Metabolic requirements of Th17 cells and of B cells: Regulation and defects in health and in inflammatory diseases.

The immune system protects from infections and cancer through complex cellular networks. For this purpose, immune cells require well-developed mechanisms of energy generation. However, the immune system itself can also cause diseases when defective regulation results in the emergence of autoreactive lymphocytes. Recent studies provide insights into how differential patterns of immune cell responses are associated with selective metabolic pathways. This review will examine the changing metabolic requirements of Th17 cells and of B cells at different stages of their development and activation. Both cells provide protection but can also mediate diseases through the production of autoantibodies and the production of proinflammatory mediators. In health, B cells produce antibodies and cytokines and present antigens to T cells to mount specific immunity. Th17 cells, on the other hand, provide protection against extra cellular pathogens at mucosal surfaces but can also drive chronic inflammation. The latter cells can also promote the differentiation of B cells to plasma cells to produce more autoantibodies. Metabolism-regulated checkpoints at different stages of their development ensure the that self-reactive B cells clones and needless production of interleukin (IL-)17 are limited. The metabolic regulation of the two cell types has some similarities, e.g. the utility of hypoxia induced factor (HIF)1α during low oxygen tension, to prevent autoimmunity and regulate inflammation. There are also clear differences, as Th17 cells only are vulnerable to the lack of certain amino acids. B cells, unlike Th17 cells, are also dependent of mechanistic target of rapamycin 2 (mTORC2) to function. Significant knowledge has recently been gained, particularly on Th17 cells, on how metabolism regulates these cells through influencing their epigenome. Metabolic dysregulation of Th17 cells and B cells can lead to chronic inflammation. Disease associated alterations in the genome can, in addition, cause dysregulation to metabolism and, thereby, result in epigenetic alterations in these cells. Recent studies highlight how pathology can result from the cooperation between the two cell types but only few have so far addressed the key metabolic alterations in such settings. Knowledge of the impact of metabolic dysfunction on chronic inflammation and pathology can reveal novel therapeutic targets to treat such diseases.

Investigation of the allele frequency of the G>A intron 10 ADAMTS17 mutation causing primary lens luxation in the Portuguese Podengo breed.

OBJECTIVE: To establish the allele frequency of the PLL-causing G>A intron 10 ADAMTS17 mutation in the Portuguese Podengo population in the UK and investigate a possible correlation between the mutation and short stature. METHODS: Two groups of dogs (Group 1 and Group 2) were recruited for the purpose of the study. Group 1 (n = 40) consisted of dogs which were genotyped only and Group 2 (n = 42) consisted of dogs which were genotyped, underwent a full ophthalmological examination and also had their height measured at the withers. RESULTS: In Group 1, genotyping for the ADAMTS17:c.1473+1G>A mutation confirmed 1/40 homozygous for the mutated allele (-/-), 7/40 heterozygous for the mutated allele (+/-), and 32/40 homozygous for the wild-type allele (+/+) dogs. In Group 2, genotyping of the dogs confirmed 6/42 heterozygous for the mutated allele (+/-) and homozygous for the wild-type allele (+/+) dogs. In total, 1/82 (1.2%) dogs were confirmed to be homozygous for the mutated allele, 13/82 (15.8%) heterozygous for the mutated allele and 68/82 (83%) homozygous for the wild-type allele. The frequency of the mutated allele across both groups was calculated as 0.09. A statistically significant correlation between the mutation and short stature could not be established (p = .590). CONCLUSIONS: The frequency of the mutation calculated in this study (0.09) is high. Genetic testing should be considered for each dog prior to breeding with a view of selective breeding.

Melting corneal ulcers (keratomalacia) in dogs: A 5-year clinical and microbiological study (2014-2018).

