Focused ultrasound facilitated thermo-chemotherapy for targeted retinoblastoma treatment: a modeling study.

Retinoblastoma is the most common type of intraocular tumors in children. Currently, with early detection and improved systemic chemo-adjuvant therapies, treatment paradigm has shifted from survival to globe salvation/vision preservation. The objective of our work has been to explore the possible application of focused ultrasound (FUS) for targeted drug delivery in the posterior pole retinoblastoma. Specifically, theoretical models were implemented to evaluate the feasibility of using FUS to generate localized hyperthermia in retinal tumor areas, for potential triggering the chemotherapeutic agent deployment from heat-sensitive drug carriers. In-vitro experiments were conducted in tissue-mimicking phantoms with embedded excised rabbit eyes to validate the reliability of the modeling results. After confirming the reliability of our model, various FUS transducer parameters were investigated to induce maximal hyperthermia coverage in the tumor, while sparing adjacent eye structures (e.g. the lens). The evaluated FUS parameters included operating frequency, total acoustic power, geometric dimensions, transducer f-number, standoff distance, as well as different pulsing scenarios. Our modeling results suggest that the most suitable ultrasound frequency for this type of treatments was in the range of 2-3.5 MHz depending on the size of retinoblastoma. Appropriate transducer f-number (close to 1) and standoff distance could be selected to minimize the risks of over-heating undesired regions. With the total acoustic power of 0.4 W, 56.3% of the tumor was heated to hyperthermic temperature range (39-44 °C) while the temperature in lens was maintained below 41 °C. In conclusion, FUS-induced hyperthermia for targeted drug delivery may be a viable option in treatments of juxta-foveal or posterior pole retinoblastomas. Future in-vivo studies will allow us to determine the effectiveness and safety of the proposed approach.

Gene expression profiling in autoimmune noninfectious uveitis disease.

Noninfectious uveitis is a predominantly T cell-mediated autoimmune, intraocular inflammatory disease. To characterize the gene expression profile from patients with noninfectious uveitis, PBMCs were isolated from 50 patients with clinically characterized noninfectious uveitis syndrome. A pathway-specific cDNA microarray was used for gene expression profiling and real-time PCR array for further confirmation. Sixty-seven inflammation- and autoimmune-associated genes were found differentially expressed in uveitis patients, with 28 of those genes being validated by real-time PCR. Several genes previously unknown for autoimmune uveitis, including IL-22, IL-19, IL-20, and IL-25/IL-17E, were found to be highly expressed among uveitis patients compared with the normal subjects with IL-22 expression highly variable among the patients. Furthermore, we show that IL-22 can affect primary human retinal pigment epithelial cells by decreasing total tissue resistance and inducing apoptosis possibly by decreasing phospho-Bad level. In addition, the microarray data identified a possible uveitis-associated gene expression pattern, showed distinct gene expression profiles in patients during periods of clinical activity and quiescence, and demonstrated similar expression patterns in related patients with similar clinical phenotypes. Our data provide the first evidence that a subset of IL-10 family genes are implicated in noninfectious uveitis and that IL-22 can affect human retinal pigment epithelial cells. The results may facilitate further understanding of the molecular mechanisms of autoimmune uveitis and other autoimmune originated inflammatory diseases.

Eradication of tumor colonization and invasion by a B cell-specific immunotoxin in a murine model for human primary intraocular lymphoma.

Human primary intraocular lymphoma (PIOL) is predominantly a B cell-originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell-specific immunotoxin therapy may have clinical implications in treating human PIOL.

HTLV-1 infection of human retinal pigment epithelial cells and inhibition of viral infection by an antibody to ICAM-1.

