Identification of Altered miRNAs in Cerumen of Dogs Affected by Otitis Externa.

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes.

IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis.

IL-36R signaling plays an important role in the pathogenesis of psoriasis. We ought to assess the specific function of IL-36R in keratinocytes for the pathology of Aldara-induced psoriasis-like dermatitis. Il36r ΔK mice presenting deletion of IL-36R in keratinocytes were similarly resistant to Aldara-induced ear inflammation as Il36r -/- mice, but acanthosis was only prevented in Il36r -/- mice. FACS analysis revealed that IL-36R signaling in keratinocytes is mandatory for early neutrophil infiltration in Aldara-treated ears. RNASeq and qRT-PCR experiments demonstrated the crucial role of IL-36R signaling in keratinocytes for induction of IL-23, IL-17, and IL-22 at early time points. Taken together, our results demonstrate that IL-36R signaling in keratinocytes plays a major role in the induction of Aldara-induced psoriasis-like dermatitis by triggering early production of IL-23/IL-17/IL-22 cytokines and neutrophil infiltration.

Serum Malassezia-specific IgE in dogs with recurrent Malassezia otitis externa without concurrent skin disease.

Immediate-type hypersensitivity (ITH), mediated by IgE, to Malassezia pachydermatis is recognized in atopic dogs with recurrent yeast dermatitis and otitis externa (OE). Malassezia-associated OE commonly occurs in dogs without other signs of atopic dermatitis (AD). The aim of this study was to detect Malassezia-specific IgE in the sera of dogs with recurrent Malassezia OE without concurrent skin disease. Sera from healthy dogs were used for comparison. An FcεRIα-based ELISA was used to measure Malassezia-specific IgE. There was no significant difference between number of positive affected dogs (6/21, 29%) and number of positive unaffected dogs (15/86, 17%) (P=0.36). There was also no significant difference in the concentrations of Malassezia-specific IgE between the two groups (P=0.97). Malassezia-specific IgE did not distinguish between patient groups so, as with other canine allergens, serum IgE reactivity for Malassezia could not be used to differentiate between diseased and healthy patients. The presence of Malassezia-specific IgE in some of the affected dogs might indicate ITH to Malassezia in those dogs. Evaluation of ITH via intradermal test reactivity and response to allergen-specific immunotherapy might clarify the role of Malassezia-associated ITH in similarly affected dogs.

Topical anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica.

Opuntia ficus-indica (OFI) has been widely used in Mexico as a food and for the treatment of different health disorders such as inflammation and skin aging. Its biological properties have been attributed to different phytochemicals such as the isorhamnetin glycosides which are the most abundant flavonoids. Moreover, these compounds are considered a chemotaxonomic characteristic of OFI species. The aim of this study was to evaluate the effect of OFI extract and its isorhamnetin glycosides on different inflammatory markers in vitro and in vivo. OFI extract was obtained by alkaline hydrolysis of OFI cladodes powder and pure compounds were obtained by preparative chromatography. Nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor- (TNF-) α, and interleukin- (IL-) 6 production were measured. NO production was tested in lipopolysaccharide-stimulated RAW 264.7 cells while in vivo studies were carried on croton oil-induced ear edema model. OFI extract and diglycoside isorhamnetin-glucosyl-rhamnoside (IGR) at 125 ng/mL suppressed the NO production in vitro (73.5 ± 4.8% and 68.7 ± 5.0%, resp.) without affecting cell viability. Likewise, IGR inhibited the ear edema (77.4 ± 5.7%) equating the indomethacin effects (69.5 ± 5.3%). Both IGR and OFI extract significantly inhibited the COX-2, TNF-α, and IL-6 production. IGR seems to be a suitable natural compound for development of new anti-inflammatory ingredient.

Ceruminal diffusion activities and ceruminolytic characteristics of otic preparations - an in-vitro study.

