Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation.

Evaluation of sources cited by an exotic animal formulary for supporting drug dosages and reference intervals in mammals.

OBJECTIVES: Formularies are intended to simplify clinical decision-making by collecting evidence-based information on drugs and their dosages. This study assessed the characteristics of sources used to support drug dosages and reference intervals for mammals in a specific exotic animal formulary, and how the sources had changed over five editions. METHODS: Each reference supporting drug dosages and reference intervals in the sections for ferrets, rabbits, rodents, hedgehogs and miniature pigs in all five editions of the formulary was evaluated and classified by two independent investigators in terms of the type of source cited. Univariable and multi-variable logistic regression models were built to evaluate changes between editions and sections. RESULTS: In total, 1338 references supporting drug dosages and 180 references supporting reference intervals were included from all editions of the formulary. Primary sources were cited by 525 (39.2%) and 39 (21.7%) of the drug and reference interval references, respectively. For drug dosages, the current edition of the formulary (2018) cited a higher proportion of primary rather than secondary sources compared with the first edition (odds ratios 3.4, 95% confidence interval 2.1 to 5.6), while for reference intervals there were no significant changes between editions. In the current edition of the formulary, the 168 secondary sources cited for drug dosages included 78 (46.4%) textbooks, 63 (37.5%) reviews, 14 (8.3%) personal communications and 7 (4.2%) other formularies. CLINICAL SIGNIFICANCE: A large proportion of references supporting drug dosages and reference intervals in the evaluated sections cited secondary sources. Although modest improvements have been observed over time, practitioners should be aware that the evidence supporting several drugs and dosages was limited, and assess the information within the formulary critically.

Ultraviolet radiation-induced cytokines promote mast cell accumulation and matrix metalloproteinase production: potential role in cutaneous lupus erythematosus.

OBJECTIVE: To examine the role of mast cells (MCs), cytokines, and matrix metalloproteinases (MMPs) following ultraviolet B (UVB) radiation in cutaneous lupus erythematosus (CLE). METHODS: Immunohistochemistry was used to determine the presence of MCs and the expression of MMP-1, MMP-9, interleukin (IL)-15, and CCL5/RANTES in skin from patients with CLE. Human keratinocytes were exposed to varying doses of UVB and supernatants were collected and assessed for IL-15, CCL5, MMP-1, and MMP-9 by protein assays. MC migration was determined against supernatants from UVB-treated keratinocytes. RESULTS: MCs in the skin of patients with CLE were significantly increased. MMP-1 and MMP-9 expression was abundant in these lesions. Intense reactivity for IL-15 and CCL5 was found in skin, particularly in epidermal keratinocytes, from patients with CLE. UVB irradiation induced IL-15, CCL5, MMP-1, and MMP-9 production from keratinocytes in a dose- and time-dependent manner. Supernatants obtained from UVB-treated keratinocytes induced MC migration, which was attenuated by anti-IL-15 and anti-CCL5 neutralizing antibodies. IL-15 induced MC-derived MMP production. CONCLUSIONS: Our results indicate that MCs and MMPs may play a role in the skin inflammation in CLE. MC recruitment as well as MMP production may be perpetuated by UV irradiation through locally released mediators.

Rectal temperature is a prognostic indicator in client-owned guinea pigs.

OBJECTIVES: To determine whether rectal temperature was associated with patient mortality in client-owned guinea pigs upon presentation to a veterinary hospital. MATERIALS AND METHODS: The medical record database at a veterinary teaching hospital was searched for records of guinea pigs from January 2016 through June 2019. Guinea pigs were included in the study if a rectal temperature was measured at presentation and there was data on survival status 7 days post-presentation. If survivor status was not documented in the medical record, follow-up information was obtained from the client via telephone or email. The data was ultimately collected from 201 client-owned guinea pigs who presented for 388 independent examinations. Univariable, multivariable and sensitivity analyses were performed. RESULTS: Guinea pigs with hypothermia (<37.9°C) at presentation had a relative risk of mortality within 7 days of presentation almost 3 times greater than guinea pigs without hypothermia (relative risk: 2.88; 95% confidence interval: 1.86 to 4.48). For each 0.55°C decrease in rectal temperature, the odds of death increased 1.6 times (odds ratio: 1.64; 95% confidence interval: 1.42 to 2.89). Sensitivity analyses confirmed the robustness of the finding. CLINICAL SIGNIFICANCE: Rectal temperature was a predictor of death for guinea pigs presenting for care at a veterinary hospital. Obtaining a rectal temperature recording should be considered for patient guinea pigs.

