Outcomes and complications of cataract surgery in patients with chronic ocular graft-versus-host-disease-a multicenter, retrospective analysis.

PURPOSE: To evaluate the outcome of phacoemulsification in patients with chronic ocular Graft-versus-host disease (oGVHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). METHODS: Retrospective, observational multicenter study from 1507 oGVHD patients. From the patient files, data were collected including best-corrected visual acuity (BCVA), intraocular pressure (IOP), Schirmer's test I, tear film break-up time (TFBUT), corneal fluorescein staining score, postoperative complications, and pre- and post-operative topical therapy. RESULTS: Seventy-three patients underwent cataract surgery in 104 eyes. In n = 84 eyes, the oGVHD NIH grade was documented; 12% (n = 12) of analyzed eyes were staged oGVHD NIH grade 1, 31% (n = 32) NIH 2 and 39% (n = 41) NIH 3. The mean BCVA improved in 82% of the eyes (n = 86 eyes). BCVA significantly increased from 0.7 ± 0.5 to 0.4 ± 0.4 LogMAR after surgery independent from oGVHD severity. The mean IOP decreased from 14 ± 4 to 13 ± 4 mmHg after surgery. Visual acuity was moderately correlated to the pre-operative degree of corneal staining (Pearson p = 0.26, p = 0.002, Cohen's effect size f = 0.29). The visual acuity decreased by 0.078 LogMar units (95% CI = 0.027-0.141) with each increase of corneal staining by one grade (p = 0.05). After surgery, corneal epitheliopathy increased significantly in 42% (n = 44) of the eyes. Postoperative complications included corneal perforation (n = 6, 6%), cystoid macular edema (n = 4, 4%), and endophthalmitis (n = 1, 1%). CONCLUSION: Phacoemulsification in patients with chronic oGVHD significantly improves visual acuity, but is associated with an increased risk of complications in particular corneal epitheliopathy and corneal perforations.

Detection of systemic immunosuppressants in autologous serum eye drops (ASED) in patients with severe chronic ocular graft versus host disease.

PURPOSE: Chronic graft versus host disease is a major consequence after allogeneic stem cell transplantation (allo-SCT) and has great impact on patients' morbidity and mortality. Besides the skin, liver, and intestines, the eyes are most commonly affected, manifesting as severe ocular surface disease. Treatment protocols include topical steroids, cyclosporine, tacrolimus, and ASED. Since these patients often receive systemic immunosuppressant therapy from their oncologists, a topical re-administration of these drugs via ASED with potentially beneficial or harmful effects is possible. The purpose of the study was to determine whether and to which extent systemic immunosuppressants are detectable in ASED. METHODS: A total of 34 samples of ASED from 16 patients with hemato-oncological malignancies after allo-SCT were collected during the manufacturing process and screened for levels of cyclosporine, mycophenolic acid, everolimus, and tacrolimus via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The study followed the tenets of the Declaration of Helsinki and informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. RESULTS: Cyclosporine was found in 18 ASED samples in concentrations ranging from 6.5-105.0 ng/ml (32.0 ± 22.8 ng/ml, mean ± SD). The concentration range of mycophenolic acid in 19 samples was 0.04-25.0 mg/l (4.0 ± 5.4 mg/l, mean ± SD). Everolimus and tacrolimus concentrations were well below the respective limits of quantification (< 0.6 and < 0.5 ng/ml) of the established LC-MS/MS method in all samples. CONCLUSIONS: Our study suggests that orally administered cyclosporine and mycophenolic acid for the treatment of systemic GvHD, but not everolimus and tacrolimus, are distinctly detectable in ASED in relevant concentrations. It is highly likely that these agents affect topical therapy of ocular GvHD. However, the extent of this effect needs to be evaluated in further studies.

Tolerance and Adherence to Cationic 0.1% Cyclosporine in Ocular Graft-versus-Host Disease.

