An Infrared Dye-Conjugated Virus-like Particle for the Treatment of Primary Uveal Melanoma.

The work outlined herein describes AU-011, a novel recombinant papillomavirus-like particle (VLP) drug conjugate and its initial evaluation as a potential treatment for primary uveal melanoma. The VLP is conjugated with a phthalocyanine photosensitizer, IRDye 700DX, that exerts its cytotoxic effect through photoactivation with a near-infrared laser. We assessed the anticancer properties of AU-011 in vitro utilizing a panel of human cancer cell lines and in vivo using murine subcutaneous and rabbit orthotopic xenograft models of uveal melanoma. The specificity of VLP binding (tumor targeting), mediated through cell surface heparan sulfate proteoglycans (HSPG), was assessed using HSPG-deficient cells and by inclusion of heparin in in vitro studies. Our results provide evidence of potent and selective anticancer activity, both in vitro and in vivo AU-011 activity was blocked by inhibiting its association with HSPG using heparin and using cells lacking surface HSPG, indicating that the tumor tropism of the VLP was not affected by dye conjugation and cell association is critical for AU-011-mediated cytotoxicity. Using the uveal melanoma xenograft models, we observed tumor uptake following intravenous (murine) and intravitreal (rabbit) administration and, after photoactivation, potent dose-dependent tumor responses. Furthermore, in the rabbit orthotopic model, which closely models uveal melanoma as it presents in the clinic, tumor treatment spared the retina and adjacent ocular structures. Our results support further clinical development of this novel therapeutic modality that might transform visual outcomes and provide a targeted therapy for the early-stage treatment of patients with this rare and life-threatening disease. Mol Cancer Ther; 17(2); 565-74. ©2017 AACR.

In vivo high-frequency contrast-enhanced ultrasonography of choroidal melanoma in rabbits: imaging features and histopathologic correlations.

PURPOSE: To evaluate the use of in vivo imaging of rabbit model of choroidal melanoma using high-frequency contrast-enhanced ultrasound (HF-CE-US) with two-dimensional (2D) or three-dimensional (3D) modes and to correlate the sonographic findings with histopathologic characteristics. METHODS: Five New Zealand white rabbits, which were immunosuppressed with daily cyclosporin A (CsA), were inoculated into their right eyes with aliquots of 1.5×10(6)/50 µl of 92.1 human uveal melanoma cells cultured in RPMI. At week 4, the tumour-bearing eyes were imaged using high-frequency ultrasound (HF-US) with microbubble contrast agent to determine the 2D tumour size and relative blood volume and by 3D mode to determine tumour volume. Histologic tumour burden was quantified in enucleated eyes by ImageJ software, and mean vascular density (MVD) was determined by counting vascular channels in periodic acid Schiff (PAS) without haematoxylin sections. RESULTS: Using HF-CE-US, melanomas were visualised as relatively hyperechoic regions in the images. The correlation coefficients of sonographic size and volume compared with histologic area were 0.72 and 0.70, respectively. The sonographic tumour relative blood volume correlated with the histologic tumour vascularity (r(2)=0.92, p=0.04). CONCLUSIONS: There is a positive correlation between in vivo sonographic tumour volume/size and histologic tumour size in our rabbit choroidal melanoma model. HF-CE-US corresponds to MVD and blood volume.

In vivo high-frequency, contrast-enhanced ultrasonography of uveal melanoma in mice: imaging features and histopathologic correlations.