Surgical resolution of uterine prolapse in three pet rabbits.

This report describes a surgical technique for resolution of uterine prolapse in rabbits. Three pet rabbits presented within 24 hours of parturition with a red mass protruding from the vagina, which was diagnosed as uterine prolapse. In the first case, an attempt to reduce the prolapse by manual compression was ineffective. A laparotomy was used to apply internal uterine traction while simultaneously using gentle external pressure with cotton-tip applicators and resulted in successful resolution. After repositioning, an ovariohysterovaginectomy was performed in all three rabbits. All rabbits recovered uneventfully. Laparotomic repositioning of the uterus and ovariohysterovaginectomy, not previously described in rabbits, was easy to perform and permitted resolution of uterine prolapse.

Evaluation of point-of-care analysers for blood gas and clinical chemistry in Hermann's tortoises (Testudo hermanni).

OBJECTIVE: To assess the agreement between point-of-care and laboratory analysers in measuring biochemical and blood gas analytes in venous samples from tortoises and to define preliminary reference intervals for venous blood gas analysis in Hermann's tortoises (Testudo hermanni). MATERIALS AND METHODS: Jugular venous blood samples from 47 Hermann's tortoises underwent paired analysis with a portable gas analyser (i-STAT 1, Abaxis), a portable chemical analyser (VetScan VS2, Abaxis), and with the respective reference analysers. Agarose gel electrophoresis was used to determine albumin concentrations on 12 specimens. Agreement was evaluated with Bland-Altman plots and regression analysis using the Passing-Bablok method. RESULTS: Point-of-care analysers had variable agreement with the reference analysers, presenting constant or proportional bias depending on the analyte. Relevant analytes in reptiles, such as ionised and total calcium, had acceptable agreement. The method for determining albumin concentration currently available in both point-of-care and laboratory analysers significantly overestimated albumin concentrations as compared to protein electrophoresis. CLINICAL SIGNIFICANCE: While the use of POC analysers is extremely advantageous in small animal primary care facilities, agreement between point-of-care and laboratory analysers varies depending on the analyte. For certain analytes, interchangeability of results is limited and specific reference intervals for point-of-care analysers are required. Veterinarians should be aware of the size and the direction of the bias of each analyte.

Epidemiology of bacterial conjunctivitis in chinchillas (Chinchilla lanigera): 49 cases (2005 to 2015).

OBJECTIVES: To evaluate the anamnesis, clinical signs, diagnostic test results, treatment and outcome of chinchillas diagnosed with bacterial conjunctivitis. METHODS: Medical records of 49 chinchillas diagnosed with bacterial conjunctivitis were retrospectively reviewed. Association between clinical signs and type of bacteria involved was determined by means of univariate logistic regression. RESULTS: 61·5% of the isolated bacteria were Gram-negative, and the most common bacterial species was Pseudomonas aeruginosa (50%), followed by Staphylococcus species (26·9%). Chinchillas with acute conjunctivitis (1 to 3 days) were much more commonly affected by Gram-negative organisms. The majority of chinchillas that presented with concurrent respiratory signs were diagnosed with P. aeruginosa. Clinical resolution of conjunctivitis was reported in 87·8% chinchillas with a median time to clinical resolution of 17·5 days. Susceptibility of P. aeruginosa isolates to potentiated sulphonamides, enrofloxacin, ciprofloxacin, gentamicin, amikacin and polymyxin B was 8·3, 36, 62·5, 88·5, 100 and 100%, respectively. CLINICAL SIGNIFICANCE: P. aeruginosa is the predominant bacterial species associated with bacterial conjunctivitis in chinchillas. With the exception of duration of clinical signs, information on the anamnesis or physical examination findings cannot aid in distinguishing conjunctivitis caused by P. aeruginosa or other Gram-negative bacteria from the ones caused by Gram-positive bacteria. Gentamicin- or polymyxin B-containing antibiotic formulations are recommended for empirical topical therapy.

In veterinary trials reporting and communication regarding randomisation procedures is suboptimal.