INTRODUCTION: Anti-inflammatory, topical therapy of severe keratitis in dry eye disease (DED) and ocular graft-versus-host disease (oGvHD) includes steroids, cyclosporine (Cs), and others. In Germany, a commercial product containing 0.1% Cs in a cationic formulation is available since 2015. OBJECTIVE: The aim of this study was to present real-life data using cationic 0.1% Cs in oGvHD patients. METHODS: This was a retrospective study of 26 oGvHD and 41 DED patients with corneal staining of at least Oxford grade III. Parameters analyzed were Ocular Surface Disease Index, corneal staining, intraocular pressure, tear film break-up time, Schirmer, and visual acuity. In addition, it was evaluated how different Cs formulations were tolerated. RESULTS: Corneal staining improved significantly in 1 eye in DED but not in oGvHD. In DED, cationic 0.1% Cs was not tolerated by 32% of the patients, in contrast to 0.05% Cs in castor oil not tolerated by 47% and liposomal 0.05% Cs by 63%. In oGvHD patients, cationic 0.1% Cs was not tolerated by 62%, 0.05% Cs in castor oil by 33%, and liposomal 0.05% Cs by 39% of the patients. CONCLUSIONS: This study demonstrates differences between the tolerance of different Cs formulations depending on the underlying cause of severe keratitis. Cationic 0.1% Cs is considerably less tolerated in oGvHD, and its use should be considered with care.

Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis.

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic "window of opportunity" is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool.

Disease-Specific Expression of Conjunctiva Associated Lymphoid Tissue (CALT) in Mouse Models of Dry Eye Disease and Ocular Allergy.

Conjunctiva-associated tissue (CALT) is assumed to play a crucial role in the immune system of the ocular surface. Its function in several ocular surface diseases (OSD) is still not fully understood. This study investigates the function of CALT in mouse models of dry-eye disease and ocular allergy. Since antigen-presentation is the central similarity in the pathologies, this study focuses on antigen-presentation in CALT Morphology and the expression of CALT, which was investigated in mice after induction of dry-eye, ocular allergy, topical antigen-stimulation, and after local depletion of phagocytic cells. Antigen uptake was investigated after the application of fluorescent ovalbumin (OVA). OSD influences the appearance and morphology of CALT in a disease-dependent manner. Ocular allergy leads to an increase and dry-eye disease to a decrease in number and size of CALT. The development of CALT is dependent on the presence of APCs. Professional APCs are present in CALT, and soluble antigen is transported into the follicle. CALT appearance is disease-specific and indicative of differing functions. Although the specific involvement of CALT in OSD needs further study, the existence of functional APCS and antigen-uptake supports the hypothesis that CALT is an immunological key player at the ocular surface.

A semifluorinated alkane (F4H5) as novel carrier for cyclosporine A: a promising therapeutic and prophylactic option for topical treatment of dry eye.

PURPOSE: Cyclosporine A (Cs) has been used as effective topical therapy for inflammatory dry eye disease since more than a decade. However, due to its lipophilic character, Cs is formulated as emulsions or oily solutions for topical application. This experimental study aimed to test if the use of semifluorinated alkanes (SFAs) as a preservative-free, well-tolerated non-stinging or burning vehicle maintains or even improves the benefits of Cs in the topical therapy of dry-eye disease. METHODS: Desiccating stress was applied to C57BL/6 mice for 14 consecutive days to induce experimental dry-eye. Cs dissolved in SFA (perfluorobutylpentane = F4H5with 0.5% Ethanol), F4H5 with 0.5% ethanol only, 0.05% Cs (Restasis®), and dexamethasone (Monodex®) were applied three times daily beginning either at day 4 or day 11 of desiccating stress for up to 3 weeks after end of dry-eye induction. RESULTS: In comparison to other groups, Cs/F4H5 demonstrated high efficacy and earlier reduction of corneal staining. In this study, Cs/F4H5 had the ability to maintain conjunctival goblet cell density once applied on day 4. Flow cytometry analysis from cervical lymphnodes demonstrated a significantly lower CD4+ and CD8+ T-cells in the Cs/F4H5 group following 3 weeks of therapy than at baseline, but no difference in regulatory T cells from regional lymphnodes were seen. CONCLUSIONS: Overall, compared to a commercially available Cs formulation (Restasis®) and dexamethasone, Cs/F4H5 was shown to be equally effective but with a significantly faster therapeutic response in reducing signs of dry-eye disease in an experimental mouse model.