OBJECTIVES: To identify bacterial microorganisms associated with canine keratomalacia, review their antimicrobial sensitivity, and evaluate clinical outcomes compared to results of microbial culture. METHODS: Retrospective analysis of clinical records of dogs diagnosed with a melting corneal ulcer presented to a referral hospital in Hertfordshire, UK between 2014 and 2018. RESULTS: One hundred and ten melting corneal ulcers were sampled in 106 dogs. The most common pure bacterial isolate was Pseudomonas aeruginosa (n = 26) followed by ß-hemolytic Streptococcus (n = 12). Melting corneal ulcers that cultured coagulase-positive Staphylococcus, coliform bacteria, Pasteurella multocida, Enterococcus, and Streptococcus viridans presented in smaller numbers and were analyzed together (n = 16). Multiple cultures were identified in nine cases (n = 9). Forty-seven cultures yielded no bacterial growth (n = 47). The susceptibility to fluoroquinolones remained high with the exception of ß-hemolytic Streptococci. There was no significant difference in the ulcer severity at presentation in regard to the cultured bacteria. Overall, 63 eyes (57%) received surgical grafting in addition to medical treatment. In 14 cases (13%), the progression of corneal melting despite medical ± surgical treatment resulted in enucleation. Fifty-seven percent (8/14) of the enucleated eyes cultured pure Pseudomonas aeruginosa isolates. In contrast, all ß-hemolytic Streptococcus-associated ulcers healed. CONCLUSIONS: The most common bacterial species associated with canine keratomalacia were Pseudomonas aeruginosa and ß-hemolytic Streptococcus. Because of the variation in antibacterial sensitivity between these two species, bacterial culture and sensitivity testing should be performed in all dogs presenting with keratomalacia. Melting corneal ulcers associated with pure Pseudomonas infection were significantly more likely to result in globe loss than melting corneal ulcers associated with other cultures.

Biodegradable Defined Shaped Printed Polymer Microcapsules for Drug Delivery.

This work describes the preparation and characterization of printed biodegradable polymer (polylactic acid) capsules made in two different shapes: pyramid and rectangular capsules about 1 and 11 µm in size. Obtained core-shell capsules are described in terms of their morphology, loading efficiency, cargo release profile, cell cytotoxicity, and cell uptake. Both types of capsules showed monodisperse size and shape distribution and were found to provide sufficient stability to encapsulate small water-soluble molecules and to retain them for several days and ability for intracellular delivery. Capsules of 1 µm size can be internalized by HeLa cells without causing any toxicity effect. Printed capsules show unique characteristics compared with other drug delivery systems such as a wide range of possible cargoes, triggered release mechanism, and highly controllable shape and size.

Polylactic Acid-Based Patterned Matrixes for Site-Specific Delivery of Neuropeptides On-Demand: Functional NGF Effects on Human Neuronal Cells.

The patterned microchamber arrays based on biocompatible polymers are a versatile cargo delivery system for drug storage and site-/time-specific drug release on demand. However, functional evidence of their action on nerve cells, in particular their potential for enabling patterned neuronal morphogenesis, remains unclear. Recently, we have established that the polylactic acid (PLA)-based microchamber arrays are biocompatible with human cells of neuronal phenotype and provide safe loading for hydrophilic substances of low molecular weight, with successive site-specific cargo release on-demand to trigger local cell responses. Here, we load the nerve growth factor (NGF) inside microchambers and grow N2A cells on the surface of patterned microchamber arrays. We find that the neurite outgrowth in local N2A cells can be preferentially directed towards opened microchambers (upon-specific NGF release). These observations suggest the PLA-microchambers can be an efficient drug delivery system for the site-specific delivery of neuropeptides on-demand, potentially suitable for the migratory or axonal guidance of human nerve cells.

Magnetically responsive layer-by-layer microcapsules can be retained in cells and under flow conditions to promote local drug release without triggering ROS production.

Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm-2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery.

Glaucoma-causing ADAMTS17 mutations are also reproducibly associated with height in two domestic dog breeds: selection for short stature may have contributed to increased prevalence of glaucoma.

BACKGROUND: In humans, ADAMTS17 mutations are known to cause Weill-Marchesani-like syndrome, which is characterised by lenticular myopia, ectopia lentis, glaucoma, spherophakia, and short stature. Breed-specific homozygous mutations in ADAMTS17 are associated with primary open angle glaucoma (POAG) in several dog breeds, including the Petit Basset Griffon Vendeen (PBGV) and Shar Pei (SP). We hypothesised that these mutations are associated with short stature in these breeds. METHODS: Two hundred thirty-three PBGV and 66 SP were genotyped for their breed-specific ADAMTS17 mutations. The height of each dog was measured at the withers. We used linear (per allele) regression to assess the association between ADAMTS17 mutations and height as a continuous variable, and linear regression and likelihood ratio tests to assess the shape of the association by comparing a general model with a linear (per allele) model. RESULTS: The adjusted mean heights of affected, carrier, and clear PBGV were 33.49 cm (n = 21, 95% CI 32.78-34.19 cm), 34.88 cm (n = 85, 95% CI 34.53-35.25 cm), and 34.92 cm (n = 121, 95% CI 34.62-35.21 cm), respectively. The mean heights of affected, carrier, and clear SP were 43.96 cm (n = 9, 95% CI 41.88-46.03 cm), 47.56 cm (n = 28, 95% CI 45.50-48.63 cm), and 48.95 cm (n = 23, 95% CI 47.80-50.11 cm), respectively. There was a significant difference between the height of affected and clear animals in the PBGV (P = 0.001) and the SP (P = < 0.0001). CONCLUSIONS: ADAMTS17 POAG mutations are significantly associated with height in these breeds.

