Brian C. Leonard

Bifidobacterium bifidum in a rat model of necrotizing enterocolitis: antimicrobial peptide and protein responses

Introduction:Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear.Methods:To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF + BIF). All groups were exposed to asphyxia and cold stress.Results:Like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. The expression of lysozyme, secretory phospholipase A2 (sPLA2), pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF group with a high incidence of NEC, as compared with the DF and FF + BIF groups where the disease was attenuated.Discussion:We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.

Activity, Expression and Genetic Variation of Canine β-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs

The skin functions as more than a physical barrier to infection. Epithelial cells of the skin can synthesize antimicrobial peptides, including defensins, which exhibit direct antimicrobial activity. Here we characterize the expression pattern, genetic variation and activity of the major β-defensin expressed in canine skin, canine β-defensin 103 (CBD103). The gene encoding CBD103 exhibits two forms of polymorphism: a common 3-basepair deletion allele and a gene copy-number variation. Golden retrievers and Labrador retrievers were the only breeds that encoded the variant allele of CBD103, termed CBD103ΔG23. Both these breeds also exhibited a CBD103 gene copy-number polymorphism that ranged from 2 to 4 gene-copies per diploid genome. Recombinant CBD103 and CBD103ΔG23, as well as the human ortholog human β-defensin 3 (hBD3) and hBD3ΔG23, showed potent and comparable antimicrobial killing against both methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius. Skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls and comparable at lesional and nonlesional sites. This expression pattern in dogs differs from the previously reported reduced expression of the human ortholog in atopic dermatitis. Overall, the similarities of CBD103 and its human ortholog reported here support the notion that the domestic dog may serve as a valuable model for studying β-defensin biology in the skin.

Interfacial Phenomena and the Ocular Surface

Ocular surface disorders, such as dry eye disease, ocular rosacea, and allergic conjunctivitis, are a heterogeneous group of diseases that require an interdisciplinary approach to establish underlying causes and develop effective therapeutic strategies. These diverse disorders share a common thread in that they involve direct changes in ocular surface chemistry as well as the rheological properties of the tear film and topographical attributes of the cellular elements of the ocular surface. Knowledge of these properties is crucial to understand the formation and stability of the preocular tear film. The study of interfacial phenomena of the ocular surface flourished during the 1970s and 1980s, but after a series of lively debates in the literature concerning distinctions between the epithelial and the glandular origin of ocular surface disorders during the 1990s, research into this important topic has declined. In the meantime, new tools and techniques for the characterization and functionalization of biological surfaces have been developed. This review summarizes the available literature regarding the physicochemical attributes of the ocular surface, analyzes the role of interfacial phenomena in the pathobiology of ocular surface disease, identifies critical knowledge gaps concerning interfacial phenomena of the ocular surface, and discusses the opportunities for the exploitation of these phenomena to develop improved therapeutics for the treatment of ocular surface disorders.

Antimicrobial peptides: agents of border protection for companion animals

Over the past 20 years, there have been significant inroads into understanding the roles of antimicrobial peptides in homeostatic functions and their involvement in disease pathogenesis. In addition to direct antimicrobial activity, these peptides participate in many cellular functions, including chemotaxis, wound healing and even determination of canine coat colour. Various biological and genetic approaches have helped to elucidate the role of antimicrobial peptides with respect to innate immunity and host defense. Associations of antimicrobial peptides with various skin diseases, including psoriasis, rosacea and atopic dermatitis, have been documented in humans. In the longer term, therapeutic modulation of antimicrobial peptide expression may provide effective new treatments for disease. This review highlights current knowledge about antimicrobial peptides of the skin and circulating leukocytes, with particular focus on relevance to physiology and disease in companion animals.