BACKGROUND: An in-vitro setup was established in order to determine a) the diffusion activities of eight otic preparations (Aurizon®, Eas Otic®, Epi Otic®, Otifree®, Otomax®, Panolog®, Posatex®, Surolan®) through synthetic cerumen, and b) the ceruminolytic capacity and impregnation effects of these products. The main lipid classes of canine cerumen produced with moderate, non-purulent otitis externa were determined by thin layer chromatography and were subsequently used to produce a standardised synthetic cerumen (SCC). SCC was filled into capillary tubes, all of which were loaded with six commercially available multipurpose otic medications and two ear cleaners, each mixed with two markers in two experimental setups. These two marker compounds (Oil red O and marbofloxacin) were chosen, since they exhibit different physicochemical drug characteristics by which it is possible to determine and verify the diffusion activity of different types of liquids (i.e. the otic preparations). A synthetic cerumen described in the literature (JSL) was also used for comparison as its lipid composition was different to SCC. The diffusion activities of the otic preparations through both types of synthetic cerumen were studied over 24 hours. A second in-vitro experiment determined both the ceruminolytic activity and impregnation effect of the otic preparations by comparing the weight loss or weight gain after repeated incubation of JSL. RESULTS: Canine cerumen is mainly composed of triglycerides, sterol esters, fatty acid esters and squalene. The diffusion experiments showed a high diffusion efficacy along with a high impregnation effect for one test product. All the other products exhibited a lower diffusion activity with a mild to moderate impregnation effect. A mild ceruminolytic activity was observed for the two ear cleaners but not for any of the otic medications. CONCLUSIONS: The present study demonstrates that there are significant differences in the diffusion characteristics and ceruminolytic properties of the eight tested otic preparations.

Cantharidin-induced inflammation in mouse ear model for translational research of novel anti-inflammatories.

The murine model of cantharidin-induced ear inflammation was profiled in detail for its alignment with the human model and to explore the mechanism of anti-inflammatory activity of the macrolide antibiotics, clarithromycin and azithromycin. Ear swelling in CD1 mice persisted for 7 days, with peak intensity at 16 h after inflammation induction. As in humans, cantharidin (12.5 µg/ear) generated macrophage-inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, keratinocyte-derived chemokine (KC), interleukin (IL)-6, IL-1ß, and myeloperoxidase (MPO) production, as well as neutrophil accumulation in mouse ear tissue. The tested macrolides, clarithromycin and azithromycin, administered orally (2 × 150 mg/kg) 0.5 h before and 5 h after cantharidin challenge, reduced MIP-2, MCP-1, KC, and MPO concentrations and thereby decreased ear swelling. Our results suggest that cantharidin-induced acute inflammation represents an excellent model for translational research of novel anti-inflammatories.

Bismuth iodoform paraffin paste hypersensitivity reactions in mastoid cavities following isolation of mucosal lining: a series of 587 patients.

AIM: (1) To assess hypersensitivity to bismuth iodoform paraffin paste impregnated ribbon gauze following its use in packing canal wall down mastoidectomy cavities; (2) to determine if isolation of the skin and mucosa from the pack, using thin Silastic sheeting and Cortisporin ointment, reduces hypersensitivity reactions, compared with a previous series; and (3) to review the literature and to determine if bismuth iodoform paraffin paste hypersensitivity precludes the consumption of seafood (due to its high iodine content). MATERIALS AND METHODS: All patients undergoing canal wall down mastoidectomy with intra-operative bismuth iodoform paraffin paste packing between 1985 and 2009 were identified and reviewed. RESULTS: Of 587 patients identified, the overall bismuth iodoform paraffin paste reaction rate was 1 per cent. All reactions were in patients undergoing revision mastoidectomy procedures, giving a reaction rate for revision procedures of 2.4 per cent. CONCLUSION: Reactions are an uncommon event following post-operative mastoid cavity packing using bismuth iodoform paraffin paste. Reaction rates may be lowered by preparing the cavity with Silastic sheeting and Cortisporin ointment prior to packing, thus isolating the skin and mucosal surfaces. Development of such a reaction does not preclude the consumption of seafood.