Magnetically targetable microcapsules display subtle changes in permeability and drug release in response to a biologically compatible low frequency alternating magnetic field.

High frequency alternating magnetic fields (AMF) have been widely used as a non-invasive method to induce local hyperthermia for antitumor treatment and to efficiently trigger drug release from various carriers. However, few studies have exploited the potential of targeted drug delivery to healthy cells or tissue and the use of low frequency AMF (LF-AMF) for intracellular triggered release. To achieve this goal, doxycycline was delivered with the layer-by-layer (LbL) assembled magnetic microcapsules, and AMF with low frequency (50 Hz) was applied. The low frequency AMF had little effect on morphology of microcapsules, which upon exposure for 360 min caused no significant damage and had the advantage of minimizing heating effects. Nonetheless, microcapsule permeability increased as a function of exposure time when assessed using FITC-dextran (70 kDa) with the number of permeable microcapsules increased from 13.5% (20 min) to 52.8% (360 min). Increased permeability also enhanced in vitro doxycycline release in genetically engineered myoblast cells where EGFP expression is regulated by the tetracycline system, while targeted EGFP expression was observed by magnetically navigating the microcapsules to a site of interest. Upon LF-AMF exposure of 30 min, no cytotoxicity was observed, but intracellular doxycycline release was promoted and enhanced EGFP expression as demonstrated by EGFP fluorescence intensity measurement. This study reveals the possibility of targeted drug delivery and using LF-AMF as a non-cytotoxic intracellular trigger of drug release from microcapsules without alteration in cell viability.

Ceria Nanoparticles-Decorated Microcapsules as a Smart Drug Delivery/Protective System: Protection of Encapsulated P. pyralis Luciferase.

The design of novel, effective drug delivery systems is one of the most promising ways to improve the treatment of socially important diseases. This article reports on an innovative approach to the production of composite microcontainers (microcapsules) bearing advanced protective functions. Cerium oxide (CeO2) nanoparticles were incorporated into layer-by-layer polyelectrolyte microcapsules as a protective shell for an encapsulated enzyme (luciferase of Photinus pyralis), preventing its oxidation by hydrogen peroxide, the most abundant type of reactive oxygen species (ROS). The protective effect depends on CeO2 loading in the shell: at a low concentration, CeO2 nanoparticles only scavenge ROS, whereas a higher content leads to a decrease in access for both ROS and the substrate to the enzyme in the core. By varying the nanoparticle concentration in the microcapsule, it is possible to control the level of core shielding, from ROS filtering to complete blocking. A comprehensive analysis of microcapsules by transmission electron microscopy, scanning electron microscopy, atomic force microscopy, confocal laser scanning microscopy, and energy-dispersive X-ray spectroscopy techniques was carried out. Composite microcapsules decorated with CeO2 nanoparticles and encapsulated luciferase were shown to be easily taken up by rat B-50 neuronal cells; they are nontoxic and are able to protect cells from the oxidative stress induced by hydrogen peroxide. The approach demonstrated that the active protection of microencapsulated substances by CeO2 nanoparticles can be used in the development of new drug delivery and diagnostic systems.

In-situ NIR-laser mediated bioactive substance delivery to single cell for EGFP expression based on biocompatible microchamber-arrays.