PURPOSE: To examine whether human T-cell leukemia virus type 1 (HTLV-1) could infect a human retinal pigment epithelial (RPE) cell line, ARPE-19, in vitro and to investigate its regulation. METHODS: A coculture system with ARPE-19 and irradiated cells of an HTLV-1-producing T-cell line, MT2 was used to determine the permissivity of RPE to HTLV-1 infection in vitro. The susceptibility to HTLV-1 was assessed by detection of viral DNA using the polymerase chain reaction (PCR), viral mRNA transcripts with reverse transcription PCR (RT-PCR) and viral antigen by immunofluorescence staining. An HTLV-1 Tax-activated HTLV-LTR-luciferase reporter assay was developed to measure viral infection quantitatively. The ICAM-1 expression on cocultured ARPE-19 cells was detected by flow cytometry and an ICAM-1-neutralizing antibody was used to test ICAM-1's role in the HTLV-1 infection of ARPE-19 cells. The regulation of HTLV-1 infection was investigated by culturing ARPE-19 cells with proinflammatory cytokines. RESULTS: HTLV-1 infected ARPE-19 cells in vitro. The infection correlated with elevated expression of intercellular adhesion molecule (ICAM)-1 on the surface of ARPE-19 cells. ICAM-1-neutralizing antibody dramatically inhibited viral infection. Furthermore, proinflammatory cytokines dramatically suppressed HTLV-1 viral infection. CONCLUSIONS: The tropism of HTLV-1 to retinal pigment epithelium could provide an explanation for the pathogenesis of HTLV-1-related ophthalmic diseases. A better understanding of specific roles of proinflammatory cytokines in the development of ophthalmic diseases may be beneficial for treatment.

Murine model of primary intraocular lymphoma.

PURPOSE: Primary intraocular lymphoma (PIOL) is a subtype of central nervous system lymphoma. Although this lymphoma is rare, its incidence has tripled in the past 15 years. Currently, the only available model is a murine metastatic malignant lymphoma that occurs after intraperitoneal inoculation of Rev-2-T-6 lymphoma cells into newborn syngeneic mice. The current study was conducted to develop a new experimental model for PIOL. METHODS: Rev-2-T-6 cells (0.5 x 10(5) or 1.0 x 10(5)) were inoculated into the vitreous of adult BALB/c mice. Mice were monitored clinically every other day and under fundoscopic examination weekly. They were euthanatized on weeks 3, 5, 6, 7, or 8, after inoculation. All eyes were processed for histology. Immunohistochemistry was performed with an antibody (p14) specific for Rev-2-T-6 cells. Cytokine mRNA expression (IL-2, -4, -6, -10, and IFN-gamma and CC chemokine receptor-1 [CCR1]) was assayed in the lymphoma cells by microdissection and RT-PCR. IL-10 and -6 levels in the vitreous were measured by ELISA. RESULTS: Within 2 to 4 weeks, tumor cells from the vitreous migrate through the retina and gather between the RPE cell and retina. Rarely (>2 months after inoculation), Rev-2-T-6 cells may break through the RPE and infiltrate the choroid and sclera. Tumor localization was confirmed by immunohistochemistry. The intraocular lymphoma cells produce high levels of IL-10, IFN-gamma, and CCR1 transcripts. A high level of IL-10 was detected in the vitreous inoculated with Rev-2-T-6 cells. CONCLUSIONS: The data suggest that RPE cells constitute a barrier to the spread of intraocular lymphoma. Intravitreal injection of Rev-2-T-6 cells is a novel model of PIOL in immune-competent hosts that will aid in understanding the molecular mechanisms of the disease.

Survival of donor epithelial cells after limbal stem cell transplantation.

PURPOSE: To determine the long-term fate of donor epithelial cells after limbal allograft transplantation. METHODS: Corneal buttons and peripheral blood leukocytes were obtained at the time of penetrating keratoplasty from three patients who had undergone a successful limbal allograft transplantation. Microdissection was used to remove the corneal epithelial cells from the button. The presence of donor and recipient epithelial cells in each sample was determined by using PCR for DNA microsatellites. Phenotypic analysis of the epithelium was performed by immunohistochemistry. RESULTS: Various patterns of DNA microsatellites were observed. Nonrecipient cells (presumed to be donor) were consistently detected in all three corneal buttons. In two of the three cases, recipient cells were also detected, whereas in the third case, exclusively donor epithelial cells were found at 3.5 years after limbal allograft transplantation. Mild T-lymphocytes and macrophages were observed in one of the corneal buttons. CONCLUSIONS: This study provides evidence for the persistence of donor epithelial cells up to 3.5 years after limbal allograft transplantation and supports the use of systemic immunosuppressive therapy.