[Influence of Aging on Severity and Anti-Inflammatory Treatment of Experimental Dry Eye Disease]. / Einfluss von Alterung auf Schweregrad und antientzündliche Therapie des experimentellen Trockenen Auges.

Purpose Aging is an important factor in dry-eye disease that has not been studied in the context of therapeutic measures. Aging-associated modifications of the ocular immune system implicate that anti-inflammatory therapies may act differently among younger individuals in terms of onset and effect of different substances. The goal of this study was to determine differences in clinical phenotype and topical anti-inflammatory therapy using a desiccating stress mouse model. Methods An experimental dry-eye disease (desiccating stress model) was induced in 12-week and 12-month-old female BALB/c mice. Topical therapy included 0.05% cyclosporine/F4H5 (Novaliq), F4H5, 0.05% cyclosporine (Restasis®, Allergan) and dexamethasone (Monodex®, Thea Pharma) for 3 consecutive weeks. A control group received no therapy whatsoever. Readout parameters included tear secretion, corneal fluorescein staining at 5 timepoints and histological analysis of goblet cell count at the end of the experiments. Results The older mice demonstrated a significantly stronger dry eye phenotype than the younger mice. Following therapy, the older mice responded to topical anti-inflammatory therapy significantly later than the younger individuals. Regarding the different substances used, cyclosporine/F4H5 showed a significantly faster decrease in corneal fluoresceine staining after only 1 week of therapy in comparison to all other groups. This substance was also superior regarding tear secretion and goblet cell count in age matched groups and in comparison to younger mice. Conclusions These experimental data support the implication that aging should be considered as an important factor in daily clinical practice. Furthermore, the differences found between substance classes, such as calcineurin antagonists and steroids, as well as different drug formulations, should be considered in future pre-clinical and clinical trials.

Detection of graft detachments immediately following Descemet membrane endothelial keratoplasty (DMEK) comparing time domain and spectral domain OCT.

PURPOSE: Correct early graft attachment is believed to be crucial for final visual outcome after Descemet membrane endothelial keratoplasty (DMEK). Nonetheless, it is not yet known which imaging technique gives superior results for examining early postoperative graft adherence status. We compared imaging data taken with two different OCT devices to examine the development of graft adherence immediately after DMEK and to determine the superior device in terms of visualization of graft adherence. METHODS: Ten consecutive patients (1 man/9 women) were examined three times postoperatively within the first 7 h after DMEK surgery using spectral domain OCT (SD-OCT) and time domain OCT (TD-OCT), as prospective case series and retrospective image data analyses. The parameters analyzed were localization and number, visibility and size of graft detachments. RESULTS: TD-OCT was able to detect a greater number of graft detachments after DMEK; however, SD-OCT provided better resolution of minor detachments. Graft detachments varied in position and degree at different time points immediately after surgery. All patients had some graft detachment within the first 7 h after DMEK surgery. CONCLUSIONS: TD-OCT enabled better overall analysis of graft detachments, even in the periphery, whereas SD-OCT allowed for the detection of even minor detachments, which suggests that a combination of the two techniques is optimal. Our results indicate that dynamic processes affecting the DMEK graft immediately after transplantation are responsible for changes in the attachment of donor tissue at an early postoperative stage. Modulation of early graft attachment may improve the final graft attachment.

Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro.

BACKGROUND: Early and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H. METHODS: Using non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally. RESULTS: Decrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models. CONCLUSIONS: Fluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.

Topical application of cannabinoid-ligands ameliorates experimental dry-eye disease.

