Modeling Chronic Graft-versus-Host Disease in MHC-Matched Mouse Strains: Genetics, Graft Composition, and Tissue Targets.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen-mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naïve CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4+ or CD8+ cells. Furthermore, co-transferred naïve and effector memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period.

Topical corticosteroids as adjunctive therapy for bacterial keratitis.

BACKGROUND: Bacterial keratitis is a serious ocular infectious disease that can lead to severe visual disability. Risk factors for bacterial corneal infection include contact lens wear, ocular surface disease, corneal trauma, and previous ocular or eyelid surgery. Topical antibiotics constitute the mainstay of treatment in cases of bacterial keratitis, whereas the use of topical corticosteroids as an adjunctive therapy to antibiotics remains controversial. Topical corticosteroids are usually used to control inflammation using the smallest amount of the drug. Their use requires optimal timing, concomitant antibiotics, and careful follow-up. OBJECTIVES: The objective of the review was to assess the effectiveness and safety of corticosteroids as adjunctive therapy for bacterial keratitis. Secondary objectives included evaluation of health economic outcomes and quality of life outcomes. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (2014, Issue 6), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2014), EMBASE (January 1980 to July 2014), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to July 2014), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 14 July 2014. We also searched the Science Citation Index to identify additional studies that had cited the only trial included in the original version of this review, reference lists of included trials, earlier reviews, and the American Academy of Ophthalmology guidelines. We also contacted experts to identify any unpublished and ongoing randomized trials. SELECTION CRITERIA: We included randomized controlled trials (RCTs) that had evaluated adjunctive therapy with topical corticosteroids in people with bacterial keratitis who were being treated with antibiotics. DATA COLLECTION AND ANALYSIS: We used the standard methodological procedures expected by The Cochrane Collaboration. MAIN RESULTS: We found four RCTs that met the inclusion criteria of this review. The total number of included participants was 611 (612 eyes), ranging from 30 to 500 participants per trial. One trial was included in the previous version of the review, and we identified three additional trials through the updated searches in July 2014. One of the three smaller trials was a pilot study of the largest study: the Steroids for Corneal Ulcers Trial (SCUT). All trials compared the treatment of bacterial keratitis with topical corticosteroid and without topical corticosteroid and had follow-up periods ranging from two months to one year. These trials were conducted in the USA, Canada, India, and South Africa.All trials reported data on visual acuity ranging from three weeks to one year, and none of them found any important difference between the corticosteroid group and the control group. The pilot study of the SCUT reported that time to re-epithelialization in the steroid group was 53% slower than the placebo group after adjusting for baseline epithelial defect size (hazard ratio (HR) 0.47; 95% confidence interval (CI) 0.23 to 0.94). However, the SCUT did not find any important difference in time to re-epithelialization (HR 0.92; 95% CI 0.76 to 1.11). For adverse events, none of the three small trials found any important difference between the two treatment groups. The investigators of the largest trial reported that more patients in the control group developed intraocular pressure (IOP) elevation (risk ratio (RR) 0.20; 95% CI 0.04 to 0.90). One trial reported quality of life and concluded that there was no difference between the two groups (data not available). We did not find any reports regarding economic outcomes.Although the four trials were generally of good methodological design, all trials had considerable losses to follow-up (10% or more) in the final analyses. Further, three of the four trials were underpowered to detect treatment effect differences between groups and inconsistency in outcome measurements precluded meta-analyses for most outcomes relevant to this review. AUTHORS' CONCLUSIONS: There is inadequate evidence as to the effectiveness and safety of adjunctive topical corticosteroids compared with no topical corticosteroids in improving visual acuity, infiltrate/scar size, or adverse events among participants with bacterial keratitis. Current evidence does not support a strong effect of corticosteroid, but may be due to insufficient power to detect a treatment effect.

Radiographic Course of Medically Managed Pediatric Orbital Subperiosteal Abscesses.