Controlled drug delivery and gene expression is required for a large variety of applications including cancer therapy, wound healing, cell migration, cell modification, cell-analysis, reproductive and regenerative medicine. Controlled delivery of precise amounts of drugs to a single cell is especially interesting for cell and tissue engineering as well as therapeutics and has until now required the application of micro-pipettes, precisely placed dispersed drug delivery vehicles, or injections close to or into the cell. Here we present surface bound micro-chamber arrays able to store small hydrophilic molecules for prolonged times in subaqueous conditions supporting spatiotemporal near infrared laser mediated release. The micro-chambers (MCs) are composed of biocompatible and biodegradable polylactic acid (PLA). Biocompatible gold nanoparticles are employed as light harvesting agents to facilitate photothermal MC opening. The degree of photothermal heating is determined by numerical simulations utilizing optical properties of the MC, and confirmed by Brownian motion measurements of laser-irradiated micro-particles exhibiting similar optical properties like the MCs. The amount of bioactive small molecular cargo (doxycycline) from local release is determined by fluorescence spectroscopy and gene expression in isolated C2C12 cells via enhanced green fluorescent protein (EGFP) biosynthesis.

Effect of methadone and acepromazine premedication on tear production in dogs.

OBJECTIVES: To evaluate the combined effect of intramuscular acepromazine and methadone on tear production in dogs undergoing general anaesthesia for elective, non-ocular procedures. DESIGN: Prospective, non-randomised, pre-post treatment study. SETTING: Patients were recruited from a referral practice in the UK. METHODS: Thirty client-owned dogs were enrolled in this study and received a combined intramuscular premedication of methadone (0.3 mg/kg) and acepromazine (0.02 mg/kg) before general anaesthesia for elective, non-ocular procedures. Full ophthalmic examination was performed and tear production was quantified using the Schirmer tear test-1 (STT-1). On the day of general anaesthesia, an STT-1 was performed before (STT-1a) and after (STT-1b) intramuscular premedication with methadone/acepromazine. RESULTS: Using a general linear model, a significant effect on STT-1 results was found for premedication with methadone/acepromazine (P=0.013), but not eye laterality (P=0.527). Following premedication, there was a significant reduction observed in the mean STT-1 readings of left and right eyes between STT-1a (20.4±2.8 mm/min) and STT-1b (16.9±4.1 mm/min; P<0.001). Significantly more dogs had an STT-1 reading less than 15 mm/min in one or both eyes after premedication (30 per cent; 9/30 dogs) compared with before premedication (6.7 per cent; 2/30 dogs; P=0.042). CONCLUSIONS: An intramuscular premedication of methadone and acepromazine results in a decrease in tear production in dogs before elective general anaesthesia. This may contribute to the risk of ocular morbidities, such as corneal ulceration, particularly in patients with lower baseline tear production.

Immediate effects of diamond burr debridement in patients with spontaneous chronic corneal epithelial defects, light and electron microscopic evaluation.

PURPOSE: To evaluate immediate effects of diamond burr debridement (DBD) on the cornea of canine patients diagnosed with spontaneous chronic corneal epithelial defects (SCCEDs). ANIMALS STUDIED: Eight client owned dogs with SCCEDs. METHODS: Nine eyes from eight dogs with SCCEDs underwent superficial keratectomy (SK). The ulcerated area was divided into quadrants with a 300-micron restricted depth knife. Two of four quadrants underwent DBD for 40-60 s. A SK followed immediately. One burred section and one nonburred section were fixed with formaldehyde 10% and underwent light microscopy (LM). The remaining quadrants from five eyes were fixed with glutaraldehyde 2.5% and underwent transmission electron microscopy (TEM). Masked pathologists evaluated the samples. A student's paired t-test was used to analyze the data. RESULTS: With LM all nonburred samples had a superficial stromal hyaline acellular zone (HAZ), seven of the burred samples had an intermittent HAZ and in two burred samples this zone was absent. The HAZ thickness of burred samples (1.062 ± 0.664 µm) was significantly thinner than that of the nonburred samples (4.309 ± 1.348 µm) (P < 0.0001). Transmission electron microscopy showed an absence of basement membrane and the presence of an amorphous, fine fibrillar material in the superficial stroma in nonburred samples. This material was intermittent or absent in burred samples. CONCLUSION: DBD significantly reduces the superficial stromal HAZ in SCCEDs. A reduction of its thickness may be responsible for the healing rates reported with DBD.

Electrospun poly(lactic acid) fibers containing novel chlorhexidine particles with sustained antibacterial activity.

