Adjunctive canakinumab reduces peripheral inflammation markers and improves positive symptoms in people with schizophrenia and inflammation: A randomized control trial.

BACKGROUND: Clinical trials of anti-inflammatories in schizophrenia do not show clear and replicable benefits, possibly because patients were not recruited based on elevated inflammation status. Interleukin 1-beta (IL-1ß) mRNA and protein levels are increased in serum, plasma, cerebrospinal fluid, and brain of some chronically ill patients with schizophrenia, first episode psychosis, and clinical high-risk individuals. Canakinumab, an approved anti-IL-1ß monoclonal antibody, interferes with the bioactivity of IL-1ß and interrupts downstream signaling. However, the extent to which canakinumab reduces peripheral inflammation markers, such as, high sensitivity C-reactive protein (hsCRP) and symptom severity in schizophrenia patients with inflammation is unknown. TRIAL DESIGN: We conducted a randomized, placebo-controlled, double-blind, parallel groups, 8-week trial of canakinumab in chronically ill patients with schizophrenia who had elevated peripheral inflammation. METHODS: Twenty-seven patients with schizophrenia or schizoaffective disorder and elevated peripheral inflammation markers (IL-1ß, IL-6, hsCRP and/or neutrophil to lymphocyte ratio: NLR) were randomized to a one-time, subcutaneous injection of canakinumab (150 mg) or placebo (normal saline) as an adjunctive antipsychotic treatment. Peripheral blood hsCRP, NLR, IL-1ß, IL-6, IL-8 levels were measured at baseline (pre injection) and at 1-, 4- and 8-weeks post injection. Symptom severity was assessed at baseline and 4- and 8-weeks post injection. RESULTS: Canakinumab significantly reduced peripheral hsCRP over time, F(3, 75) = 5.16, p = 0.003. Significant hsCRP reductions relative to baseline were detected only in the canakinumab group at weeks 1, 4 and 8 (p's = 0.0003, 0.000002, and 0.004, respectively). There were no significant hsCRP changes in the placebo group. Positive symptom severity scores were significantly reduced at week 8 (p = 0.02) in the canakinumab group and week 4 (p = 0.02) in the placebo group. The change in CRP between week 8 and baseline (b = 1.9, p = 0.0002) and between week 4 and baseline (b = 6.0, p = 0.001) were highly significant predictors of week 8 change in PANSS Positive Symptom severity scores. There were no significant changes in negative symptoms, general psychopathology or cognition in either group. Canakinumab was well tolerated and only 7 % discontinued. CONCLUSIONS: Canakinumab quickly reduces peripheral hsCRP serum levels in patients with schizophrenia and inflammation; after 8 weeks of canakinumab treatment, the reductions in hsCRP are related to reduced positive symptom severity. Future studies should consider increased doses or longer-term treatment to confirm the potential benefits of adjunctive canakinumab in schizophrenia. Australian and New Zealand Clinical Trials Registry number: ACTRN12615000635561.

The microbiome in HLA-B27-associated disease: implications for acute anterior uveitis and recommendations for future studies.

The pathogenesis of human leukocyte antigen (HLA)-B27-associated diseases such as acute anterior uveitis (AAU) and ankylosing spondylitis (AS) remains poorly understood, though Gram-negative bacteria and subclinical bowel inflammation are strongly implicated. Accumulating evidence from animal models and clinical studies supports several hypotheses, including HLA-B27-dependent dysbiosis, altered intestinal permeability, and molecular mimicry. However, the existing literature is hampered by inadequate studies designed to establish causation or uncover the role of viruses and fungi. Moreover, the unique disease model afforded by AAU to study the gut microbiota has been neglected. This review critically evaluates the current literature and prevailing hypotheses on the link between the gut microbiota and HLA-B27-associated disease. We propose a new potential role for HLA-B27-driven altered antibody responses to gut microbiota in disease pathogenesis and outline recommendations for future well-controlled human studies, focusing on AAU.

Improved performance of confirmatory assays for detecting dense fine speckled (DFS) 70 antibodies.

