levels. The tapetum lucidum is presented here according to a classification based on the location, as well as the composition, of this reflective layer. Finally, the physical and chemical properties, as well as the origins of the different tapeta lucida, are discussed and compared. METHODS: The anatomic and biochemical aspects of the tapetum lucidum in various vertebrates are examined. Morphologic observations were made from paraffin and plastic embedded specimens. Specimens were treated with traditional stains and observed by light and transmission electron microscopy. RESULTS: Some species (primates, squirrels, birds, red kangaroo and pig) do not have this structure and they usually are diurnal animals. In vertebrates, the tapetum lucidum exhibits diverse structure, organization and composition. Therefore, the retinal tapetum (teleosts, crocodilians, marsupials, fruit bat), the choroidal guanine tapetum (elasmobranchs), the choroidal tapetum cellulosum (carnivores, rodents, cetacea), and the choroidal tapetum fibrosum (cow, sheep, goat, horse) are described. CONCLUSIONS: The tapetum lucidum represents a remarkable example of neural cell and tissue specialization as an adaptation to a dim light environment and, despite these differences, all tapetal variants act to increase retinal sensitivity by reflecting light back through the photoreceptor layer. These variations regarding both its location and structure, as well as the choice of reflective material, may represent selective visual adaptations associated with their feeding behavior, in response to the use of specific wavelengths and amount of reflectance required.

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We have shown previously that the antiepileptic phenytoin impairs transfer in an instrumental learning task (Banks et al., 1999). The present study examined the effects of contextual alterations on appetitive-to-aversive transfer performance of rats treated with either phenytoin or tang. Adult rats were tested in tone-signaled appetitive and aversive instrumental tasks, where the animal bar-pressed to obtain a food reward (sugar pellet) or to avoid shock. Rats were trained on the appetitive task for 31 days. Beginning on the twenty-first day, rats were gavaged with either phenytoin or tang twice daily. Animals were then transferred to aversive training, with the phenytoin or tang treatment continuing throughout the 25 testing days. For some animals, contextual changes were introduced as they shifted from appetitive to aversive training, while for other animals these changes were not made. Phenytoin-treated rats that were presented with changes in context as they transferred from the appetitive to the aversive task learned the avoidance response to levels substantially higher than drug-treated rats not presented with the contextual changes. These results indicate that phenytoin impairs avoidance learning following transfer from the appetitive task, and that this impairment can be eliminated by introducing changes in context at the point of transfer. In the tang-treated control subjects, on the other hand, there was no improvement in transfer learning performance associated with the changes in contextual cues. This pattern of results suggests that contextual encoding processes in rats being trained in an instrumental appetitive-to-aversive paradigm are dramatically affected by phenytoin.

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PURPOSE: To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. METHODS: Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age-matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate-chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. RESULTS: Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. CONCLUSIONS: The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.

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The objective of this study was to determine the effectiveness of testosterone in suppressing estrus in the bitch, and of cabergoline in shortening the length of the subsequent anestrous period. In Experiment 1, 12 diestrual Beagle bitches were randomly divided into two groups when plasma progesterone (P(4)) concentration was <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with testosterone cypionate every 14 days for a total of 239 days, and bitches in Group 2 served as untreated controls. On Day 274, bitches in both groups were treated with cabergoline for 40 days and blood samples were obtained on Days 274, 276 and 279 for determination of plasma prolactin (PRL) concentrations using RIA. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 2, 12 Greyhound bitches previously treated with testosterone within the last 6 months were randomly divided into two groups. At the initiation of this experiment, P(4) concentration was determined to verify that all bitches had a concentration of <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with cabergoline for 36 days, and bitches in Group 2 (n=6) served as untreated controls. Blood samples were obtained on Days 0, 2 and 5 to determine PRL concentrations. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 1, one bitch (Group 1) exhibited estrus after treatment with testosterone (1mg/kg body weight) for 43 days, and one bitch (Group 1) exhibited estrus after treatment with testosterone (2mg/kg body weight) for 113 days. None of the other four bitches in Group 1 exhibited estrus during the period of testosterone treatment (239 days). All bitches in Group 2 (control) exhibited estrus during the 239 days of the study. In addition, five of the six testosterone-treated bitches showed signs of proestrual bleeding within an average of 12.6 days (range of 5-25 days) after treatment with cabergoline; and, four of the six nontestosterone bitches showed signs of proestrual bleeding within an average of 28 days (range of 6-46 days). Prolactin concentrations in bitches in both Groups 1 and 2 significantly decreased after treatment with cabergoline. In Experiment 2, one of the six bitches showed signs of proestrual bleeding within 15 days after treatment with cabergoline. From the results of this study, it was concluded that exogenous testosterone was moderately effective (66%) in suppressing estrus in Beagle bitches, and cabergoline was effective in shortening the length of the anestrous period of Beagle bitches whose estrous cycle was previously suppressed with exogenous testosterone, but less effective in shortening the length of the anestrous period in Greyhound bitches previously treated with testosterone to suppress estrus.