PURPOSE: To describe the natural radiographic course of subperiosteal orbital abscesses that were managed medically in pediatric patients. METHODS: A retrospective case review was undertaken at Children's Mercy Hospitals and Clinics of Kansas City, Missouri. All patients admitted to the hospital and diagnosed as having orbital cellulitis or subperiosteal abscess from 2008 to 2017 were included in the study. Of the 418 patients identified, 15 patients had repeat imaging and did not undergo surgery prior to the second scan. The initial size of the empyema, size of the empyema on repeat imaging, and clinical course were recorded for each patient. RESULTS: The size of the empyemas increased 240% on average in the first 2 to 3 days. Imaging up to 11 days after the diagnosis showed that 9 cases persisted; meanwhile, 4 cases had radiographic resolution, with the earliest by 21 days. Two cases recurred months later. The largest increase in size was 500% over 3 days, but the initial empyema was only 0.3 cm3. CONCLUSIONS: This review describes the natural history of radiographically reimaged subperiosteal empyema. Empyema size will increase for a few days prior to a gradual resolution in 1 to 3 weeks. An ultimate resolution of radiographic evidence of an empyema takes up to 21 days. This information will help guide clinical management and decision making in caring for patients with pediatric orbital cellulitis with subperiosteal empyema. [J Pediatr Ophthalmol Strabismus. 2018;55(6):387-392.].

Botulinum toxin B-induced mouse model of keratoconjunctivitis sicca.

PURPOSE: To develop a mouse model of human chronic dry eye (keratoconjunctivitis sicca [KCS]). METHODS: Under direct visualization with an operating microscope, CBA/J mice received a transconjunctival injection of saline or 1.25, 5, or 20 milliunits (mU) of botulinum toxin B (BTX-B) into the lacrimal gland. The mice were either left unstressed or were subjected to an air blower for 5 h/d, 5 d/wk in fixed temperature and humidity conditions. Tear production and corneal fluorescein staining were evaluated in all groups before injection and at several time points after. Tear production was measured with phenol red-impregnated cotton threads. Corneal fluorescein staining was photographed under cobalt blue light with a digital camera fitted with a macro lens. RESULTS: BTX-B-injected mice displayed significantly decreased tear production until the 4-week time point. Throughout all time points, the addition of environmental blower stress did not appear to alter tear production significantly. Linear regression models, used to evaluate the effects of various doses of BTX-B on tear production, showed that doses higher than 1.25 mU did not provide significantly different outcomes. After 3 days, saline-injected mice showed no corneal staining, whereas BTX-B-injected mice displayed various amounts of staining. At the early time point (day 3), there did not appear to be an additional effect of the blower on corneal fluorescein staining. However, at 1, 2, and 4 weeks, the blower stress appeared to increase the amount of corneal fluorescein staining at each BTX-B dose, although not significantly. Furthermore, at 8 to 10 weeks, in the BTX B-injected groups, corneas had persistent staining, even though tear production had already returned to normal levels. Histopathologic analyses revealed no inflammatory cell infiltration of the stroma or acini of the lacrimal glands and conjunctivae of both saline-injected and BTX-B-injected animals. CONCLUSIONS: Intralacrimal gland injection of BTX-B resulted in persistent corneal fluorescein staining within 3 days, and a significant decrease in aqueous tear production that persisted for 1 month. Intralacrimal gland injection of BTX-B suppressed lacrimation, thereby establishing a dry eye state. This animal model could be a useful tool for investigating the pathogenesis of the chronic condition KCS in humans.

Modified microkeratome-assisted posterior lamellar keratoplasty using a tissue adhesive.

OBJECTIVE: To compare graft stability and astigmatic change using suture vs tissue adhesive in an experimental model of microkeratome-assisted posterior lamellar keratoplasty. METHODS: A 300-microm-thick partial flap keratectomy was performed in human donor corneoscleral rims using an artificial anterior chamber and a manual microkeratome. The flap stopped at the left central opening border, providing a wide hinge to add stability. After flap reflection, a 6.25-mm trephination was performed to obtain a disc of posterior stroma, Descemet membrane, and endothelium. The disc was positioned in a sutureless fashion, and the flap secured with either 5 interrupted sutures or a chondroitin-sulfate-aldehyde-based adhesive. Increasing intrachamber pressures were created to detect graft stability. Videokeratographic data were recorded to evaluate astigmatic change. RESULTS: The mean (SD) astigmatic change was 3.08 (0.84) diopters (D) in the sutured group and 1.13 (0.55) D in the glued group (P = .008). Mean (SD) resisted pressures were 95.68 (27.38) mm Hg and 82.45 (18.40) mm Hg in the sutured and glued groups, respectively (P = .97). CONCLUSION: This modified technique of microkeratome-assisted posterior lamellar keratoplasty showed excellent graft stability in both groups. Flaps sealed with the novel tissue adhesive had reduced astigmatic changes in our experimental model. CLINICAL RELEVANCE: Sutureless microkeratome-assisted posterior lamellar keratoplasty using tissue adhesive may become a new alternative in the surgical treatment of corneal endothelial disorders.

Use of topical human amniotic fluid in the treatment of acute ocular alkali injuries in mice.