Proliferative and necrotising otitis in a kitten: first demonstration of T-cell-mediated apoptosis.

Otitis externa in cats is relatively uncommon. This report describes a case of a rare, visually distinctive, proliferative and necrotising otitis in a three-month-old Persian kitten. The cat had proliferative, erythematous and necrotic tissue covering most of the proximal pinnae and vertical ear canals. On histopathological examination, the most striking feature was the existence of scattered apoptotic-appearing keratinocytes within severely hyperplastic epithelium. For the first time, immunohistochemistry was used to show a closed association between CD3(+) T cells and caspase-3 stained keratinocytes, consistent with a keratinocyte apoptosis by epidermal-infiltrating T cells. Treatment was initiated using topical tacrolimus twice daily and an ear cleanser once daily. A marked improvement was observed after 10 days of treatment and the lesions completely resolved over a period of three weeks. The origin of T cells directed against keratinocytes is currently unknown.

Evaluation of a spontaneous canine model of immunoglobulin E-mediated food hypersensitivity: dynamic changes in serum and fecal allergen-specific immunoglobulin E values relative to dietary change.

The purpose of the pilot study reported here was to evaluate serum and fecal total and allergen-specific immunoglobulin E (IgE) responses to dietary change in five Maltese x beagle dogs with suspected food hypersensitivity, compared with those of five clinically normal dogs. Clinical parameters (pruritus, otitis, and diarrhea) improved in the Maltese x beagle dogs during feeding of a novel diet, and signs were exacerbated by oral allergen provocation. Relative concentrations of serum and fecal wheat-, corn-, and milk-specific IgE were determined by use of an ELISA. The onset of clinical signs of disease was accompanied by an increase in serum allergen-specific IgE concentrations. In contrast, changes in clinical signs of disease or allergen-specific IgE values were not seen in the control group undergoing the same regimen. Total serum IgE concentration was measured by use of the ELISA, and comparison with known quantities of a monoclonal IgE allowed absolute values to be reported. Values were high in the Maltese x beagle colony (7 to 34 microg/ml), compared with those in the control dogs (0.7 to 6 microg/ml). Total serum and total fecal IgE concentrations did not change in either group during the study. Although allergen-specific IgE was detected in the feces of both groups, significant interassay variability made interpretation of the results difficult. The authors concluded that these Maltese x beagle dogs satisfied the currently recognized clinical criteria for the diagnosis of canine food hypersensitivity. Furthermore, the clinical and serologic responses seen in these dogs in response to oral allergen provocation suggest that this may be a useful model for the study of spontaneous food hypersensitivity.

Allergy for the otologist. External canal to inner ear.

Allergy may affect the outer, the middle, or the inner ear. Although the otologic manifestations of allergy are not by themselves diagnostic, the history, including family history and associated symptoms in other target organs, will often help lead to the correct diagnosis and institution of therapy. Patients with significant and chronic symptoms, including those with labyrinthine symptoms of allergy, will respond well to specific immunotherapy and/or dietary elimination.

Characteristics of cerumen in diabetic patients: a key to understanding malignant external otitis?

Malignant externa otitis is a potentially fatal disease in diabetic and other immunocompromised patients. Cerumen contains defense properties that protect the patient against infection. We tested the hypothesis that patients with diabetes mellitus have abnormalities in their cerumen that affect the environment of their external auditory canals and may predispose them to malignant externa otitis.

Contact hypersensitivity in patients with chronic otitis externa.