Constitutive and cytokine-induced GITR ligand expression on human retinal pigment epithelium and photoreceptors.

PURPOSE: The glucocorticoid-induced TNF-related receptor (GITR) plays a pivotal role in regulating the suppressive function of CD4+CD25+ regulatory T cells. GITR is also involved in the inhibition of T-cell receptor-induced apoptosis and the upregulation of inducible nitric oxide synthase (iNOS). GITR expression on CD4+ T cells has been shown to correlate with the disease course of noninfectious uveitis. The current study was conducted to examine the expression of glucocorticoid-induced TNF-related receptor ligand (GITRL) and its regulation by ocular tissue. METHODS: Immunohistochemistry and confocal immunofluorescence microscopy were performed on human ocular tissue to examine the in vivo protein expression of GITRL. The regulation of GITRL was investigated by culturing retinal pigment epithelium (RPE) with proinflammatory cytokines and performing immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The in vivo mRNA expression of GITRL was studied by RT-PCR on RNA from microdissected tissue sections. RESULTS: Both immunohistochemistry and confocal immunofluorescence microscopy demonstrated that GITRL was expressed constitutively on RPE and photoreceptor inner segments. Immunocytochemistry demonstrated that in vitro stimulated RPE cells expressed GITRL at the protein level, and RT-PCR showed that GITRL was upregulated at 24 hours by proinflammatory cytokines. Constitutive GITRL mRNA expression in vivo was confirmed by RT-PCR analysis of microdissected tissue. CONCLUSIONS: GITRL is expressed constitutively on RPE and in high levels on photoreceptor inner segments. The upregulation of GITRL by proinflammatory cytokines suggests that GITRL may play an important role in ocular immunity. The high level of constitutive GITRL expression on photoreceptor inner segments suggests that photoreceptors participate in the regulation of ocular inflammation.

Utility of Heidelberg retinal tomography as a screening tool for analyzing retinal nerve fiber layer defects.

CONTEXT: Although Heidelberg retinal tomography (HRT)-generated topographic images have been studied extensively for the detection of retinal nerve fiber layer (RNFL) defects, little is known about the role of HRT-generated surface reflectivity images in the detection of RNFL defects in either patients with glaucoma or glaucoma suspects. AIMS: To evaluate the effectiveness of HRT version II (HRT II) optic nerve reflectivity images in uncovering RNFL defects in an outpatient population evaluated for glaucoma. STUDY DESIGN/MATERIALS AND METHODS: In 102 consecutive eyes from 60 patients evaluated for glaucoma in an academic-based practice, HRT II optic nerve images were prospectively imaged and compared with clinical optic nerve exam techniques to see if HRT II was able to detect RNFL defects overlooked in clinical practice. RESULTS: Nine eyes (8.8%) were found to have RNFL defects recognized by screening with HRT II. Of these nine eyes, eight (88.9%) were recognized to demonstrate RNFL defects by conventional examination techniques. One additional eye had an RNFL defect seen on physical exam that was not detected by HRT. CONCLUSION: In academic practice, HRT II may be helpful in complementing conventional exam techniques in the recognition and documentation of acquired RNFL loss.

Ultrasound-enhanced delivery of antibiotics and anti-inflammatory drugs into the eye.