PURPOSE: To evaluate the usefulness of in vivo imaging of uveal melanoma in mice using high-frequency contrast-enhanced ultrasound (HF-CE-US) with 2D or 3D modes and to correlate the sonographic findings with histopathologic characteristics. METHODS: Fourteen 12-week-old C57BL6 mice were inoculated into their right eyes with aliquots of 5 × 10(5)/2.5 µL B16LS9 melanoma cells and were randomly assigned to either of two groups. At 7 days after inoculation, tumor-bearing eyes in group 1 (n = 8) were imaged using HF-CE-US to determine the 2D tumor size and relative blood volume; eyes in group 2 (n = 6) were imaged by 3D microbubble contrast-enhanced ultrasound, and the tumor volume was determined. Histologic tumor burden was quantified in enucleated eyes by image processing software, and microvascular density was determined by counting von Willebrand factor-positive vascular channels. Ultrasound images were evaluated and compared with histopathologic findings. RESULTS: Using HF-CE-US, melanomas were visualized as relatively hyperechoic regions. The intraobserver variability of sonographic measurements was 9.65% ± 7.89%, and the coefficient of variation for multiple measurements was 7.33% ± 5.71%. The correlation coefficient of sonographic volume or size and histologic area was 0.71 (P = 0.11) and 0.79 (P = 0.32). The relative blood volume within the tumor demonstrated sonographically correlated significantly with histologic tumor vascularity (r = 0.83; P < 0.001). CONCLUSIONS: There was a positive linear correlation between sonographic tumor measurements and histologic tumor burden in the mouse ocular melanoma model. Contrast-enhanced intensity corresponded with microvascular density and blood volume. HF-CE-US is a real-time, noninvasive, reliable method for in vivo evaluation of experimental intraocular melanoma tumor area and relative blood volume.

Animal models of choroidal and retinal neovascularization.

There have been numerous types of animal models of choroidal neovascularization (CNV) and retinal neovascularization (RNV). Understanding the pathobiology of CNV and RNV is important when evaluating and utilizing these models. Both CNV and RNV are dynamic processes. A break or defect in Bruchs' membrane is necessary for CNV to develop. This may be induced with a laser, mechanically via surgery, or in the setting of transgenic mice. Some of the transgenic mouse models spontaneously develop RNV and/or retinal angiomatous proliferation (RAP)-like lesions. The pathogenesis of RNV is well-known and is generally related to ischemic retinopathy. Models of oxygen-induced retinopathy (OIR) closely resemble retinopathy of prematurity (ROP). The streptozotocin (STZ) rat model develops features similar to diabetic retinopathy. This review summarizes general categories and specific examples of animal models of CNV and RNV. There are no perfect models of CNV or RNV and individual investigators are encouraged to choose the model that best suits their needs.

Subconjunctival nanoparticle carboplatin in the treatment of murine retinoblastoma.

OBJECTIVE: To evaluate the efficacy of subconjunctival nanoparticle carboplatin in the treatment of transgenic murine retinoblastoma. METHODS: Dendrimeric nanoparticles loaded with carboplatin were prepared. Forty LHbeta-Tag mice were randomly assigned into 4 groups and treated at 10 weeks of age. Each mouse received a single subconjunctival injection in one eye, and the opposite eye was left untreated as a control. Group 1 (high-dose nanoparticle carboplatin) received 37.5 mg/mL of nanoparticle carboplatin; group 2 (low-dose nanoparticle carboplatin) received 10 mg/mL of nanoparticle carboplatin; group 3 (conventional carboplatin) received 10 mg/mL of carboplatin in aqueous solution; and group 4 (phosphate-buffered saline) received phosphate-buffered saline. Mice were killed on day 22 after treatment. Eyes were serially sectioned, and retinal tumor burden was quantified by histopathologic analysis. RESULTS: Mean tumor burden in the treated eyes was significantly smaller compared with the untreated eyes in the same mice in both nanoparticle carboplatin groups (group 1, P = .02; group 2, P = .02) and the treated eyes in the conventional carboplatin group (group 1 vs group 3, P < .01; group 2 vs group 3, P = .01) and phosphate-buffered saline group (group 1 vs group 4, P < .01; group 2 vs group 4, P = .01). The untreated eyes in the high-dose nanoparticle carboplatin group showed significantly smaller tumor mass compared with the conventional carboplatin (P = .03) and PBS (P = .04) groups. No toxic effects were observed in any of the groups. CONCLUSION: A single injection of subconjunctival nanoparticle carboplatin was effective in the treatment of transgenic murine retinoblastoma, with no associated toxic effects. The higher dose of subconjunctival nanoparticle carboplatin decreased the tumor burden in the contralateral eye. CLINICAL RELEVANCE: This model provides a basis to test carboplatin nanoparticles for the treatment of human retinoblastoma.

Modulation of choroidal neovascularization by subretinal injection of retinal pigment epithelium and polystyrene microbeads.