Expression and Activity of a Novel Cathelicidin from Domestic Cats

Cathelicidins are small cationic antimicrobial peptides found in many species including primates, mammals, marsupials, birds and even more primitive vertebrates, such as the hagfish. Some animals encode multiple cathelicidins in their genome, whereas others have only one. This report identifies and characterizes feline cathelicidin (feCath) as the sole cathelicidin in domestic cats (Felis catus). Expression of feCath is predominantly found in the bone marrow, with lower levels of expression in the gastrointestinal tract and skin. By immunocytochemistry, feCath localizes to the cytoplasm of neutrophils in feline peripheral blood. Structurally, the mature feCath sequence is most similar to a subgroup of cathelicidins that form linear α-helices. feCath possesses antimicrobial activity against E. coli D31, Salmonella enterica serovar Typhimurium (IR715), Listeria monocytogenes and Staphylococcus pseudintermedius (clinical isolate) similar to that of the human ortholog, LL-37. In contrast, feCath lacks the DNA binding activity seen with LL-37. Given its similarity in sequence, structure, tissue expression, and antimicrobial activity, the cathelicidin encoded by cats, feCath, belongs to the subgroup of linear cathelicidins found not only in humans, but also non-human primates, dogs, mice, and rats.

Species variation and spatial differences in mucin expression from corneal epithelial cells.

Mucins are large glycoproteins expressed by epithelial cells of both the conjunctiva and cornea, and principle components of the glycocalyx. They are thought to play an important role in determining the interactions between the cornea/conjunctiva and the overlying tear film. The purpose of this study was to characterize the membrane-associated corneal mucin expression pattern from multiple species commonly used in ophthalmic research and drug development to better define the biochemical attributes of the ocular surface. Humans, rhesus macaques and dogs were found to have a very similar pattern of mucin expression, with mucin 16 (MUC16) being the most prevalent mucin transcript. In contrast, the rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively similar levels. To determine if there were spatial differences in expression, peripheral and central corneal epithelium were individually isolated and evaluated for mucin expression. In all species examined, MUC1, MUC4 and MUC16 had higher peripheral corneal expression when compared with central, which reached statistical significance in MUC1 (rhesus and dog). The data demonstrated variation in corneal epithelial membrane-associated mucin expression between species, with the rabbit having a distinct expression pattern. These differences may be reflective of the environment, pathogen exposure or tear film dynamics of the respective species. The species differences, as well as regional mucin expression patterns, characterized in this study further define the biochemical composition of the ocular surface and may play an important role in tear film stability.

Medical management of deep ulcerative keratitis in cats: 13 cases

Case series summary Described are 13 cats diagnosed with deep ulcerative keratitis and successfully managed medically without grafting procedures. Typical treatment involved frequent topical application of serum and antibiotics (usually a fluoroquinolone and a cephalosporin). Seven cats also received systemic antibiotics. Analgesia was achieved using various combinations of topical atropine and systemic buprenorphine, robenacoxib or corticosteroids. Six cats were hospitalized for a median (range) period of 2.5 (1–8) days, typically because of frequent medication administration. Median (range) follow-up time was 41.5 (9–103) days. Median (range) number of recheck examinations was 4 (2–6). Median (range) time to corneal re-epithelialization was 21 (9–103) days. Median (range) topical antibiotic course was 29.5 (16–103) days. Median (range) duration of Elizabethan collar use was 28 (13–73) days. At the time of writing, no further recheck examinations were recommended for 10 cats; median (range) time between initial to final examinations in these cats was 35 (20–103) days. All cats retained the affected globes and were apparently comfortable and visual at the latest recheck examination. Relevance and novel information These cases reveal that aggressive medical management is highly successful in select cats with deep ulcerative keratitis, and can result in a cosmetically acceptable, apparently comfortable and visual globe. However, therapy is intensive with frequent administration of multiple topical and sometimes systemic medications, and requires multiple veterinary visits over many weeks. Referral to a veterinary ophthalmologist for consideration of surgical stabilization is recommended, as not all cases may be amenable to the medical therapy described here.