Delivery of sufficient amounts of therapeutic drugs into the eye is often a challenging task. In this study, ultrasound application (frequencies of 400 KHz to 1 MHz, intensities of 0.3-1.0 W/cm(2) and exposure duration of 5 min) was investigated to overcome the barrier properties of cornea, which is a typical route for topical administration of ophthalmic drugs. Permeability of ophthalmic drugs, tobramycin and dexamethasone and sodium fluorescein, a drug-mimicking compound, was studied in ultrasound- and sham-treated rabbit corneas in vitro using a standard diffusion cell setup. Light microscopy observations were used to determine ultrasound-induced structural changes in the cornea. For tobramycin, an increase in permeability for ultrasound- and sham-treated corneas was not statistically significant. Increase of 46%-126% and 32%-109% in corneal permeability was observed for sodium fluorescein and dexamethasone, respectively, with statistical significance (p < 0.05) achieved at all treatment parameter combinations (compared with sham treatments) except for 1-MHz ultrasound applications for dexamethasone experiments. This permeability increase was highest at 400 kHz and appeared to be higher at higher intensities applied. Histologic analysis showed structural changes that were limited to epithelial layers of cornea. In summary, ultrasound application provided enhancement of drug delivery, increasing the permeability of the cornea for the anti-inflammatory ocular drug dexamethasone. Future investigations are needed to determine the effectiveness and safety of this application in in vivo long-term survival studies.

Differentiation of malignant B-lymphoma cells from normal and activated T-cell populations by their intrinsic autofluorescence.

Patients with active posterior and intermediate uveitis have inflammatory cells in their vitreous; those with primary intraocular lymphoma have malignant B-lymphoma cells concomitantly. These cell types cannot be distinguished clinically. The goal of this study was to investigate intrinsic autofluorescence as a noninvasive way of differentiating immune and lymphomatous cell populations. Human primary T cells were stimulated with or without anti-CD3 plus anti-CD28 stimulation. B-lymphoma cells (CA46) were cultured separately. Five experimental groups were prepared: unstimulated T cells, stimulated T cells, CA46 cells, and stimulated T cells mixed with CA46 cells at a ratio of 1:3 or mixed at a ratio of 3:1. Samples were excited with three wavelengths and imaged with a confocal microscope. For each condition, the autofluorescent emissions from the sample were measured. In separate experiments, T cells or CA46 cells were injected into the anterior chamber of a BALB/c mouse eye and autofluorescence was measured. Pure T-cell and lymphoma populations were clearly distinguishable based on autofluorescence intensity spectra. CA46 cells were the least fluorescent when excited with 351-nm light, but most fluorescent when excited with longer wavelengths like 488 nm. Mixed populations of T cells and CA46 cells had emission intensities that fell predictably in between those of the pure populations. An ex vivo study showed that CA46 cells could be detected based on their intrinsic autofluorescence. Our studies showed that normal activated and malignant lymphocyte populations can be distinguished based on their intrinsic autofluorescent properties. Future work with in vivo models may prove useful in facilitating the diagnosis of uveitis and other ocular diseases.

Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis.

Glucocorticoid-induced tumor necrosis factor receptor (GITR), found constitutively expressed on human primary natural killer (NK) cells at low levels was up-regulated upon stimulation by either Toll-like receptor ligand or NK cell growth factor, interleukin (IL)-15. cDNA microarray analysis showed that engagement of GITR primarily suppressed the activation of NF-KB pathway of NK cells and up-regulated anti-inflammatory genes heme oxygenase-1 and IL-10. Further analysis revealed that GITR activation suppressed NK cell proliferation in response to IL-15. GITR activation also suppressed proinflammatory cytokine secretion and increased NK cell apoptosis. GITR activation resulted in blocked phosphorylation of Stat5 and Akt, which may have contributed to the observed antiproliferative effect of GITR on NK cells. Increased apoptosis was independent of the Fas-FasL pathway, but Bcl-XL and phospho-Bad protein expressions were diminished, suggesting involvement of the mitochondrial apoptosis pathway. The results suggest that although GITR is an activation marker for NK cells similar to that for T cells, GITR serves as a negative regulator for NK cell activation. Our studies demonstrate a novel physiological role of GITR on NK cells.