To evaluate randomisation mechanisms in the veterinary literature, all trials defined as 'randomised' were extracted from five leading veterinary journals for the year 2013. Three blinded investigators evaluated (1) if the random sequence generation was actually non-random, and (2) whether method (CONSORT item 8A) and (3) type of randomisation (CONSORT item 8B) were reported. Trialists were contacted via email to establish (1) willingness to respond to questions on randomisation procedures, (2) whether reporting of randomisation improved following a suggestion to use the CONSORT 2010 guideline. Seven per cent ((95 per cent CI 2 to 12 per cent); 8/114) of the trials defined as 'randomised' explicitly used methods that are considered non-random. Almost half of the trials (49 per cent (40 to 59 per cent); 52/106) did not report any mechanism of randomisation. Only 13 trials (12.3 per cent (6 to 19 per cent); 13/106) reported both items. 39 of 114 (34.2 per cent) trialists contacted were willing to respond to further questions on randomisation mechanisms; 4 (3.5 per cent) trialists were unwilling and 71 (62.3 per cent) trialists did not respond. Email correspondence resulted in a mean clarification of 0.7 items (95 per cent CI 0.4 to 1.0) for the 15 trials for trialists that replied. Improved adherence to CONSORT guidelines and trialists communication is imperative to increase the quality of published evidence in veterinary medicine and to reduce research waste.

Vertebral heart size in chinchillas (Chinchilla lanigera) usingradiography and CT.

OBJECTIVE: To determine the vertebral heart size in chinchillas using right and left lateral radiographic views and CT images. To evaluate the agreement between radiographic and CT modalities. METHODS: Twenty-one clinically healthy chinchillas and seven chinchillas with cardiovascular abnormalities underwent cardiovascular examination before thoracic radiographs and thoracic CT obtained under dexmedetomidine-ketamine anaesthesia. Two observers calculated vertebral heart size for radiographic and CT studies. Reference intervals were calculated with the robust method. Agreement between radiographic and CT-derived vertebral heart size was evaluated with Bland-Altman plots and Deming regression. RESULTS: Mean ±sd vertebral heart size for lateral radiographs was 8·9 ±0·62 (reference interval: 7·5 to 10·2) and for CT-derived vertebral heart size was 8·2 ±0·55 (reference interval: 7·1 to 9·4). CT significantly underestimated the radiographic vertebral heart size by 0·66 vertebrae. There was no significant difference between vertebral heart size for right and left lateral radiographic views, or between female and male chinchillas. CLINICAL SIGNIFICANCE: Radiographic vertebral heart size for chinchillas is larger than that reported for similar rodents. Vertebral heart size can be calculated using radiography or CT in chinchillas, but these techniques are not interchangeable.

Polyostotic Lymphoma in a Ferret (Mustela putorius furo).

An 8.5-year-old, neutered female ferret (Mustela putorius furo) was presented with tachypnoea, polyuria, polydipsia, anorexia and depression. Radiographs revealed multiple osteolytic lesions of the bone, characterized cytologically by the infiltration of medium- to large-sized lymphocytes. The animal was humanely destroyed and post-mortem examination revealed multifocal masses obliterating the bone marrow of the mandible, right and left humeri and femur, and consisting of an infiltrative population of neoplastic lymphocytes. Immunohistochemical labelling for CD3 and CD79a revealed a CD3-positive neoplastic population. A diagnosis of polyostotic T-cell lymphoma was made, which is the first report of this condition in a ferret.

Porous CS based membranes with improved antimicrobial properties for the treatment of infected wound in veterinary applications.

Recently, much attention has been given to the use of innovative solution for the treatment of infected wounds in animals. Current applied treatments are often un-effective leading to infection propagation and animal death. Novel engineered membranes based on chitosan (CS) can be prepared to combine local antimicrobial effect, high flexibility and easy manipulation. In this work, CS crosslinked porous membranes with improved antimicrobial properties were prepared via freeze-drying technique to promote wound healing and to reduce the bacterial proliferation in infected injuries. Silver nanoparticles (AgNPs) and gentamicin sulfate (GS) were incorporated into the CS matrices to impart antibacterial properties on a wild range of strains. CS based porous membranes were tested for their physicochemical, thermal, mechanical as well as swelling and degradation behavior at physiological condition. Additionally, GS release profile was investigated, showing a moderate burst effect in the first days followed by a decreasing release rate which it was maintained for at least 56 days. Moreover, porous membranes loaded with GS or AgNPs showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. The bacterial strains used in this work were collected in chelonians after carapace injuries to better mimic the environment after trauma.

Functional activity of plasma fibronectin in patients with diabetes mellitus.