PURPOSE: Dry eye disease (DED) is a multifactorial disease, with limitations regarding efficacy and tolerability of applied substances. Among several candidates, the endocannabinoid system with its receptors (CB1R and CB2R) were reported to modulate inflammation, wound healing and pain, which are also core DED pathomechanisms. This study is to investigate the therapeutic responses of Δ-9 tetrahydrocannabinol (a non-selective agonist) and two selective antagonists, SR141716A (CB1R antagonist) and SR144528 (CB2R antagonist), as a topical application using a DED mouse model. METHOD: Experimental DED was induced in naïve C57BL/6 mice. Expression of CBR at the ocular surface of naïve and DED mice was determined by qPCR and in-situ hybridization. Either THC or CBR antagonists were compounded in an aqueous solution and dosed during the induction of DED. Tear production, cornea sensitivity, and cornea fluorescence staining were tested. At the end of each experiment, corneas were stained with ß3-tubulin for analysis of corneal nerve morphology. Conjunctiva was analyzed for CD4+ and CD8+ infiltration. RESULTS: CB1R and CB2R are present at the ocular surface, and desiccating stress increased CBR expressions (p < 0.05). After 10 days of DED induction, treated groups demonstrated a reduced CBR expression in the cornea, which was concurrent with improvements in the DED phenotype including fluorescence staining & inflammation. Applying THC protected corneal nerve morphology, thus maintained corneal sensitivity and reduced CD4+ T-cell infiltration. The CB1R antagonist maintained cornea sensitivity without changing nerve morphology. CONCLUSIONS: Endocannabinoid receptor modulation presents a potential multi-functional therapeutic approach for DED.

Olive Pomace Phenolic Compounds: From an Agro-Industrial By-Product to a Promising Ocular Surface Protection for Dry Eye Disease.

Dry eye (DED) is a prevalent disease with immune-mediated inflammation as the principal pathophysiological etiology. Olive pomace, the major by-product of the olive oil industry, is rich in high-value polyphenols. Their anti-inflammatory and immunomodulatory activities were determined on human CD4+ T cells (hTCD4+) and in a DED animal model. The viability of hTCD4+ cells isolated from peripheral blood and activated with phytohemagglutinin-M was evaluated after treatment for 48 h with an olive pomace extract (OPT3, 0.10-0.40 mg/mL) and its major compound, hydroxytyrosol (25-100 µM). Regarding the DED animal model, 100 µM hydroxytyrosol, 0.20 mg/mL OPT3, or vehicle (borate buffer) were topically administered to 14 days-desiccating stress-exposed (constant airflow/scopolamine administration) C57BL/6 mice. Tear volume, corneal fluorescein staining (CFS), CD4+, and CD8+ T cell count in lymph nodes (flow cytometry), and IP-10 and TNF-α gene expression (qRT-PCR) in the cornea, conjunctiva, and lacrimal glands were evaluated. OPT3 (0.2-0.4 mg/mL) and hydroxytyrosol (100 µM) significantly reduced hTCD4+ proliferation. In mice, both treatments reduced lacrimal gland IP-10 gene expression. OPT3 also decreased CFS, and conjunctival IP-10 and corneal TNF-α gene expression. In lymph nodes, hydroxytyrosol reduced CD3+, OPT3, and CD8+ count. Thus, a high-value application as a promising DED protection was proposed for olive pomace.

Desiccating Stress Significantly Increases the Risk for Chronic Ocular Graft-versus-Host-Disease.

Desiccating stress (DS) is known to induce dry eye disease but has not been studied in the context of ocular graft-versus-host disease (oGVHD). Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are exposed to DS on transplantation wards, which are highly climate-regulated for hygienic purposes. Because oGVHD demonstrates features of dry eye disease, this retrospective study aimed to analyze DS as a risk factor for chronic oGVHD. A total of 444 patients undergoing allo-HSCT were investigated with a maximum follow-up of 5.8 years post-transplantation. Relative humidity (%rH) on the transplantation ward was monitored, and data were correlated with the occurrence, severity, and onset of chronic oGVHD, as well as the occurrence of acute skin GVHD. A logistic regression model was used to predict the development of oGVHD. One hundred three of 213 surviving patients developed oGVHD. oGVHD was significantly correlated with a lower %rH (r = .2; P = .03), and more patients (73%) developed oGVHD after transplantation under DS compared with patients after transplantation under high-humidity conditions (30%; P = .02). Reduced humidity increased the relative risk for oGVHD by 4% for each %rH, but it did not affect the severity or time of first diagnosis of oGVHD. In this study, we demonstrate that DS is an independent risk factor for oGVHD. Adjusting air humidity during allo-HSCT has the potential to serve as a preventive measure with clinical relevance.