PURPOSE: The study was conducted to create a rapidly developing and reproducible animal model of subretinal choroidal neovascularization (CNV) that allows a time-dependent evaluation of growth dynamics, histopathologic features, and cytokine expression. METHODS: C57BL/6 and chemoattractant leukocyte protein-2 deficient (DeltaCcl-2) mice were studied. Mice received single or combined subretinal injections of cultured retinal pigment epithelium (RPE; C57BL/6-derived), polystyrene microbeads, or phosphate buffer solution (PBS). Fluorescence angiograms were performed over a period of 3 weeks. Mice were euthanized on post inoculation day 3, 7, 10, 14, or 21, and their eyes were evaluated by light, confocal, and electron microscopy. RESULTS: CNV membranes occurred in all study groups with an overall incidence of 94.3%. They extended in the subretinal space through central breaks in Bruch's membrane. CNV lesions were characterized by dynamic changes such as initiation, active inflammatory, and involution stages. CNV thickness peaked around PI day 7 and was greater in mice that received combined injections of RPE and microbeads or RPE cells alone. Small lesions developed in the control groups (microbeads or PBS only), in DeltaCcl-2, and old C57BL/6 mice. Variable expression of cytokines and growth factors was detected within the membranes. CONCLUSIONS: Our murine model represents a reliable approach inducing CNV growth by subretinal injection of either RPE cells alone or RPE cells and microbeads. The development of CNV lesions is a dynamic process that relies in part on macrophage trafficking and age.

Histologic evaluation of human posterior lamellar discs for femtosecond laser Descemet's stripping endothelial keratoplasty.

PURPOSE: To evaluate the histologic changes in corneal structure after femtosecond laser preparation of posterior lamellar discs, more specifically, the smoothness of the stromal bed and the accuracy of the predicted depth of the horizontal lamellar cut. MATERIALS AND METHODS: Nineteen human donor eyes unsuitable for transplantation were used. Femtosecond laser was used to prepare a horizontal lamellar cut in donor corneas at a depth of 400 microm. Transmission electron microscopy images were used to evaluate the changes in the corneal structure and to measure the damage zone. Scanning electron microscopy images were used to determine the relative depth of the horizontal lamellar cut, and the stromal bed was examined to determine the smoothness of the surface. RESULTS: Transmission electron microscopy images showed a mean damage zone of 6.8 +/- 3.1 microm, which consisted of irregularly oriented collagen fibrils and electron-dense granular material. The collagen lamellae, both anteriorly and posteriorly of the damaged zone, showed a regular parallel configuration. The relative depth of the horizontal lamellar cut as percentage of the total corneal thickness in the center and periphery was 70.4% +/- 4.5% and 55.6% +/- 5.9%. Scanning electron microscopy images of the stromal bed showed a relatively smooth surface. CONCLUSION: The femtosecond laser is effective to prepare a deep horizontal lamellar cut in a standardized method. The stromal bed is smooth and without extensive adjacent tissue damage. The is thinner in the center and thicker at the edges, which may produce a mild hyperopic shift after femtosecond laser-assisted Descemet's stripping endothelial keratoplasty.

Proliferating trichilemmal cyst of the eyelid.

PURPOSE: To report a case of proliferating trichilemmal cyst in a 55-year-old woman. DESIGN: Observational case report. METHODS: A 55-year-old woman sought treatment for a mass in the right upper eyelid. The lesion was excised twice previously, and recurred. The mass was reexcised and examined histopathologically. RESULTS: Light microscopy showed a cystic lesion lined by stratified squamous epithelium with a compact layer of eosinophilic keratin without granular cell layer. There was no atypia, mitosis, or stromal invasion. CONCLUSIONS: Although proliferating trichilemmal cyst shows benign histopathologic features, clinical manifestations may mimic those of more aggressive tumors with local recurrences or distant metastasis. Wide excision of the lesion and close long-term follow-up is recommended.

Composite lymphoma of the orbit treated with rituximab.

We describe an unusual case of composite lymphoma (CL) in the orbit. The clinical history and biopsy specimen of an 82-year-old woman with a right orbital mass were evaluated. The orbital biopsy contained a dense lymphocytic infiltration and nodules of large lymphocyte that immunostained positive for CD20. Flow cytometric immunophenotyping showed two distinct populations of cells, confirming the diagnosis of a simultaneous follicular lymphoma (FL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The patient was treated with CD20 antibody (rituximab) and is in remission at 3 months follow-up.