The presence of monospecific dense fine speckled 70 (DFS70) pattern in indirect immunofluorescence assay (IFA) without concomitant systemic autoimmune related diseases (SARD)-associated antibodies could be an exclusion biomarker for SARD. Since interpretation of DFS pattern on IFA is subjective, an assay for confirming the presence of anti-DFS70 is required. We evaluated the performance of two commercial assays [fluorescence enzyme immunoassay (FEIA) and line immunoassay (LIA)] for detecting anti-DFS70. Sera with monospecific DFS (n=176) and without DFS (n=179) pattern from referred patients for ANA testing, in two independent laboratories and healthy donors, were investigated for anti-DFS70 by FEIA (Phadia EliA) and LIA (Euroimmun). The assay performance including sensitivity and specificity at different cut-offs was evaluated, and optimal cut-offs were determined. The newly established optimal cut-offs (2.7 U/mL on EliA, band intensity of 28 on LIA) showed significantly better assay performance in detecting anti-DFS70 and confirming monospecific DFS pattern. A relative sensitivity of 93.7% with relative specificity of 100% on EliA and a relative sensitivity of 96.6% with relative specificity of 95.3% on LIA were achieved. Superior Cohen's Kappa agreements with IFA were also achieved for both assays (0.936 for EliA and 0.906 for LIA). Application of an optimal cut-off can significantly increase the assay performance for anti-DFS70 and enhance the accuracy in reporting DFS pattern by IFA.

Adalimumab in patients with vision-threatening uveitis: real-world clinical experience.

OBJECTIVES: Biologics are rapidly emerging as an effective vision saving addition to systemic uveitis therapy. The aim of this multicentre retrospective study is to review the outcomes of a large group of patients treated with adalimumab. METHODS: A retrospective chart review of patients with refractory non-infectious, active uveitis treated with adalimumab was conducted. The main outcome measures were ability to reduce prednisolone dose, ability to control uveitis, final visual acuity and time to treatment failure. RESULTS: Forty-six patients with uveitis, treated with adalimumab were included in the study. The most common anatomical uveitis phenotype was panuveitis (n=17, 37.0%). The most common diagnosis was idiopathic uveitis (n=19, 41.3%). At their latest review (mean: 4.46 years; median 4.40 years), 35 (76.1%) patients were able to discontinue corticosteroids, 11 (23.9%) patients were able to taper to <7.5 mg/day and only 1 (2.2%) patient required 10 mg of prednisone. The mean visual acuity at the latest follow-up of the worse eye was logarithm of the minimum angle of resolution (logMAR) 0.42 (SD 0.72), while the mean visual acuity of the better eye was logMAR 0.19 (SD 0.34). Of the 89 eyes, 21 (23.6%) eyes improved by at least 2 lines, 5 eyes (5.6%) deteriorated by ≥2 lines while vision was unchanged in the remaining 63 (70.8%) eyes. The time to recurrence was 1 in 12.47 person-years for adalimumab, with a 17.4% (8 patient) relapse rate. There were no serious adverse events. CONCLUSIONS: This study highlights the efficacy of adalimumab in patients with vision-threatening non-infectious uveitis, preserving vision and allowing reduction of corticosteroid dose.

Inducing dry eye disease using a custom engineered desiccation system: Impact on the ocular surface including keratin-14-positive limbal epithelial stem cells.

PURPOSE: Dry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its' effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs. METHODS: A mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs. RESULTS: Custom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4+ cell infiltration compared to controls. DS elicited K14+ epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50-100 µm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317]. CONCLUSIONS: Application of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic.

Regulation of the Acute Sickness Response by the P2RX7 Receptor.

BACKGROUND: The acute sickness response to infection is a stereotyped set of illness manifestations initiated by proinflammatory signals in the periphery but mediated centrally. P2RX7 is a highly polymorphic gene encoding an ATP-gated cationic pore, widely expressed on immune cells and the brain, and regulating the NLRP3 inflammasome, as well as diverse neural functions. METHODS: Associations between P2RX7 genotype, pore activity, and illness manifestations were examined in a cohort with acute viral and bacterial infections (n = 484). Genotyping of 12 P2RX7 function-modifying single-nucleotide polymorphisms (SNPs) was used to identify haplotypes and diplotypes. Leucocyte pore activity was measured by uptake of the fluorescent dye, YO-PRO-1, and by ATP-induced interleukin-1ß (IL-1ß) release. Associations were sought with scores describing the symptom domains, or endophenotypes, derived from principal components analysis. RESULTS: Among the 12 SNPs, a 4-SNP haplotype block with 5 variants was found in 99.5% of the subjects. These haplotypes and diplotypes were closely associated with variations in pore activity and IL-1ß production. Homozygous diplotypes were associated with overall illness severity as well as fatigue, pain, and mood disturbances. CONCLUSIONS: P2RX7 signaling plays a significant role in the acute sickness response to infection, likely acting in both the immune system and the brain.