Treatment of severe chronic ocular graft-versus-host disease using 100% autologous serum eye drops from a sealed manufacturing system: a retrospective cohort study.

BACKGROUND/AIMS: To analyse patients with chronic ocular graft-versus-host disease (GvHD) under treatment with 100% autologous serum eye drops from a sealed manufacturing system. METHODS: 17 patients with chronic ocular GvHD received 100% autologous serum eye drops from single use vials manufactured in a sealed system. Retrospective analysis included visual acuity, corneal staining, frequency of artificial tears, ocular symptoms by means of a questionnaire and information on subjective side effects and cost compensation. RESULTS: Data of prior to autologous serum eye drops therapy and at a 6-month follow-up were obtained. They demonstrated a significant increase in visual acuity (logMAR oculus dexter/right eye (OD) 0.5±0.32 to 0.4±0.3; oculus sinister/left eye (OS) 0.6±0.35 to 0.3±0.35; p=0.177/0.003) and significant improvement in corneal staining (Oxford grading scheme: OD from 3±1.03 to 2±1.43, OS from 4±1.0 to 2±1.09, p=0.004/0.001) and ocular symptoms (ocular surface disease index: 88±20.59 to 63±22.77; p=0.02). Frequency of artificial tears was reduced and no side effects were reported. Patient satisfaction was 100%, and cost compensation by health insurance reached 80%. CONCLUSIONS: 100% autologous serum eye drops using a sealed manufacturing system were efficient in improving the ocular surface, patient symptoms and visual acuity without side effects. It seems to be safe to use 100% autologous serum despite earlier suspicions regarding immune complex accumulations and exacerbation of ocular surface inflammation. The potential effects of serum levels of systemic immunosuppressives through readministration onto the ocular surface need to be elucidated.

Non-Invasive Multi-Dimensional Two-Photon Microscopy enables optical fingerprinting (TPOF) of immune cells.

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients. Two photon based autofluorescence features of immune cells enables non-invasive differentiation.

Optimising deep anterior lamellar keratoplasty (DALK) using intraoperative online optical coherence tomography (iOCT).

BACKGROUND/AIMS: To describe the use of intraoperative online optical coherence tomography (iOCT) for improving deep anterior lamellar keratoplasty (DALK) surgery. METHODS: Retrospective case series of 6 eyes of 6 male patients with keratokonus, corneal dystrophy or herpetic stromal scars undergoing DALK were investigated using intraoperative optical coherence tomography and postsurgical image/video analysis. Main outcome measures were: visibility of surgical steps, especially, assessment of placement depth of injection needle, preparation of bare Descemet's membrane and drainage of interface fluid. RESULTS: iOCT enables real-time visualisation of all surgical steps of DALK procedure in all patients. Placement of air injection needle above Descemet's membrane was reliably monitored as was presence of bare Descemet's membrane and potential interface fluid. CONCLUSIONS: iOCT assists with visualisation of injection needle placement and with assessment of bare Descemet's membrane as well as interface fluid during the DALK procedure. Overall iOCT may be a helpful device that supports surgeons in all steps of DALK procedure.

Optimizing descemet membrane endothelial keratoplasty using intraoperative optical coherence tomography.

IMPORTANCE: Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. OBJECTIVE: To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. DESIGN: Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. SETTING: Department of Ophthalmology, University of Cologne. PARTICIPANTS: Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. EXPOSURE: Descemet membrane endothelial keratoplasty. MAIN OUTCOMES AND MEASURES: Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. RESULTS Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling (R2 = 0.5 [P = .006]). CONCLUSIONS AND RELEVANCE: Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Development, alteration and real time dynamics of conjunctiva-associated lymphoid tissue.

PURPOSE: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. METHODS: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. RESULTS: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. CONCLUSIONS: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.