PURPOSE: To evaluate the efficacy of topical human amniotic fluid (HAF) in the treatment of ocular acute alkali burns in mice. DESIGN: Experimental study. METHODS: A chemical burn with 2 microl of sodium hydroxide 0.15 mol/l was created in one eye of 30 mice. The animals were divided into gender- and age-matched groups according to the topical treatment that was administered: group 1 was treated with preterm HAF (n = 10 mice); group 2 was treated with term HAF (n = 10 mice), and group 3 was treated with saline solution (n = 10 mice). Treatment consisted of one drop that was applied to the burned eye five times per day (week one), and three times per day (week two). The epithelial defect was photographed and measured on days two and four. Ocular burn damage was assessed at days two, seven, and 14 after a pre-established classification. On day 14, both eyes of each mouse were enucleated and assessed histopathologically. RESULTS: Median epithelial defect (interquartile range [IQR], 25th, 75th percentile) at day four was 9.93% (IQR, 8.57, 11.27) for group 1, 7.30% (IQR, 5.96, 8.97) for group 2, and 18.92% (IQR, 11.71, 27.64) for group 3 (P < .0076). The overall change (difference in slope) in ocular burn score between days 2 and 14 was -0.127 (P = .009) in group 1 vs 3, -0.134 (P = .012) in group 2 vs 3, and 0.007 (P = .88) in group 1 vs 2. On histologic examination saline solution-treated corneas had more inflammatory cells and blood vessels than HAF-treated corneas. CONCLUSION: Topical preterm/term HAF was an effective topical therapy for limiting the damage after acute alkali burns of the eye in this animal model.

A modified chondroitin sulfate aldehyde adhesive for sealing corneal incisions.

PURPOSE: To compare a modified chondroitin sulfate aldehyde adhesive with standard sutures for sealing corneal incisions. METHODS: A keratome knife was used to create non-self-sealing, uniplanar, 3-mm, clear corneal incisions in enucleated rabbit eyes (n = 18). The wounds were sealed with either a chondroitin sulfate-aldehyde adhesive (n = 8), three 10-0 nylon sutures (n = 5), or one 10-0 nylon suture (n = 5). Wound stability was tested by filling the globes with balanced salt solution through an anterior chamber port and slowly increasing the IOP. The pressure changes were monitored with a digital manometer connected to the anterior chamber, and leak pressure was recorded for each eye. Confocal microscopy was performed on the glued eyes, to document the glue distribution along the wound. RESULTS: The mean leak pressures in the single-suture and three-suture subgroups were 26.4 +/- 6.0 and 44.3 +/- 8.2 mm Hg (SD), respectively. The maximum IOP achieved in eyes that received the glue was 104.7 mm Hg with a mean of 101.4 +/- 3.2 mm Hg. None of the eyes in which glue was used showed leakage. At confocal microscopy, the glue was distributed inside the wound edges as a homogeneous thin layer of a less dense signal than that of the stroma. CONCLUSIONS: A novel chondroitin sulfate-aldehyde adhesive was shown to be effective ex vivo for sealing corneal incisions in rabbit eyes and was superior to sutures for this purpose.

Inflow of ocular surface fluid into the anterior chamber after phacoemulsification through sutureless corneal cataract wounds.

PURPOSE: To report inflow of extraocular fluid after phacoemulsification with use of sutureless corneal incisions. DESIGN: Interventional case series. METHODS: setting: Wilmer Eye Institute, Johns Hopkins Hospital, Baltimore, Maryland. patients: Eight patients (three women), aged 58 to 91 years, showing minimal bleeding from the limbal capillary bed during phacoemulsification. intervention: Surgery was performed through a 2.8-mm limbal incision. External pressure simulating patient manipulation was applied before and after wound hydrosealing with an irrigation cannula. main outcome measures: Inflow of blood-tinged tear fluid into the anterior chamber through the wound was monitored by using digital video. RESULTS: Inflow of extraocular fluid was observed in all eyes when the cannula was released, even after wound hydrosealing. Two patients showed spontaneous fluid inflow. CONCLUSIONS: Tested sutureless corneal incisions allow inflow of extraocular fluid into the anterior chamber after phacoemulsification. This may permit intraocular contamination leading to endophthalmitis.

Recruitment of Donor T Cells to the Eyes During Ocular GVHD in Recipients of MHC-Matched Allogeneic Hematopoietic Stem Cell Transplants.