The severity of the pathogen-induced acute sickness response is affected by polymorphisms in genes of the NLRP3 inflammasome pathway.

The acute sickness response (ASR) is a stereotyped set of symptoms including fatigue, pain, and disturbed mood, which are present in most acute infections. The immunological mechanisms of the ASR are conserved, with variations in severity determined partly by the pathogen, but also by polymorphisms in host genes. The ASR was characterised in three different serologically-confirmed acute infections in Caucasians (n = 484) across four symptom domains or endophenotypes (termed 'Fatigue', 'Musculoskeletal pain', 'Mood disturbance', and 'Acute sickness'). Correlations were sought with functional single nucleotide polymorphisms in the NLRP3 inflammasone pathway and severity of the endophenotypes. Individuals with severe Fatigue, Musculoskeletal pain, or Mood endophenotypes were more likely to have prior episodes of significant fatigue (11.4 vs. 3.8%, p = 0.07), pain (14.3 vs. 1.2%, p = 0.001), or Mood disturbance (13 vs 1%, p=0.001), suggesting trait characteristics. The high functioning allele of the rs35829419 SNP in NLRP3 was more common in those with severe Fatigue (OR = 13.3, 95% CI: 1.7-104), particularly in a dominant inheritance pattern (OR = 13.4, 95% CI: 1.8-586.3). In a multivariable analysis assuming dominant inheritance, both rs35829419 and the rs4848306 SNP in Interleukin(IL)-1ß, were independently associated with severe Fatigue (OR = 29.6, 95% CI: 2.6-330.9 and OR = 13, 95% CI: 2.7-61.8, respectively). The severity of fatigue in acute infection is influenced by genetic polymorphisms in NLRP3 and IL-1ß.

Is glaucoma an autoimmune disease?

There is increasing evidence from animal and human studies that glaucoma is an autoimmune disease. Evidence for this hypothesis includes the fact that antibodies as well as T-cell responses to heat-shock proteins (HSPs) are detectable in some patients with glaucoma and in an animal model of the disease. As in the human disease, experimental animal models of glaucoma have been found to demonstrate neurodegenerative changes in the optic nerve associated with immunoglobulin and T-cell infiltration. Although there is still insufficient evidence in humans to classify all cases of glaucoma as autoimmune diseases, the implications of this hypothesis have major impact on the diagnosis and treatment of glaucoma.

Genomewide Association Study of Acute Anterior Uveitis Identifies New Susceptibility Loci.

Purpose: Acute anterior uveitis (AAU) is a common intraocular inflammatory disease. AAU occurs in 30% to 50% of patients with ankylosing spondylitis (AS), and both conditions are strongly associated with human leukocyte antigen (HLA)-B27, implying a shared etiology. This study aims to apply genomewide association study (GWAS) to characterize the genetic associations of AAU and their relationship to the genetics of AS. Methods: We undertook the GWAS analyses in 2752 patients with AS with AAU (cases) and 3836 patients with AS without AAU (controls). There were 7,436,415 single-nucleotide polymorphisms (SNPs) available after SNP microarray genotyping, imputation, and quality-control filtering. Results: We identified one locus associated with AAU at genomewide significance: rs9378248 (P = 2.69 × 10-8, odds ratio [OR] = 0.78), lying close to HLA-B. Suggestive association was observed at 11 additional loci, including previously reported AS loci ERAP1 (rs27529, P = 2.19 × 10-7, OR = 1.22) and NOS2 (rs2274894, P = 8.22 × 10-7, OR = 0.83). Multiple novel suggestive associations were also identified, including MERTK (rs10171979, P = 2.56 × 10-6, OR = 1.20), KIFAP3 (rs508063, P = 5.64 × 10-7, OR = 1.20), CLCN7 (rs67412457, P = 1.33 × 10-6, OR = 1.25), ACAA2 (rs9947182, P = 9.70 × 10-7, OR = 1.37), and 5 intergenic loci. The SNP-based heritability is approximately 0.5 for AS alone, and is much higher (approximately 0.7) for AS with AAU. Consistent with the high heritability, a genomewide polygenic risk score shows strong power in identifying individuals at high risk of either AS with AAU or AS alone. Conclusions: We report here the first GWAS for AAU and identify new susceptibility loci. Our findings confirm the strong overlap in etiopathogenesis of AAU with AS, and also provide new insights into the genetic basis of AAU.