Decreased wound healing and increased infection are major problems in patients with diabetes mellitus. Fibronectin plays a fundamental role in wound healing and acts as an opsonin for the phagocytosis of foreign antigens. The aim of this study was to ascertain the functional activity of plasma fibronectin from patients with diabetes mellitus. Initially, a modified Boyden chamber technique was used to measure cell migration on fibronectin purified from patient's plasma and an enzyme-linked immunosorbent assay was used to measure the binding of gelatin. A sandwich assay was developed that enabled the capture of fibronectin directly from patient's plasma without prior purification. With the use of a 96-well format, the binding of two different monoclonal antibodies could be compared simultaneously with the binding of gelatin and cell adhesion. In this way, differences in the function of particular domains of fibronectin from diabetic patients and control subjects could be measured. Results showed no difference between fibronectin from diabetic patients and control subjects with respect to the monoclonal antibodies binding in 1) the cell adhesion domain and 2) the heparin-binding domain. Furthermore, no detectable differences were noted with respect to cell adhesion, cell migration, or gelatin binding. These results suggest that diabetic patients receiving insulin treatment show no modulation of plasma fibronectin function, despite raised levels of circulating glucose.

Expression of TNF-alpha by human plasma cells in chronic inflammation.

Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory cytokine and mediator of the inflammatory response. It has been implicated in the pathogenesis of many inflammatory disorders, including rheumatoid arthritis (RA), septic shock, and Crohn's disease. Using a specific anti-human TNF-alpha antibody we detected immunoreactivity for this cytokine in the cytoplasm of inflammatory cells in several chronic inflammatory disorders, including RA, scleritis, and polyarteritis nodosa. These cells were identified predominantly as IgG-expressing plasma cells. Lymph nodes from patients with Hodgkin's lymphoma and breast cancer, but not from control subjects, were also found to contain TNF-alpha-positive plasma cells. Cultured EBV-B lymphocytes and a human plasma cell line (ARH-77) when stimulated with phorbol myristate acetate demonstrated cytoplasmic TNF-alpha immunoreactivity. Western blot analysis of cell membranes and conditioned media from both cell types revealed the presence of the 26-kDa membrane-bound from and the 17-kDa soluble from of TNF-alpha, respectively. TNF-alpha was quantitated by enzyme-linked immunosorbent assay and found to be biologically active as determined by the L929 cytotoxicity assay. This is the first demonstration that plasma cells may be capable of modulating immune and inflammatory responses, not only by antibody production, but also by their secretion of a key inflammatory mediator, TNF-alpha.

Proinflammatory properties of the human S100 protein S100A12.

S100 proteins represent a new class of chemoattractants. Here we extend earlier evidence for the proinflammatory properties of human S100A12. A12 induced migration of monocytoid cells, with optimal activity at 10(-10) M and potency of >10(-9) M C5a. Neutrophils were poorly responsive, and lymphocyte migration was not affected. Actin polymerization in monocytoid cells was accompanied by a sustained [Ca(2+)]i flux of a magnitude comparable with C5a. A12 elicited a transient infiltration of neutrophils (4-8 h) and more delayed recruitment of monocytes (8-24 h) in vivo. A12 (approximately 70 nM) was present in synovial fluid (SF) from rheumatoid arthritis patients, and synovium contained A12-positive neutrophils in the sublining and interstitial region, often surrounding the perivasculature but rarely in the synovial lining layer, although some macrophages were positive. The A12 gene was transiently up-regulated in monocytes by tumor necrosis factor alpha (6 h); induction by lipopolysaccharide (LPS) was sustained (12-48 h). A12 may contribute to leukocyte migration in chronic inflammatory responses.

Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis.