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OBJECTIVE: The canine iridocorneal angle contains an operculum, which is similar to that in nonhuman primates and consists of a peripheral extension of the inner cornea that overlies the anterior-most portion of the corneoscleral trabecular meshwork. This region contains cells, the Schwalbe line's (SL) cells, that have been found to have secretory and epithelial characteristics. This region of the iridocorneal angle represents the nonfiltering portion and becomes altered early during spontaneous glaucoma in the Beagle. The present study describes the SL cell for the first time in the dog and changes associated with canine primary open angle glaucoma. PROCEDURES: The iridocorneal angles from 18 Beagles with inherited glaucoma (3 months-8 years old) and 17 normal, age-matched Beagles were placed in 10% buffered formalin for light microscopic evaluation, or 2.5% glutaraldehyde for ultrastructural evaluation. Using at least three fields from each region of the iridocorneal angle (opercular, corneoscleral, and uveal) at x 1000 magnification, trabecular cell nuclei were counted. RESULTS: The operculum in the canine iridocorneal angle consisted of the peripheral extension of the corneal endothelium and underlying anterior-most corneoscleral meshwork, having no direct contact with the angular aqueous plexus. The SL cells associated with operculum-retained epithelial morphology (polyhedral in shape with rER, Golgi apparatus, and secretory vesicles) in both normal, pre-and early glaucomatous dogs. In animals with moderate and advanced stages the SL cells often became less epithelial and secretory in appearance. The number of SL cells in normal dogs declined by approximately one-third by the end of their first year with gradual loss thereafter. In the glaucoma group the decline was more substantial and continuous through the first three years. CONCLUSIONS: The SL cell is morphologically a distinct cell type within the canine iridocorneal angle that is specifically associated with the nonfiltering portion of the corneoscleral trabecular meshwork. Changes in the SL cells of the glaucomatous dog occurred with regard to age and progression of the disease.

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Light damage research began during the early years of laser light exploration. There is a clear and significant literature that identifies an easily demonstrated retina-pigment epithelium pathology which is associated with short wavelength exposures below 520 nm. Recent interest has expanded because of the growing evidence for a blue light contribution to the retina aging process by way of a poorly understood chemical process(es) that involve circulation, oxidative reactions and the spectral absorption properties of the pigment epithelium. New powerful sources of relatively inexpensive blue energy have become available as a family of light emitting diodes. In this experiment, we examined funduscopic, angiographic and scanning laser tomographic measures of the retinal-pigment epithelium response to LED and laser spectral blue and infrared emissions closely matched in wavelengths and delivered under carefully matched circumstances. Ten retinas in normal young rhesus monkeys were locally exposed to various energy density values at 458 nm (Argon laser) ranging from 5 to 54 J cm(-2). Eight rhesus eyes were exposed to LED irradiation with a peak wavelength of 460 nm ranging from 9 to 62 J cm(-2). Similarly, a matched infrared (IR) laser and IR LED pair were used to expose an additional ten eyes for comparison of the long wavelengths. IR irradiance ranged from 21 to 306 J cm(-2). There was no response to IR exposure in any of the eyes. Blue light exposure results were measured from the color fundus photographs, scanning laser tomographs and early- and late-phase fluorescein angiogram responses at 2 and 30 days after the exposure. Results scores were accumulated for the four measures at the two time periods. The resulting lesion scores when plotted against the exposure in J cm(-2)showed no demonstrable effect at irradiance lower than 10 J cm(-2)and near 100% effectiveness for irradiance greater than 30 J cm(-2). The most sensitive and enduring indicator of change was the late fluorescein angiograms. Nonparametric statistical analysis of the scores from the two samples support the conclusion that there is no difference in the consequences of LED and laser light exposures under these matched conditions.