Recent Developments in HLA B27 Anterior Uveitis.

There has been steady progress in understanding the pathogenesis, clinical features, and effective treatment of acute anterior uveitis (AU) over the past 5 years. Large gene wide association studies have confirmed that AU is a polygenic disease, with overlaps with the seronegative arthropathies and inflammatory bowel diseases, associations that have been repeatedly confirmed in clinical studies. The role of the microbiome in AU has received increased research attention, with recent evidence indicating that human leukocyte antigen B27 (HLA B27) may influence the composition of the gut microbiome in experimental animals. Extensive clinical investigations have confirmed the typical features of acute AU (AAU) and its response to topical, regional and systemic immunosuppressive treatment. Increased understanding of the role of cytokines has resulted in studies confirming the value of anti-cytokine therapy [anti-tumor necrosis factor (anti-TNF) and interleukin 6 (IL-6) therapy] in severe and recurrent cases of AAU, particularly in subjects with an associated spondyloarthopathy (SpA) and in juvenile idiopathic arthritis (JIA)-associated AAU.

Dose-dependent benzalkonium chloride toxicity imparts ocular surface epithelial changes with features of dry eye disease.

PURPOSE: Inclusion of the preservative benzalkonium chloride (BAC) in ophthalmic solutions is prevalent, despite the noted potential for exacerbating dry eye disease (DED). Whilst studies incorporating BAC have assessed its' effects as a mouse model of DED, the impact on limbal epithelia is under-studied. Our investigation aimed to comprehensively assess the impact of different BAC dosing regimens and their suitability as a mouse model of DED. METHODS: C57BL/6J mice (n = 72) were administered topical BAC (0.05-0.2%) over 7 days. Fluorescein staining, corneal smoothness index, and immuno-histological analyses were applied to determine architectural and cellular changes on the ocular surface following BAC treatment. The effect of BAC (0.0001-0.01%) on cultivated primary mouse corneo-limbal epithelial cells (CLECs) (n = 6) was examined using morphological and functional assays. RESULTS: Whilst 0.2% BAC induced severe corneal epithelial defects, 0.1% BAC dispensed once daily over 7 days, induced punctate fluorescein staining without detriment to corneal smoothness. Histochemical staining revealed disorganized basal corneal epithelial cells with enlarged cytoplasmic halos. Furthermore, PAS+ goblet cells were decreased. BAC treatment also modulated K14 expression and distribution within the limbus. In cultured CLEC, BAC triggered cell contraction and vacuolation, increased LDH release and elevated cell necrosis by 4.1-fold. Concentrations of BAC as low as 0.0001% decreased colony formation. CONCLUSIONS: This study describes how exposing C57BL/6 mice to BAC induce some clinicopathological features of DED seen in humans, and therefore provides the foundations to explore the consequences on the ocular surface, particularly on limbal epithelia and its' stem cells.

Contribution of individual psychological and psychosocial factors to symptom severity and time-to-recovery after naturally-occurring acute infective illness: The Dubbo Infection Outcomes Study (DIOS).

BACKGROUND: Substantial heterogeneity exists in both the severity of symptoms experienced as part of the sickness response to naturally-occurring infections, and the time taken for individuals to recover from these symptoms. Although contributing immunological and genetic factors have been previously been explored, less is known about the role of individual psychological and psychosocial factors, which may modulate the host immune response, or contribute independently, to symptom severity and duration. METHODS: Longitudinally-collected data from 484 Caucasian participants (mean age: 33.5 years; 51% women) experiencing a naturally-occurring acute infective illness enrolled in the prospective Dubbo Infection Outcome Study (DIOS) were analysed. At intake and subsequent follow-up assessments, self-report questionnaires were used to ascertain individual psychological and psychosocial characteristics and symptom information. Principal component analysis was applied to symptom data to derive endophenotype severity scores representing discrete symptom domains (fatigue, mood, pain, neurocognitive difficulties) and an overall index of severity. The contribution of individual psychological (trait neuroticism, locus of control, and illness behaviours) and psychosocial factors (relative socioeconomic advantage) to endophenotype severity at baseline were examined using multivariable linear regression models; interval-censored flexible parametric proportional hazards survival models were used to explore time to recovery (defined using within-sample negative threshold values). RESULTS: After controlling for time since symptom onset, greater levels of trait neuroticism consistently predicted greater symptom severity across all symptom domains (all p's < 0.015). Similarly, greater relative socioeconomic disadvantage was significantly associated with greater severity across all endophenotypes (p's < 0.025) except neurocognitive disturbance. Locus of control and illness behaviours contributed differentially across endophenotypes. Reduced likelihood of recovery was significantly predicted by greater initial symptom severity for all endophenotypes (all p's < 0.001), as well as higher levels of trait neuroticism. CONCLUSIONS: Individual psychological and psychosocial factors contribute to the initial severity and to the prolonged course of symptoms after naturally-occurring infective illnesses. These factors may play an independent role, represent a bias in symptom reporting, or reflect increased stress responsivity and a heightened inflammatory response. Objective metrics for severity and recovery are required to further elucidate their roles.