PURPOSE: Pterygia are invasive, proliferative fibrovascular growths, with the matrix metalloproteinase (MMP) family of enzymes strongly implicated in the pathogenesis of these lesions. The purpose of this study was to determine the cellular distribution and activation status of matrilysin (MMP-7) in pterygia. METHODS: Resected pterygia (n = 8) and normal conjunctiva (n = 8) were sectioned and analyzed immunohistochemically with two different epitope-specific anti-MMP-7 monoclonal antibodies (Abs) which differentiate pro- and active MMP-7. The specificity of each Ab was confirmed by Western blot analysis of p-aminophenylmercuric acetate (APMA)-activated and latent recombinant MMP-7. Pterygia (n = 4) and autologous normal conjunctiva (n = 4) were placed in organ culture to determine the activation status of secreted MMP-7. RESULTS: Precursor and active forms of MMP-7 were detected in epithelial cells from both pterygia and normal conjunctiva. Intense immunoreactivity for pro- and active MMP-7 was also observed in the pterygium vasculature, but was essentially absent from conjunctival vessels. Pro-MMP-7 was also identified in the epithelial basement membrane and associated with matrix components in pterygia. The 141-7B2 Ab reacted with the 30-kDa latent MMP-7, and the IM47L Ab precipitated a 19-kDa active enzyme, thus confirming the differential specificity of each Ab. Pro- and active MMP-7 were increased 1.4- and 2.7-fold, respectively, in the supernatants from organ-cultured pterygia compared with conjunctiva. CONCLUSIONS: This study is the first to specifically localize an active MMP species in pterygia and strengthens the hypothesis that these enzymes are involved in the pathogenesis of this disease. The data also suggest that MMP-7 may play a significant role in the angiogenesis that characterizes this lesion. Future studies will be directed at determining whether targeting MMP activity may be useful for treatment of pterygia.

Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head.

PURPOSE: Pterygia are a proliferative and inflammatory growth of limbal epithelial stem cell origin, characterized by corneal tissue invasion and extensive matrix remodeling including the destruction of Bowman's layer (BL). The purpose of this study was to determine the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) at the advancing pterygium edge. METHODS: Formalin-fixed, paraffin-embedded whole eyes (n = 11) with pterygia attached, were serially sectioned and analyzed immunohistochemically to determine the spatial distribution of four MMPs and three TIMPs. Tear samples were collected from other patients with pterygia (n = 11) and displayed by gelatin zymography. RESULTS: Collagenase-1 was expressed by pterygium epithelial cells, corneal stromal fibroblasts and pterygium fibroblasts that had migrated between the epithelium and BL at the advancing pterygium edge. Collagenase-3 and gelatinases A and B were detected in all pterygia, intensely staining columnar epithelial cells directly adjacent to the denatured BL. In addition, gelatinase A immunoreactivity was observed on BL. Immunoreactivity for TIMP-1 and -3 paralleled that of the gelatinases, with more intense staining in epithelial cells and fibroblasts where BL was absent. TIMP-2 was faintly detected in pterygium epithelial cells but intensely stained pterygium fibroblasts. Gelatinase B was the most abundant gelatinolytic enzyme present in tears, elevated approximately twofold in eyes with pterygia versus the contralateral control eyes. CONCLUSIONS: This investigation is the first to identify the expression pattern of MMPs and TIMPs at the advancing pterygium edge in specimens of human eyes and in tears derived from patients with pterygia. These enzymes may be responsible for the destruction of BL, and their pattern of differential expression suggests that each may play a selective role in the pathogenesis of pterygia.

Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells.

PURPOSE: Pterygia are a common, benign, fibrovascular, and infiltrative process of the corneal-conjunctival junction of unknown pathogenesis. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes active against all components of the extracellular matrix, whose activity is specifically neutralized by tissue inhibitors of MMPs (TIMPs). In the current study the hypothesis was that MMPs and TIMPs may actively participate in the formation and progression of pterygia. METHODS: In this study, 25 pterygium specimens and 15 normal conjunctival biopsies obtained from subjects undergoing surgery for glaucoma and cataract, were processed for immunohistochemistry or in situ hybridization. Pterygium epithelial cells (PECs) were cultured under serum-free conditions and exposed to proinflammatory cytokines to determine both the mRNA and protein expression profiles of MMPs and TIMPs. RESULTS: Collagenase-1 and gelatinase A were expressed in all pterygia examined, specifically localized to the epithelium (directly adjacent to collagen type III), with gelatinase B expression exclusively associated with neutrophils. No collagenase-1 or gelatinase A was detected in normal conjunctiva. TIMP-1 and -3 were localized to epithelial cells with additional TIMP-3 immunoreactivity detected in the extracellular matrix, endothelial cells and leukocytes of all diseased tissue. TIMP-3 protein was evident in 4 of 15 normal conjunctiva. Induction of collagenase-1, gelatinase A, and TIMP-1 mRNA and protein was demonstrated in epithelial cells treated with tumor necrosis factor-alpha and interleukin-1alpha, whereas TIMP-3 expression was unaltered. CONCLUSIONS: This is the first study to document the cellular expression of MMPs and TIMPs in pterygia and cultured human PECs. MMPs and TIMPs may contribute to the inflammation, tissue remodeling, and angiogenesis that characterize pterygia. Understanding the role these proteins play may lead to novel therapies intended to reduce the progressive nature of pterygia.