The treatment of persistent infections often requires a high local drug concentration and sustained release of antimicrobial agents. This paper proposes the use of novel electrospinning of poly(lactic acid) (PLA) fibers containing uncoated and encapsulated chlorhexidine particles. Chlorhexidine particles with a mean (SD) diameter of 17.15 ± 1.99 µm were fabricated by the precipitation of chlorhexidine diacetate with calcium chloride. Layer-by-layer (LbL) encapsulation of the chlorhexidine particles was carried out to produce encapsulated particles. The chlorhexidine particles had a high chlorhexidine content (90%), and when they were electrospun into PLA fibers a bead-in-string structure was obtained. The chlorhexidine content in the fibers could be tuned and a sustained release over 650 h was produced, via chlorhexidine particle encapsulation. Chlorhexidine release was governed by the polyelectrolyte multilayer encapsulation as demonstrated by SEM and confocal imaging. The incorporation of uncoated and encapsulated chlorhexidine particles (0.5% and 1% wt/wt chlorhexidine) into the fibers did not cause toxicity to healthy fibroblasts or affect cell adhesion to the fibers over a period of 5 days. The chlorhexidine-containing fibers also demonstrated sustained antibacterial activity against E. coli via an agar diffusion assay and broth transfer assay. Therefore, the chlorhexidine-containing PLA fibers may be useful in the treatment of persistent infections in medicine and dentistry.

Local and Sustained Activity of Doxycycline Delivered with Layer-by-Layer Microcapsules.

Achieving localized delivery of small molecule drugs has the potential to increase efficacy and reduce off target and side effects associated with systemic distribution. Herein, we explore the potential use of layer-by-layer (LbL) assembled microcapsules for the delivery of doxycycline. Absorbance of doxycycline onto core dextran sulfate of preassembled microcapsules provides an efficient method to load both synthetic and biodegradable microcapsules with the drug. Application of an outer layer lipid coat enhances the sustained in vitro release of doxycycline from both microcapsule types. To monitor doxycycline delivery in a biological system, C2C12 mouse myoblasts are engineered to express EGFP under the control of the optimized components of the tetracycline regulated gene expression system. Microcapsules are not toxic to these cells, and upon delivery to the cells, EGFP is more efficiently induced in those cells that contain engulfed microcapsules and monitored EGFP expression clearly demonstrates that synthetic microcapsules with a DPPC coat are the most efficient for sustain intracellular delivery. Doxycycline released from microcapsules also displayed sustained activity in an antimicrobial growth inhibition assay compared with doxycycline solution. This study reveals the potential for LbL microcapsules in small molecule drug delivery and their feasible use for achieving prolonged doxycycline activity.

Survey of the incidence of pectinate ligament dysplasia and glaucoma in the UK Leonberger population.

OBJECTIVE: To determine the prevalence of pectinate ligament dysplasia (PLD) in UK Leonbergers and identify cases affected by glaucoma. Also, to define the spectrum of pectinate ligament (PL) appearance in this breed and determine whether gonioscopic monitoring should be recommended. ANIMALS STUDIED: Data were compiled from 78 prospective gonioscopy examinations performed by one author (GF) and retrospective analysis of 233 UK eye scheme certificates (2009-2014). Clinical cases of glaucoma in Leonbergers diagnosed by UK veterinary ophthalmologists, where gonioscopy of the fellow eyes or histology of affected eyes had been performed, were also reviewed. PROCEDURE: In the prospective study, intraocular pressure was recorded prior to gonioscopy using a rebound tonometer. Gonioscopy was performed using a slit-lamp biomicroscope with a Koeppe goniolens. PLD was categorized according to the percentage of the iridocorneal drainage angle affected (grade 0 = <25% affected; grade 1 = 25-50% affected; grade 2 = 51-75% affected; and grade 3 = >75% affected), and the degree of narrowing of the angle was noted. RESULTS: Of 78 dogs examined prospectively, 64/78 (82%) were grade 0, 7/78 (9%) were grade 1, 3/78 (4%) were grade 2, and 4/78 (5%) were grade 3. A large phenotypic variation was observed. Spearman's rank correlation showed a positive correlation between age and severity of PLD (P < 0.0055). 52 (22%) of Leonbergers examined under the UK eye scheme 2009-2014 were affected by PLD. Five clinical cases of glaucoma were reviewed where gonioscopy had been performed and one where histology was performed. All individuals had grade 3 PLD with gonioscopy of the contralateral eye or severe goniodysgenesis with histological sections of the affected eye. CONCLUSION: This survey suggests the prevalence of PLD is sufficient to justify ongoing screening of Leonbergers.

Improved and targeted delivery of bioactive molecules to cells with magnetic layer-by-layer assembled microcapsules.