Thirty-seven patients with chronic otitis externa were investigated for contact hypersensitivity to 49 agents using standardized patch testing. Positive reactions were found in 22 (58%) patients. This incidence is higher than the 40% found in the only previous similar study and is within the range of 32-72% found in patients with contact dermatitis. The average age of subjects showing sensitivity was 56.7 years compared with 34.9 years for those with negative tests. This significant age difference suggests that contact hypersensitivity is not an aetiological factor in the younger patient. There was no significant difference in the duration of otitis externa between those with positive and negative tests. This feature has not been previously examined or reported. Neomycin was the commonest sensitizing agent, sensitization occurring in 12 (32%) patients.

Necrotizing otitis externa due to Aspergillus in an immunocompetent patient.

An 85-year-old, nondiabetic man is described with necrotizing (malignant) otitis externa. The patient developed facial nerve paralysis in the presence of a progressive cellulitis and chondritis of the external auditory meatus. Destruction of the bony meatus and mastoid and obliteration of normal soft-tissue planes at the skull base were documented by computed tomographic scans. Aspergillus fumigatus was isolated from the external meatus and deep tissue specimens. Fungal invasion of bone and soft tissue was confirmed histopathologically. Pseudomonas aeruginosa was never isolated. This case is unusual from both the standpoint of etiology and host susceptibility in that the patient had no apparent underlying disease. Amphotericin B and rifampin therapy following radical mastoidectomy halted the progression of disease. Finally, this case illustrates the pitfalls of empiric antibacterial therapy for necrotizing otitis externa without cultural confirmation of the etiologic agent.

Immunologic aspects of otologic disease: an overview.

The immunologic explosion has now reached the field of otology. By having better techniques to measure the changes at cellular and molecular levels, it is now possible to devise experiments to show morphologic anatomic changes as well as functional changes. The demonstration in 1980 (M.S.) that tympanosclerosis could be induced immunologically represents a concrete advancement in immunologic thinking in conceptualization of otologic disease. In 1974, one of the authors (M.S.) published work dealing with the treatment of vasculitis of immunologic origin for sudden hearing loss. This was aimed at inhibiting the complement cascade from starting its destructive action. Recently, the immunologic challenge in animals demonstrated by changes in the inner ear was shown by one of the authors (T.J.Y.). Such changes were compatible with labyrinthine hydrops, or Meniere's disease, otosclerosis, and sensorineural hearing loss and vestibular dysfunction.

Malignant external otitis and polymorphonuclear leukocyte migration impairment. Improvement with ascorbic acid.

Malignant external otitis (MEO) is a rare disease due to a Pseudomonas infection of the external ear occurring in an elderly patient with uncontrolled diabetes mellitus. Its high mortality raises the question of an alteration of the defense mechanisms of the body. A 58-year-old man was affected with MEO, and after several months of unsuccessful treatment, a study of the function of his polymorphonuclear neutrophil leukocytes (PMNs) revealed a defect of the migration capability. Ascorbic acid (vitamin C) was proved in vitro to be able to improve the results of the migration test. The patient was treated for one month with ascorbic acid and, parallel to the normalization of the chemotaxis test results, the ear lesions healed. The mechanism of such an alteration of the PMN function, implying several factors (the active infection, old age, and diabetes mellitus), is still unclear. Nevertheless, it is certainly important to test the PMN function in patients with MEO and treat them with immunomodulators.

Impairment of cellular immunity in patients with malignant external otitis.

Immunological studies were performed on 4 elderly diabetic patients with Malignant External Otitis caused by Pseudomonas aeruginosa. Impairment of cellular immunity was found. Skin tests for delayed hypersensitivity to PPD, SK-SD and Mumps antigen were negative and stimulation rates of peripheral blood lymphocytes by PHA, Con-A and PWM were depressed in all 4 patients. Serum immunoglobulins and complement levels were normal except in one case in which paraprotein IgG was found. Peripheral blood lymphoid cell markers and the neutrophil nitroblue tetrazolium (NBT) test were within normal limits in the 2 patients tested. These results indicate that cellular immune deficiency predisposes to the development of Malignant External Otitis in elderly diabetic patients.