Nicorandil inhibits the release of TNFalpha from a lymphocyte cell line and peripheral blood lymphocytes.

Modulation of cytokine release may be of interest in modulating inflammatory diseases. This study determined whether nicorandil, a potassium channel opener, and nitric oxide (NO) donor could inhibit the release of tumour necrosis factor alpha (TNFalpha) from lymphocytes. Nicorandil significantly and dose-dependently inhibited the TNFalpha release from a human Epstein Barr virus-transformed B lymphocyte cell line (EBV-B) and peripheral blood B and T lymphocytes. The inhibition was reversed by the addition of both potassium channel inhibitor glibenclamide and the guanylyl cyclase inhibitor 1H-(1,2,4) oxadiazolo (4,3) quinoxalin-1-one (ODQ). Other potassium channel openers, pinacidil, or the nicorandil analogue SG-209, however, failed to demonstrate inhibition of TNFalpha release. The NO scavenger haemoglobin was unable to reverse the nicorandil-induced TNFalpha inhibition, but in contrast to this, sodium nitroprusside (SNP) partially inhibited the release, which was reversed by haemoglobin. Nicorandil is able to inhibit TNFalpha release from lymphocytes, which requires the dual modes of both potassium channel opening and the nitrate moiety. Moreover, NO donation mechanism appears to be more dominant in the nicorandil inhibitory activity in lymphocytes.The dual mechanism involved in the inhibition of this cytokines may represent a novel therapeutical approach in the modulation of inflammatory disease.

Neutrophil accumulation correlates with type IV collagenase/gelatinase activity in endotoxin induced uveitis.

BACKGROUND/AIM: Anterior uveitis is a common inflammatory ocular disease characterised by protein accumulation and leucocyte infiltration in the anterior chamber. The aim of this study was to determine the expression of gelatinases in the aqueous humour (AH) and uvea in an animal model of endotoxin induced uveitis (EIU). METHODS: EIU was established in Lewis rats following an intraperitoneal injection of lipopolysaccharide (LPS). AH and ocular tissue were obtained from control animals and those with EIU over a 1 week time course and the samples analysed immunohistochemically and by gelatin zymography. RESULTS: Matrix metalloproteinase (MMP) 2 and 9 levels were elevated in rat AH over a 1 week time course. MMP-2 and MMP-9 levels peaked at the time of maximum uveal inflammation, before returning to baseline levels as the inflammation subsided. MMP-9 was detected in the latent and functionally active form. Total protein extracted from inflamed rat uveal tissue displayed no significant gelatinolytic modulation throughout the time course of EIU. Anterior chamber neutrophils and ciliary body epithelial cells were the most abundant source of the gelatinases. CONCLUSION: This study has revealed a correlation between infiltrating neutrophils and the presence of elevated gelatinases in EIU. The results suggest that these proteolytically active enzymes may be important mediators of the inflammatory response and contribute to matrix remodelling observed in uveitis. Furthermore, the excess production of MMPs may be a mechanism by which leucocytes, such as neutrophils, gain access to uveal tissue and AH. Therapeutic strategies aimed at reducing MMP activity may be of some benefit in the treatment of uveitis.

Expression and distribution of matrix metalloproteinases and their inhibitors in the human iris and ciliary body.

AIM: To determine the expression and distribution of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the normal human iris and ciliary body. METHODS: Seven postmortem human eyes were fixed with formalin. The iris and ciliary body were dissected out and embedded in paraffin. The expression of MMPs -1, 2, 3, and 9, and TIMPs 1-4 in the iris and ciliary body was determined by a novel immunofluorescence technique and the results graded by masked observers. RESULTS: Positive staining for MMPs and TIMPs was observed in all regions of the anterior uvea, and was more intense in the ciliary body than in the iris. Most MMPs and TIMPs showed similar patterns in their distribution. In the ciliary body, staining was strongest in the epithelium, and was localised to the epithelial cell cytoplasm, except for TIMP-3 which was strongly expressed in the basement membranes. In the iris, staining was most noticeable in the anterior border and anterior epithelial layer. Blood vessels in the stroma of the iris and ciliary body also stained moderately for MMPs and TIMPs. CONCLUSION: Both MMPs and TIMPs are widely expressed in the anterior uvea, with a positive correlation between their expressions. Their differential localisation in the ciliary body suggests they may have a role in maintaining homeostasis in the uveal tract.