Ocular Toxoplasmosis in a Tertiary Referral Center in Sydney Australia-Clinical Features, Treatment, and Prognosis.

PURPOSE: The aim of this study was to provide a retrospective analysis of the presentation, demographics, and treatment regimens for ocular toxoplasmosis at a large tertiary referral uveitis center. DESIGN: Retrospective cohort study. PARTICIPANTS: A total of 48 patients with ocular toxoplasmosis who presented to Sydney Eye Hospital participated in this study. METHODS: This is a retrospective review of patient files who presented to Sydney Eye Hospital between 2007 and 2016 with clinical features consistent with ocular toxoplasmosis. Baseline risk factors and treatment details were recorded and analyzed. Main outcome measures were visual acuity and relapse rate compared with other studies in ocular toxoplasmosis. RESULTS: The median age was 35.5 (interquartile range 21-50) with 30 (60%) patients having no previous symptomatic episodes or evidence of chorioretinal scarring. Visual acuity at presentation was 0.51 or 6/19 (SE 0.096) and at follow-up 0.31 or 6/12 (SE 0.094). Nine patients experienced a recurrence during the period of observation with median time to recurrence 2.2 years (SE 0.45) and the relapse rate was 0.09/person-years. Location of lesion was predominantly within the vascular arcades (n = 44) with macular involvement in 9 patients. Most patients received clindamycin therapy (n = 34) with pyrimethamine and sulfadiazine was used for those with macula involvement. CONCLUSIONS: Patients with ocular toxoplasmosis had fewer recurrences compared with other published series and had better visual recovery. The majority of patients received clindamycin and oral prednisolone which were well tolerated with pyrimethazine and sulfadiazine reserved for those with macula-involving disease.

Visualizing the Contribution of Keratin-14+ Limbal Epithelial Precursors in Corneal Wound Healing.

It is thought that corneal epithelial injuries resolve by leading-edge cells "sliding" or "rolling" into the wound bed. Here, we challenge this notion and show by real-time imaging that corneal wounds initially heal by "basal cell migration." The K14CreERT2-Confetti multi-colored reporter mouse was employed to spatially and temporally fate-map cellular behavior during corneal wound healing. Keratin-14+ basal epithelia are forced into the wound bed by increased population pressure gradient from the limbus to the wound edge. As the defect resolves, centripetally migrating epithelia decelerate and replication in the periphery is reduced. With time, keratin-14+-derived clones diminish in number concomitant with their expansion, indicative that clonal evolution aligns with neutral drifting. These findings have important implications for the involvement of stem cells in acute tissue regeneration, in key sensory tissues such as the cornea.

Incidence, clinical features and diagnosis of cicatrising conjunctivitis in Australia and New Zealand.