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In this study, we evaluated the effects of dietary supplementation at two stages of lactation with various levels of Mepron85 (M85) and M85 plus DL-methionine (DL-Met) on milk production and composition of Holstein and Brown Swiss cows fed an alfalfa-hay and corn grain-based diet. In experiment 1, control diets were formulated to supplement, in early lactation [days in milk (DIM) = 73.2], concentrations of metabolizable methionine at 104% of the estimated requirements based on the Cornell Net Carbohydrate and Protein System. Treatment groups were fed the control diet plus 10, 20, or 30 g/d of M85 at 116, 128, or 139% of the estimated requirements for metabolizable methionine. The supplementation with 10 g/d in Brown Swiss and 30 g/d of M85 in Holstein cows increased milk yields and fat percentage, but had no effects on protein percentage. These data suggested that the estimated postruminal supply of metabolizable methionine in the control ration was limiting for maximum milk fat synthesis. Conversely, in experiment 2, the cosupplementation with M85 (15 g/d) plus DL-Met (15 g/d) to cows in midlactation (DIM = 140.5) did not influence fat percentage, but increased protein yield and percentage (+0.1%) in both Holstein and Brown Swiss, and lactose percentage (+0.18%) in Holstein cows. The supplementation with 15 g/d of M85 reduced milk and protein yields, whereas 15 g/d of DL-Met reduced protein percentage in four of the five experimental weeks for Holstein cows. We conclude that supplementation with M85, alone or in combination with DL-Met, may be used to influence milk composition, but these effects are influenced by dosage and type of supplemental methionine, breed, and stage of lactation.

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The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

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Metallothionein and zinc have been implicated in cellular defense against a number of cytotoxic agents. With respect to the free radical-generating hepatotoxin carbon tetrachloride, conclusions about a defensive role were reached from in vitro studies, in vivo studies using inducers of metallothionein and studies using injections of pharmacological amounts of zinc. Metallothionein knockout (null) and metallothionein transgenic mice are more direct models to examine the effects of metallothionein expression on induced cytotoxicity. Similarly, zinc presented via the diet is a more physiological model than that presented via injection. We examined whether metallothionein-overexpressing mice or metallothionein knockout mice had altered sensitivity to carbon tetrachloride and whether supplemental dietary zinc reduced sensitivity to carbon tetrachloride in these genotypes. Metallothionein knockout mice produced no metallothionein and were unable to sequester additional hepatic zinc in response to elevated dietary zinc. Hepatotoxicity, as measured by serum alanine aminotransferase activity, histological analyses and hepatic thiol levels, was greater in the knockout mice than in controls 12 h after carbon tetrachloride treatment but not at later time points (up to 48 h). In contrast, metallothionein-overexpressing mice produced more metallothionein and sequestered more liver zinc than control mice, but hepatotoxicity was similar between genotypes. Supplemental dietary zinc had no effect on hepatotoxicity with either genotype. These data suggest metallothionein null mice were more susceptible to carbon tetrachloride-induced hepatotoxicity than were control mice. However, neither metallothionein overexpression nor supplemental dietary zinc provided further protection.

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Sirenians, including Florida manatees, possess an array of hairs and bristles on the face. These are distributed in a pattern involving nine distinct regions of the face, unlike that of any other mammalian order. Some of these bristles and hairs are known to be used in tactile exploration and in grasping behaviors. In the present study we characterized the microanatomical structure of the hair and bristle follicles from the nine regions of the face. All follicles had the attributes of vibrissae, including a dense connective tissue capsule, prominent blood sinus complex, and substantial innervation. Each of the nine regions of the face exhibited a distinct combination of these morphological attributes, congruent with the previous designation of these regions based on location and external morphological criteria. The present data suggest that perioral bristles in manatees might have a tactile sensory role much like that of vibrissae in other mammals, in addition to their documented role in grasping of plants during feeding. Such a combination of motor and sensory usages would be unique to sirenians. Finally, we speculate that the facial hairs and bristles may play a role in hydrodynamic reception.