A multidisciplinary, minimally invasive approach combining lacrimoscopy and fluoroscopically guided stenting for management of nasolacrimal apparatus obstruction in dogs

OBJECTIVE To describe and evaluate outcomes of a multidisciplinary, minimally invasive approach combining lacrimoscopy and fluoroscopically guided stenting for management of nasolacrimal apparatus (NLA) obstruction in dogs. DESIGN Prospective, nonrandomized clinical trial. ANIMALS 16 client-owned dogs with confirmed NLA obstruction. PROCEDURES Dogs underwent CT contrast dacryocystorhinography, rhinoscopy, and lacrimoscopy. Whenever possible, the NLA was stented, typically with fluoroscopic guidance. RESULTS Median duration of clinical signs prior to treatment was 3.2 months (range, 0.2 to 14 months). Causes of NLA obstruction were a foreign body (n = 5), dacryocystitis (4), stenosis secondary to fibrosis (3), granulation tissue (1), or granulation tissue in association with a small foreign body (1); a cause was not identified in 2 dogs. Stents were placed in 14 of 16 (88%) dogs for a median duration of 5.6 weeks (range, 1.3 to 9.4 weeks). Stenting was not possible in 2 dogs with stenosis of the NLA secondary to granulation tissue or fibrosis. Owners of all 16 dogs reported at least 60% clinical improvement with median improvement rated as 95%, and owners of 8 dogs reporting complete resolution of signs. Two dogs required antimicrobial administration because of dacryocystitis that persisted after stent removal; a foreign body was not found in either dog. CONCLUSIONS AND CLINICAL RELEVANCE Overall clinical response and owner-rated improvement for dogs with NLA obstruction that underwent lacrimoscopy and fluoroscopically guided stenting were high, especially given that these dogs had failed to respond to conventional treatment.

A population study of common ocular abnormalities in C57BL/6N rd8 mice

Purpose The purpose of this study was to quantify the frequency and severity of ocular abnormalities affecting wild-type C57BL/6N mice, the most common strain used worldwide for the creation of single-gene knockouts. Methods A total of 2773 animals (5546 eyes) were examined at one colony at UC Davis and in three more colonies at the Institut Clinique de la Souris in Strasbourg, France. Mice were examined at 15 to 16 weeks postnatal age by performing anterior segment biomicroscopy, posterior segment examination by indirect ophthalmoscopy, intraocular pressure measurement, and optical coherence tomography of anterior and posterior segment structures. Results Common ocular findings in the C57BL/6N strain included corneal deposits (3%), increased optical density of the anterior lens capsule (67%), punctate nuclear cataracts (98%), vitreous crystalline deposits (61%), hyaloid vascular remnant (6%), and retinal dysplasia attributed to the rd8 mutation (58%). Interestingly, retinal dysplasia was more common in male mice in all four breeding colonies evaluated in this study. The thickness of ocular tissues and compartments were measured by spectral-domain optical coherence tomography, including the central cornea, anterior chamber, vitreous, and retinal layers. Intraocular pressure was measured by rebound tonometry. Conclusions Ocular abnormalities are common in anterior and posterior segments of the C57BL/6N mouse, the most common background on which single-gene knockout mice have been made. It is important that vision scientists understand the extent and variability of ocular findings associated with this particular genetic background of mice.

Arap1 deficiency causes photoreceptor degeneration in mice

Purpose Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the control of GTPase-activating proteins (GAPs). Arap1 is an Arf-directed GAP that inhibits the trafficking of epidermal growth factor receptor (EGFR) to the early endosome, but the diversity of its functions is incompletely understood. The aim of this study was to determine the role of Arap1 in the mammalian retina. Methods Genetically engineered Arap1 knockout mice were screened for ocular abnormalities in the National Institutes of Health Knockout Mouse Production and Phenotyping (KOMP2) Project. Arap1 knockout and wild-type eyes were imaged using optical coherence tomography and fundus photography, and analyzed by immunohistochemistry. Results Arap1−/− mice develop a normal appearing retina, but undergo photoreceptor degeneration starting at 4 weeks postnatal age. The fundus appearance of mutants is notable for pigmentary changes, optic nerve pallor, vascular attenuation, and outer retinal thinning, reminiscent of retinitis pigmentosa in humans. Immunohistochemical studies suggest the cell death is predominantly in the outer nuclear layer. Functional evaluation of the retina by electroretinography reveals amplitudes are reduced. Arap1 is detected most notably in Müller glia, and not in photoreceptors, implicating a role for Müller glia in photoreceptor survival. Conclusions Arap1 is necessary for normal photoreceptor survival in mice, and may be a novel gene relevant to human retinal degenerative processes, although its mechanism is unknown. Further studies in this mouse model of retinal degeneration will give insights into the cellular functions and signaling pathways in which Arap1 participates.