AIMS: This study aimed to determine the incidence, clinical features and management of cicatrising conjunctivitis in Australia and New Zealand, also enabling comparison with data from the United Kingdom. METHODS: A prospective surveillance study was conducted over 17 months via the Australian and New Zealand Ophthalmic Surveillance Unit with a one-year follow-up period. Practicing ophthalmologists on the Surveillance Unit's database were asked to report recently diagnosed cases of cicatrising conjunctivitis on a monthly basis. Initial and follow-up questionnaires were sent to ophthalmologists who had reported positive cases to obtain demographic and clinical data. The minimum incidence of cicatrising conjunctivitis was calculated based on cases reported during the study period and from population data. RESULTS: During the 17-month study period (December 2011-April 2013), 56 cases of cicatrising conjunctivitis were reported. Data was obtained for 35 cases (62%) with a mean age of 74 years (range, 28-94 years). The most common aetiologies were ocular mucus membrane pemphigoid (n = 18 cases, 51.4%), Stevens-Johnson Syndrome (n = 3, 8.6%) and graft versus host disease (n = 3, 8.6%). The minimum incidence of cicatrising conjunctivitis in Australia and New Zealand was 1.5 per million, comparable to incidence data from the United Kingdom. CONCLUSIONS: This study is the first to prospectively record the incidence of cicatrising conjunctivitis in Australia and New Zealand and the second worldwide. It provides novel data on demographics and management of cicatrising conjunctivitis, as reported by treating ophthalmologists.

Sarcoidosis: a state of the art review from the Thoracic Society of Australia and New Zealand.

Sarcoidosis is a systemic disease of unknown aetiology, characterised by non-caseating granulomatous inflammation. It most commonly manifests in the lungs and intrathoracic lymph nodes but can affect any organ. This summary of an educational resource provided by the Thoracic Society of Australia and New Zealand outlines the current understanding of sarcoidosis and highlights the need for further research. Our knowledge of the aetiology and immunopathogenesis of sarcoidosis remains incomplete. The enigma of sarcoidosis lies in its immunological paradox of type 1 T helper cell-dominated local inflammation co-existing with T regulatory-induced peripheral anergy. Although specific aetiological agents have not been identified, mounting evidence suggests that environmental and microbial antigens may trigger sarcoidosis. Genome-wide association studies have identified candidate genes conferring susceptibility and gene expression analyses have provided insights into cytokine dysregulation leading to inflammation. Sarcoidosis remains a diagnosis of exclusion based on histological evidence of non-caseating granulomas with compatible clinical and radiological findings. In recent years, endobronchial ultrasound-guided transbronchial needle aspiration of mediastinal lymph nodes has facilitated the diagnosis, and whole body positron emission tomography scanning has improved localisation of disease. No single biomarker is adequately sensitive and specific for detecting and monitoring disease activity. Most patients do not require treatment; when indicated, corticosteroids remain the initial standard of care, despite their adverse side effect profile. Other drugs with fewer side effects may be a better long term choice (eg, methotrexate, hydroxychloroquine, azathioprine, mycophenolate), while tumour necrosis factor-α inhibitors are a treatment option for patients with refractory disease.

Visualizing the Fate of Transplanted K14-Confetti Corneal Epithelia in a Mouse Model of Limbal Stem Cell Deficiency.

Purpose: Therapies for limbal stem cell deficiency (LSCD) include stem cell (SC) grafts that regenerate the damaged ocular surface. However, the fate of transplanted cells is ill-defined. We addressed this limitation using primary corneal epithelial cells from K14-Confetti mice. Methods: Cultures of primary corneo-limbal epithelia were generated from K14-Confetti (n = 6) and wild-type (WT) (n = 3) mice. Cell phenotype and function was ascertained by immunofluorescence, flow cytometry, quantitative PCR and colony formation. K14-Confetti cells were nurtured on fibrin and transferred onto WT mice with experimentally induced LSCD (n = 16) to determine the site of implantation, longevity, and phenotype. Results: Transgenic and WT cells derived from explanted corneal tissue displayed no phenotypic or functional differences. K14-Confetti corneo-limbal epithelia that engrafted in recipient LSCD WT mice formed 107 ± 36 fluorescent clones at 2 weeks postprocedure, which decreased to 70.0 ± 5.5 by 6 weeks (P = 0.15). Furthermore, cells commonly implanted in the periphery (P < 0.05) and some generated clones that migrated centripetally. However, a normal corneal epithelial phenotype was not restored. We speculate this is due to insufficient SCs being seeded within grafts, and shows evidence of both cell loss from the implants and transdifferentiation into K8+-conjunctival and K10+-cutaneous epithelia after transplantation. Conclusions: This study successfully tracked the fate of transplanted corneo-limbal epithelia in a mouse model of LSCD by intravital microscopy. Our data shed new light into how donor cells behave, the positions they take, how long they survive, and potential mechanisms of loss from the ocular surface. This information is important for improving future animal models, to render them clinically relevant.