Expression of GITR ligand abrogates immunosuppressive function of ocular tissue and differentially modulates inflammatory cytokines and chemokines.

The glucocorticoid-induced TNF-related receptor ligand (GITRL) was previously shown to be constitutively expressed at low levels in human eye, including retinal pigment epithelial (RPE) cells. By expressing enhanced yellow fluorescent protein-tagged human GITRL in human RPE cells, we investigated the significance of expression of GITRL on human ocular tissue. Confocal immunofluorescence microscopy and flow cytometry confirmed the surface expression of GITRL on RPE cells. However, a soluble form of GITRL was also detected. Remarkably, expression of GITRL on the RPE cells abrogated RPE-mediated immunosuppression of CD3(+) T cells, implicated as a possible mechanism for ocular immune privilege. This abrogation of immunosuppression by GITRL-RPE was dependent on GITR-GITRL interaction and could not be mimicked by anti-CD28 antibody. Analysis of cytokine profiles revealed high level of TGF-beta during the immunosuppression by RPE cells while expression of GITRL abrogated the RPE cell-induced TGF-beta secretion. Expression of GITRL also stimulates secretion of an array of proinflammatory cytokines/chemokines from T cells. GITR-GITRL interaction provides a unique proinflammatory costimulation that may signal through a different pathway than that of CD28-B7 costimulation. This study implicated that GITRL could be a potential candidate for regulation of the ocular immune privilege and the balance between immune privilege and inflammation.

Cutting edge: in vivo blockade of human IL-2 receptor induces expansion of CD56(bright) regulatory NK cells in patients with active uveitis.

In vivo blockade of the human IL-2R by mAb has been used for immunosuppression in transplantation, therapy for leukemia, and autoimmune diseases. In this study, we report that administration of a humanized IL-2R blocking Ab induced a 4- to 20-fold expansion of CD56(bright) regulatory NK cells in uveitis patients over time. The induced CD56(bright) regulatory NK cells from patients exhibited similar phenotype as those naturally occurring CD56(bright) cells. Patients with active uveitis had a significantly lower level of CD56(bright) NK cells compared with normal donors (p < 0.01). In addition, the induced CD56(bright) cells could secrete large amounts of IL-10 whereas CD56(dim) NK cells could not, suggesting that the induction of the CD56(bright) cells may have a beneficial effect on the remission of active uveitis. Our observation may have implications to IL-2R blockade therapy and for the potential role of CD56(bright) regulatory NK cells in autoimmune diseases.

Expression of glucocorticoid induced TNF receptor family related protein (GITR) on peripheral T cells from normal human donors and patients with non-infectious uveitis.

The expression of the glucocorticoid induced TNF receptor family related gene (GITR) in subsets of T lymphocytes from human peripheral blood was studied. In normal human peripheral blood mononuclear cells, GITR expression on the resting CD4+ T cells was low but markedly increased after activation. The percentage of GITR+ T cells in the CD4+CD25+ T cell subpopulation (15.1%) was significantly higher than that in the CD4+CD25- T cell subpopulation (5.2%, P<0.01), suggesting a preferential co-expression of GITR with CD25. In a group of patients with non-infectious uveitis, a proposed T helper cell mediated autoimmune ocular disease, the GITR expression on the CD4+ T cells in both the active patients (34.5%) and the inactive patients (19.6%) was significantly higher as compared to that in the normal donors (10.7%; P<0.01 vs. active, P<0.05 vs. inactive). This increased GITR expression in T cells was only seen in the CD4 positive T helper cell subpopulation but not in the CD4 negative T cell subpopulation. GITR expression on the CD4+ T cells decreased when the patients became clinically quiescent. Therefore, GITR is an activation marker for the CD4+ T cells and preferentially co-expressed with CD25 on the CD4+ T cells in human peripheral blood. Its expression correlates with the clinical course of non-infectious uveitis.