PURPOSE: The primary objective of the present study was to identify the kinetics and origin of ocular infiltrating T cells in a preclinical model of graft-versus-host disease (GVHD) that induces eye tissue damage. METHODS: Graft-versus-host disease was induced using an major histocompatibility complex (MHC)-matched, minor histocompatibility-mismatched hematopoietic stem cell transplant (HSCT) model. This approach, which utilized congenic and EGFP-labeled donor populations, mimics a matched, clinically unrelated donor (MUD) cell transplant. Systemic and ocular GVHD were assessed at varying time points using clinical examination, intravital microscopy, immune phenotype via flow cytometric analyses, and immunohistochemical staining. RESULTS: Following transplant, we observed characteristic changes in GVHD-associated immune phenotype as well as clinical signs present in recipients post transplant. Notably, the kinetics of the systemic changes and the ocular damage paralleled what is observed clinically, including damage to the cornea as well as the conjunctiva and lacrimal gland. Importantly, the infiltrate contained predominantly donor CD4 as well as CD8 T cells with an activated phenotype and macrophages together with effector cytokines consistent with the presence of a TH1 alloreactive population. CONCLUSIONS: Overall, the findings here unequivocally demonstrated that donor T cells compose part of the corneal and ocular adnexa infiltrate in animals undergoing ocular GVHD. In total, the results describe a novel and promising preclinical model characterized by both systemic and ocular changes as detected in significant numbers of patients undergoing GVHD following allo-HSCT, which can help facilitate dissecting the underlying immune mechanisms leading to damage associated with ocular GVHD.

The allure and peril of hematopoietic stem cell transplantation: overcoming immune challenges to improve success.

Since its inception in the mid-twentieth century, the complication limiting the application and utility of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat patients with hematopoietic cancer is the development of graft-versus-host disease (GVHD). Ironically, GVHD is induced by the cells (T lymphocytes) transplanted for the purpose of eliminating the malignancy. Damage ensuing to multiple tissues, e.g., skin, GI, liver, and others including the eye, provides the challenge of regulating systemic and organ-specific GVH responses. Because the immune system is also targeted by GVHD, this both: (a) impairs reconstitution of immunity post-transplant resulting in patient susceptibility to lethal infection and (b) markedly diminishes the individual's capacity to generate anti-cancer immunity--the raison d'etre for undergoing allo-HSCT. We hypothesize that deleting alloreactive T cells ex vivo using a new strategy involving antigen stimulation and alkylation will prevent systemic GVHD thereby providing a platform for the generation of anti-tumor immunity. Relapse also remains the major complication following autologous HSCT (auto-HSCT). While GVHD does not complicate auto-HSCT, its absence removes significant grant anti-tumor responses (GVL) and raises the challenge of generating rapid and effective anti-tumor immunity early post-transplant prior to immune reconstitution. We hypothesize that effective vaccine usage to stimulate tumor-specific T cells followed by their amplification using targeted IL-2 can be effective in both the autologous and allogeneic HSCT setting. Lastly, our findings support the notion that the ocular compartment can be locally targeted to regulate visual complications of GVHD which may involve both alloreactive and self-reactive (i.e., autoimmune) responses.

Proteomics of the aqueous humor in healthy New Zealand rabbits.

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.

Metabolic changes in mesenchymal stem cells in osteogenic medium measured by autofluorescence spectroscopy.

The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell-supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription-polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction-oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell-cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.

¿Queratitis difusa lamellar?: desafortunadamente un diagnóstico errado / Diffuse lamellar keratitis?: unfortunately wrong diagnosis

Reportar un caso de hipertensión ocular inducida por esteriodes con presencia de fluido en la interfase luego de queratomileusis láser (LASIK), diagnósticado de manera errónea como queratitis difusa lamelar (DLK). Paciente de sexo femenino de 53 años de edad presentó visión borrosa posterior a cirugía LASIK bilateral. La presión intraocular (PIO) era normal en ambos ojos con tonometría de aplanación. Fue reoperada 2 veces, diagnosticada con DLK de inicio tardío y tratada sin éxito con el uso de lentes de contacto terapéuticos, esteroides tópicos, antibióticos y solución salina hipertónica al 5 por ciento. Se encontró fluido en la entrecara y se sospechó la presencia y hipertensión ocular. Los esteroides fueron descontinuados y mejoró la agudeza visual. La tonometría de aplanación reveló entonces 30mmHg en ambos ojos, la cual retornó a valores normales con el uso de maleto timolol al 0.50 por ciento. El tratamiento de la DLK mal diagnósticada puede llevar al empeoramiento de una hipertensión ocular inducida por esteroides. Una cuidadosa evaluación biomicroscópica y corroboración de los valores de PIO es mandatario en estos pacientes