Subconjunctival herniated orbital fat: A benign adipocytic lesion that may mimic pleomorphic lipoma and atypical lipomatous tumor.

Prolapse of subconjunctival intraconal orbital fat is a rare cause of an intraorbital mass lesion. Over the past several years, we have seen a number of cases in which this prolapsed fat was confused pathologically with a neoplasm of adipocytic lineage, specifically pleomorphic lipoma and atypical lipomatous neoplasm (well-differentiated liposarcoma). We report the clinical, histopathologic, and immunohistochemical findings in 21 specimens from 17 patients, all of whom presented with prolapsed intraconal orbital fat. All specimens were routinely examined and processed for light microscopy. Immunohistochemistry for CD34, CD68, S100 protein, vimentin, alpha-smooth muscle actin, and Ki-67, and Giemsa, Masson trichrome, and alcian blue histochemical stains were performed. Clinical and follow-up information was extracted from a chart review. The mean age (+/-SD) of the patients was 65.6+/-11.9 years (range: 41 to 85 y); 2 were women and 15 were men. Subconjunctival prolapsed orbital fat was localized in the superotemporal quadrant or lateral canthus around the rectus muscle below the lacrimal gland. The lesions were unilateral in 10 and bilateral in 7 patients. No recurrence was clinically evident over a mean (+/-SD) follow-up time of 2.5+/-3.2 years (range: 1 mo to 13.5 y). Histopathologically, all specimens showed an admixture of mature fat, fibrous septae lacking hyperchromatic cells, adipocytes with intranuclear vacuoles (Lochkern cells), multinucleated giant cells with a wreathlike configuration of normochromatic nuclei (floret cells), and varying numbers of histiocytes, lymphocytes, plasma cells, and mast cells. "Control" sections of normal orbital fat showed occasional Lochkern cells but lacked floret cells. By immunohistochemistry, the floret cells expressed only CD34 and vimentin, whereas the Lochkern cells expressed CD34, S100 protein, and vimentin. We conclude that subconjunctival herniated orbital fat commonly contains multinucleated floretlike giant cells, fibrous septae, and Lochkern cells, features that may result in diagnostic confusion with pleomorphic lipoma and atypical lipomatous neoplasms. Importantly, specific diagnostic features, such as aggregates of bland spindled cells associated with wiry collagen, as seen in pleomorphic lipoma, and enlarged hyperchromatic cells within fibrous septae, as in atypical lipomatous neoplasms, are entirely absent in herniated orbital fat. Multinucleated floret cells present in prolapsed orbital fat likely represent a reactive phenomenon, as they are not present in normal orbital fat.

Excimer laser exacerbation of Avellino corneal dystrophy.

We review the clinical, histopathological, and ultrastructural findings and DNA phenotyping of a patient with Avellino corneal dystrophy exacerbated by laser in situ keratomileusis. The findings are reported and interpreted in the context of a literature review. The case highlights the possible difficulty of recognizing subtle dystrophic findings, as well as the importance of avoiding refractive surgical intervention in patients with Avellino corneal dystrophy to avoid exacerbation of dystrophic deposits in the cornea and subsequent reduction in vision.

Interface fluid syndrome in laser in situ keratomileusis after complicated trabeculectomy.

A 69-year-old man developed stromal edema and a pocket of fluid in the laser in situ keratomileusis (LASIK) interface wound in the left eye after acute endothelial cell loss from complicated trabeculectomy. He eventually required penetrating keratoplasty along with cataract surgery. Histologic examination of the corneal button showed an edematous 720 microm central residual stromal bed, a 54 microm empty space at the level of the central interface wound, and a 154 microm LASIK flap. The endothelial cell count was 0 to 2 cells per high-power field, corresponding to a cell density of 450 to 500 cells/mm(2). Four years after LASIK, the central interface wound was susceptible to forming a pocket of serous fluid after the corneal endothelial function was compromised.

Ischemic necrosis and atrophy of the optic nerve after periocular carboplatin injection for intraocular retinoblastoma.