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Purpose To describe the clinical appearance of corneal epithelial cell microerosions associated with keratomycosis in the horse. METHODS: Retrospective clinical study. RESULTS: Multifocal, punctate, superficial corneal opacities with positive rose bengal retention were noted in six horses with presumed 'viral keratitis'. Faint fluorescein staining was also present in three cases. Equine herpesvirus tissue culture inoculation was negative for a cytopathic effect in three cases. Aspergillus (n = 3), Curvularia (n = 1), and an unidentified fungus (n = 1) were cultured in five horses, and hyphae found on corneal cytology from the sixth. Mixed bacterial infections were present in three eyes. The eyes of two horses with Aspergillus progressed to deep melting corneal ulcers that required surgical therapy. The microerosions remained superficial, but persistent in the other four eyes. Natamycin was utilized topically in all six horses. Transmission electron microscopy from case 6 revealed mucin layer disruption, an intact corneal epithelial cell layer, and fungal attachment to degenerating epithelial cells. The visual outcome was positive in all six horses, although healing was prolonged (48.5 +/- 14.5 days on average in the horses with no surgery; 62 days on average in the two horses that required surgery). CONCLUSIONS: Complete removal or full-thickness penetration of the corneal epithelial cell barrier may not be necessary to allow fungal adherence and initiation of keratomycosis in the horse. Prior to colonization and invasion of the horse cornea, fungi may induce changes in the mucin layer of the tear film that result in or are associated with rose bengal positive microerosions of the superficial corneal epithelium. Horses with painful eyes, and eyes with superficial, multifocal corneal opacities should have their corneas stained with both fluorescein and rose bengal as fungal microerosions may stain weakly, or not at all, with fluorescein, and may thus be mistaken for presumed 'viral keratitis' of the horse.

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Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.

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BACKGROUND: We have previously shown that glutamine synthetase protein and mRNA are concentrated in the crypt region of the rat small intestine and that the activity of this enzyme is highest around the time of weaning. This anatomical location and time of peak activity are sites and periods of active enterocyte differentiation. This led to our current hypothesis that glutamine synthetase is important in the differentiation of enterocytes. METHODS: To test our hypothesis, we treated Caco-2 cells with physiologic (0.6 mM) glutamine concentrations in cell culture medium. The experimental group was treated with methionine sulfoximine, an irreversible glutamine synthetase inhibitor, and the control group with phosphate buffered saline. Three standard and well-defined markers of intestinal differentiation-sucrase-isomaltase activity, microvillus formation, and electrical impedance in transwell plates-were compared between the two groups. RESULTS: The methionine-sulfoximine-inhibited group was found to have lower sucrase-isomaltase activity, a lower density of microvilli, and lower electrical impedance values over time compared with the control group. CONCLUSION: The experimental group was found to be less differentiated by all three markers of differentiation. Therefore, glutamine synthetase is important for Caco-2 cell differentiation.

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The effects of low zinc nutrition and aging on central choroidal melanocytes were examined in the pig. Three populations of pigs (young, pregnant and aged), were maintained on either control (C) or low zinc (LZ) diets. Twenty-five weanling boars were sacrificed at 2, 4, 8, 10, and 12-month intervals, and nine pregnant sows and eight aged sows were sacrificed after a 6-month interval. Melanocytes of the central choroid were morphologically and morphometrically examined. The melanocyte was found to be conservative in its form, which was mostly elliptical longitudinal profile, throughout the different age populations that were fed the C diet. Morphometric observations revealed that this cell type increased in size in the oldest animals, having been 40% greater than that in the younger two populations. However, the overall percentage area occupied by melanocytes remained the same throughout all age groups. In the animals that were fed the LZ diets, a large subpopulation of choroidal melanocytes was oval to round in shape in the pregnant and aged groups. Many members of this subpopulation possessed less opaque pigment than the elliptically shaped cell. Measurements of the size and percentage area occupied in these oldest groups increased significantly. In addition to the change of size, shape and melanin opaqueness, unusually large melanosomes were consistently observed in the pregnant and aged LZ groups. Low zinc nutrition had a remarkable age-related impact on the usually quiescent melanocyte.