Despite our increasing knowledge of cell biology and the recognition of an increasing repertoire of druggable intracellular therapeutic targets, there remain a limited number of approaches to deliver bioactive molecules to cells and even fewer that enable targeted delivery. Layer-by-layer (LbL) microcapsules are assembled using alternate layers of oppositely charged molecules and are potential cell delivery vehicles for applications in nanomedicine. There are a wide variety of charged molecules that can be included in the microcapsule structure including metal nanoparticles that introduce physical attributes. Delivery of bioactive molecules to cells with LbL microcapsules has recently been demonstrated, so in this study we explore the delivery of bioactive molecules (luciferase enzyme and plasmid DNA) to cells using biodegradable microcapsules containing a layer of magnetite nanoparticles. Interestingly, significantly improved intracellular luciferase enzyme activity (25 fold) and increased transfection efficiency with plasmid DNA (3.4 fold) was observed with magnetic microcapsules. The use of a neodymium magnet enabled efficient targeting of magnetic microcapsules which further improved the delivery efficiency of the cargoes as a consequence of increased microcapsule concentration at the magnetic site. Microcapsules were well tolerated by cells in these experiments and only displayed signs of toxicity at a capsule : cell ratio of 100 : 1 and with extended exposure. These studies illustrate how multi-functionalization of LbL microcapsules can improve and target delivery of bioactive molecules to cells.

Validity of aqueocentesis as a component of anterior uveitis investigation in dogs and cats.

OBJECTIVE: To describe aqueocentesis cytopathology results from dogs and cats presenting for uveitis investigation and to determine whether this is a useful and safe procedure. ANIMAL STUDIED: Dogs and cats presenting for investigation of anterior uveitis (April 2008-December 2013). PROCEDURES: Aqueous was collected via limbal entry under sedation/general anesthesia, for cytopathology and occasionally bacterial culture or polymerase chain reaction (PCR) testing. Further workup included blood testing (hematology, biochemistry, and serology), diagnostic imaging, nonocular cytopathology, and available histopathology. RESULTS: Fifty-six dogs and 39 cats were included in the study. An aqueous cytopathologic diagnosis of lymphoma (or discrete cell neoplasia) was made in six dogs and seven cats, and a diagnosis of large cell carcinoma made in one dog. This diagnosis of lymphoma was confirmed by ocular histopathology in two dogs and one cat; nonocular cytopathology corroborated lymphoma in another three dogs and five cats. Lymphoma was not evident on aqueous cytopathology but confirmed on nonocular histopathology in two dogs and by cytopathology in one cat. Additionally, aqueous cytopathology in three cats suggested, but was not considered diagnostic of, lymphoma; one of these cats had a confirmatory diagnosis of lymphoma on subsequent clinical investigation. Aqueous humor cytopathology alone was not diagnostic in non-neoplastic anterior uveitis cases, but supplemented the clinical picture with other systemic diagnostic tests. No clinically important complications were reported in association with aqueocentesis. CONCLUSIONS: Aqueocentesis is performed readily with minimal risk. The results were primarily useful in aiding a diagnosis of lymphoma in both dogs and cats.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-ß.

OBJECTIVES: Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-ß with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes. METHODS: Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay. RESULTS: We demonstrate here that fusion proteins of TGF-ß, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-ß can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease. CONCLUSIONS: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Location of molecules in layer-by-layer assembled microcapsules influences activity, cell delivery and susceptibility to enzyme degradation.

Layer-by-layer assembled microcapsules have the potential to be versatile cell delivery systems incorporating multiple activities and functions. However, it is necessary to determine the influence that different capsule locations have on activity of bioactive molecules in order to optimise delivery and for generation of multifunctional capsules. In this study we examine the influence that locating the bioluminescent enzyme luciferase in different microcapsule locations has on activity in intact synthetic and biodegradable microcapsules before and after cell delivery as well as its susceptibility to protease degradation. We also examine the effect of microcapsule position on cell transfection with plasmid DNA. Based on the findings of experiments in this study we also demonstrate co-delivery of luciferase protein and plasmid DNA encoding a fluorescent protein from two different locations within the same microcapsule. Our studies confirm that, the core, subouter layer, and outer layer are optimal for cell delivery but these positions offer least protection from protease activity. By contrast middle layer molecules remain entangled with capsule layers preventing their release which is inefficient for cell delivery but this provides better protection from protease degradation. The findings of this study will enable more rationale layer-by-layer assembly of microcapsules containing biologically active molecules for cell delivery and aid in the generation of multifunctional microcapsules.