PURPOSE: To report four cases of optic nerve neuropathy in three children treated with periocular carboplatin injections for unilateral or bilateral intraocular retinoblastoma. DESIGN: Retrospective, observational case series. SETTING: University-based Ophthalmology Practice. STUDY POPULATION: Four eyes of three children with retinoblastoma enucleated after nonsuccessful multimodality treatment including periocular carboplatin injections. OBSERVATION PROCEDURES: The enucleated eyes were routinely processed and evaluated by light microscopy. A retrospective chart review of all four cases was performed. RESULTS: Three enucleated eyes (Reese-Ellsworth groups III and VB) were obtained from two children with bilateral multifocal retinoblastoma, and one eye (Reese-Ellsworth group IIB) was harvested from a child with unilateral retinoblastoma. All affected eyes underwent three to seven periocular carboplatin injections before enucleation. Additional treatment modalities included systemic chemotherapy, transpupillary thermotherapy, transscleral cryotherapy, and external beam radiotherapy. Histopathologic evaluation of the enucleated eyes revealed focal areas of ischemic necrosis or atrophy of the optic nerve along with dystrophic calcification and mild inflammation in the surrounding fibrovascular adipose tissue. CONCLUSIONS: Periocular injections of carboplatin may be a useful treatment approach in the management of patients with advanced intraocular retinoblastoma and may minimize systemic side-effects. However, ophthalmologists and pediatric oncologists should be aware of potential marked local complications with periocular carboplatin delivery, including ischemic optic neuropathy. Modifying the injection site/location (for example, subtenon space) or adding other delivery routes adjuncts (for example, fibrin sealant) deserves further study.

Conjunctival granulomas caused by synthetic fibers: report of two cases and review of literature.

PURPOSE: We sought to demonstrate the histopathologic and ultrastructural features of conjunctival foreign body granulomas because of synthetic fibers and to compare them to other cases published in the literature. METHODS: A 2- and a 7-year-old girl were referred for the surgical removal of slow-growing unilateral inferior conjunctival masses with a lack of primary trauma or surgery. In this report, we describe the light and electron microscopic findings of the 2 cases and review the literature of similar cases using the Medline database. RESULTS: Histopathologic and ultrastructural examination of both specimens revealed a granulomatous inflammatory cell response, including histiocytes and multinucleated foreign body giant cells around acellular, uniform sized, oval to round birefringent fibers with manufacturing artifacts. Thirteen other patients with conjunctival synthetic fiber granulomas were identified from the literature. CONCLUSIONS: On the basis of the findings in our cases and the review of literature, it appears that conjunctival synthetic fiber granulomas are not a rare entity but are not recognized frequently by ophthalmologists. The most reliable clinical sign to suggest this diagnosis is the presence of a unilateral inferior conjunctival mass in a child or adolescent. Histopathologic and ultrastructural evaluation appears to be the only way to specifically diagnose this condition with certainty.

Photodynamic therapy using verteporfin-induced minimal change nephrotic syndrome.

PURPOSE: To report a case of minimal change nephrotic syndrome (MCNS) after photodynamic therapy using verteporfin. DESIGN: Interventional case report. METHODS: After four cycles of photodynamic therapy, general weakness with generalized edema developed in an otherwise healthy 66-year-old woman, resulting in dyspnea and ascites. Urinalysis showed heavy proteinuria (4+) with decreased serum total protein and albumin, and increased total cholesterol levels, suggesting nephrotic syndrome. Renal biopsy and pathologic diagnosis were performed. RESULTS: Renal biopsy revealed normal glomeruli and tubulointerstitium by light microscopy, with no immunoglobin or complement deposition. Transmission electron microscopy showed diffuse effacement of the foot processes of visceral epithelial cells, which is the characteristic finding of minimal change nephrotic syndrome. CONCLUSIONS: We herein report a case of minimal change nephrotic syndrome after photodynamic therapy using verteporfin.

The effect of photodynamic therapy with rose bengal on posterior capsule opacification in rabbit eyes.

We investigated the in vivo effect of photodynamic therapy (PDT) using rose bengal on the development of posterior capsule opacification (PCO). Endocapsular phacoemulsification was performed on white rabbits, which were divided into 4 groups: control group; group 1, treated with visible light only; group 2, treated with rose bengal only, and group 3, treated with PDT. In the case of the PDT group, rose bengal dissolved in sodium hyaluronate was injected into the empty capsular bag and treated with visible light. Three months after surgery, the rabbits were sacrificed and the eyeballs enucleated. The obstruction rate of visible light caused by PCO was measured with an optical powermeter. The mean obstruction rate was 30.6% in the control group, 28.3% in group 1, 19.3% in group 2, and 14.3% in group 3. Group 3 showed a statistically significant decrease in PCO compared with the control group and group 1 (p = 0.0014). Our results suggest that PDT using rose bengal effectively decreased PCO in rabbit eyes.

Opacification of a hydrophilic acrylic intraocular lens with exacerbation of Behçet's uveitis.

Behçet's disease is 1 of the most common causes of uveitis in the Eastern world. Its common ocular complications are uveitis, cataract, and obliteration of retinal vessels. Phacoemulsification with intraocular lens (IOL) implantation in patients with Behçet's disease is known to be a safe procedure. We managed a patient with Behçet's disease who had aggravated uveitis and opacification of a hydrophilic acrylic IOL (ACRL-C160, Ophthalmed) 4 months after cataract surgery. Recalcitrant uveitis despite maximum tolerable medication and IOL opacification with vitreous opacity necessitated an IOL exchange and trans pars plana vitrectomy. After the procedure, the eye became quiescent. However, the visual acuity was 20/200 because of the obliteration of retinal vessels.

Viscoelastic protection from endothelial damage by air bubbles.

PURPOSE: To determine whether viscoelastic materials effectively protect the corneal endothelium from air bubbles. SETTING: Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. METHODS: Human eye-bank and rabbit eyes had a standardized phacoemulsification procedure with or without viscoelastic material (Healon [sodium hyaluronate 1.0%], Healon GV [sodium hyaluronate 1.4%], or Viscoat [chondroitin sulfate 4.0%-sodium hyaluronate 3.0%]). The integrity of the endothelium was examined after the procedure with F-actin staining and scanning electron microscopy. Rabbit eyes with and without viscoelastic material (Healon or Viscoat) had a standardized irrigation/aspiration (I/A) procedure. The mucinous layer of the endothelium was examined after the procedure with transmission electron microscopy. RESULTS: In the phacoemulsification experiment without viscoelastic material, with Healon, and with Healon GV, the endothelium of human and rabbit corneas had many areas of cell loss in a pattern consistent with air-bubble damage. With Viscoat, endothelial cells remained intact. In the I/A experiment, the mucinous layer of Viscoat-exposed rabbit endothelium appeared thinner. In the same experiments without viscoelastic material or with Healon, the mucinous layer of the endothelium appeared normal. CONCLUSIONS: Viscoat effectively protected the endothelium from air-bubble damage. Viscoat appears to protect the endothelium by acting as a physical barrier. Its adherence is probably related to the way it interacts with the mucinous layer of the endothelium.

Endothelial protection: avoiding air bubble formation at the phacoemulsification tip.

PURPOSE: To investigate the conditions under which bubbles form during phacoemulsification. SETTING: Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. METHODS: In the first part of the study, the partial pressure of oxygen (pO(2)) was used as a surrogate measure for the partial pressure of air. Irrigation solutions packaged in glass and plastic containers were studied. A directly vented glass bottle was also tested. The pO(2) of the various irrigation solutions was measured as the containers were emptied. In the second part, phacoemulsification procedures were performed in rabbit eyes with different power settings and different irrigation solutions. Intracameral bubble formation during the procedure was recorded. Following the phacoemulsification procedures, the corneas were stained for F-actin and examined for endothelial injury. RESULTS: The initial pO(2) in irrigation solutions packaged in glass bottles was about half that at atmospheric levels; in solutions packaged in plastic, it was at atmospheric levels. As irrigation solutions were drained from the container, the pO(2) of the solution tended to rise toward atmospheric levels. The rate of pO(2) increase was markedly reduced by using a directly vented glass bottle. In the phacoemulsification procedures, bubble formation was most likely to occur with higher pO(2) and higher power settings. Observation of bubbles by the surgeon was highly correlated with endothelial damage. CONCLUSIONS: Keeping the pO(2) low reduced the risk of endothelial damage, especially at higher phacoemulsification powers. The packaging of irrigation solutions was the most important factor in controlling the initial pO(2) of the solution. The pO(2) can be minimized throughout a phacoemulsification procedure by using a directly vented glass bottle.