Evaluation of the Effects of Fucoidans from Fucus Species and Laminaria hyperborea against Oxidative Stress and Iron-Dependent Cell Death

Fucoidans are algal polysaccharides that exhibit protective properties against oxidative stress. The aim of this study was to investigate different fucoidans from brown seaweeds for their ability to protect against iron-dependent oxidative stress (ferroptosis), a main hallmark of retinal and brain diseases, including hemorrhage. We investigated five new high-molecular weight fucoidan extracts from Fucus vesiculosus, F. serratus, and F. distichus subsp. evanescens, a previously published Laminaria hyperborean extract, and commercially available extracts from F. vesiculosus and Undaria pinnatifida. We induced oxidative stress by glutathione depletion (erastin) and H₂O₂ in four retinal and neuronal cell lines as well as primary cortical neurons. Only extracts from F. serratus, F. distichus subsp. evanescens, and Laminaria hyperborea were partially protective against erastin-induced cell death in ARPE-19 and OMM-1 cells, while none of the extracts showed beneficial effects in neuronal cells. Protective fucoidans also attenuated the decrease in protein levels of the antioxidant enzyme GPX4, a key regulator of ferroptosis. This comprehensive analysis demonstrates that the antioxidant abilities of fucoidans may be cell type-specific, besides depending on the algal species and extraction method. Future studies are needed to further characterize the health-benefiting effects of fucoidans and to determine the exact mechanism underlying their antioxidative abilities.     

Malignant melanoma of brown bullhead (Ameiurus nebulosus) in Lake Memphremagog,Vermont/Quebec

In 2012, brown bullhead (Ameiurus nebulosus) with large, raised, black growths were first reported from multiple areas within the Vermont portion of Lake Memphremagog. Subsequent surveys conducted from 2014 to 2017 at two sites within the lake indicated a prevalence of 30% in adult brown bullhead 200 mm and above total length. These lesions ranged from slightly raised smooth black areas to large nodular areas on the body surface and fins and within the oral cavity. Microscopically, these lesions were determined to be malignant melanoma with invasion into surrounding hypodermis, skeletal muscle and bone as well as metastases to gill, ovary and intestine. Liver neoplasms were also observed in 8% of the bullhead collected from Lake Memphremagog in 2015. Neither skin nor liver neoplasms were noted in Ticklenaked Pond, a site used for comparison.     

Evaluation of a Brown Seaweed Extract from Dictyosiphon foeniculaceus as a Potential Therapeutic Agent for the Treatment of Glioblastoma and Uveal Melanoma

Ingredients of brown seaweed like fucoidans are often described for their beneficial biological effects, that might be interesting for a medical application. In this study, we tested an extract from Dictyosiphon foeniculaceus (DF) to evaluate the effects in glioblastoma and uveal melanoma, looking for a possible anti-cancer treatment. We investigated toxicity, VEGF (vascular endothelial growth factor) secretion and gene expression of tumor and non-tumor cells. SVGA (human fetal astrocytes), the human RPE (retinal pigment epithelium) cell line ARPE-19, the tumor cell line OMM-1 (human uveal melanoma), and two different human primary glioblastoma cultures (116-14 and 118-14) were used. Tests for cell viability were conducted with MTS-Assay (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and the proliferation rate was determined with cell counting. VEGF secretion was assessed with ELISA (enzyme-linked immunosorbent assay). The gene expression of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2) and VEGF-A was determined with real-time qPCR (quantitative polymerase chain reaction). DF lowered the cell viability of OMM-1. Proliferation rates of ARPE-19 and OMM-1 were decreased. The VEGF secretion was inhibited in ARPE-19 and OMM-1, whereas it was increased in SVGA and 116-14. The expression of VEGFR1 was absent and not influenced in OMM-1 and ARPE-19. VEGFR2 expression was lowered in 116-14 after 24 h, whereas VEGF-A was increased in 118-14 after 72 h. The extract lowered cell viability slightly and was anti-proliferative depending on the cell type investigated. VEGF was heterogeneously affected. The results in glioblastoma were not promising, but the anti-tumor properties in OMM-1 could make them interesting for further research concerning cancer diseases in the human eye.     

Unilateral invasive intraocular melanoma with choroidal and optic nerve involvement resulting in bilateral vision loss in a Labrador Retriever

A 10-year-old male castrated Labrador Retriever cross was referred for evaluation of acute vision loss. Ophthalmic examination revealed mild left sided exophthalmos, bilateral resting mydriasis, an absent direct and reduced consensual PLR in the left eye and reduced direct and absent consensual PLR in the right eye. Examination of the cornea and anterior segment with slit lamp biomicroscopy was unremarkable. Indirect fundoscopy revealed a left optic nerve head obscured by a darkly pigmented lesion. Fundic examination in the right eye was unremarkable. Magnetic resonance imaging revealed a smoothly marginated, lobulated cone to irregularly shaped, strongly T1 hyperintense, T2 and T2 fluid-attenuated inversion recovery hypointense, strongly contrast enhancing mass closely associated with the entire left optic nerve, extending across the optic chiasm and into the right optic nerve ventrally. Full clinical staging revealed no evidence of metastasis. Exenteration of the left eye was performed. Histopathology revealed an unencapsulated, poorly demarcated, multilobulated and infiltrative pigmented mass that was effacing the posterior choroid and optic nerve. The mass was composed of a moderately pleomorphic population of heavily pigmented polygonal cells arranged in sheets and clusters, displaying moderate anisocytosis and anisokaryosis. The population of cells contained moderate amounts of abundant brown-black granular pigment consistent with melanin within the cytoplasm. Mitotic figures averaged approximately three per ten 400× fields (2.37 mm²). This is the first report of a melanocytic tumor invading along the optic nerve and tract to result in contralateral vision loss.     

Penetrating sclerokeratoplasty and autologous pinnal cartilage and conjunctival grafting to treat a large limbal melanoma in a dog

A four‐year‐old neutered male Labrador retriever presented to Portland Veterinary Specialists Ophthalmology Service for evaluation of a pigmented mass oculus sinister (OS) of approximately 4‐month duration. Complete ophthalmic examination revealed a large, pigmented, raised, well‐demarcated, epibulbar mass appearing to originate from the nasodorsal limbal region. The mass was smooth and roughly circular, extending approximately 4 mm into the sclera and 14 mm into the nasodorsal cornea. Gonioscopy directly under the mass was not possible due to mass size. The visible iridocorneal angle was normal. High‐resolution B‐scan ultrasound showed mass extension to Descemet's membrane and deep sclera, but no intraocular invasion. Penetrating sclerokeratoplasty was performed followed by autologous pinnal cartilage and conjunctival grafting to repair the corneoscleral defect (20 mm x 19 mm) and to restore globe integrity and function. Histopathology confirmed the mass to be a benign limbal melanoma with complete excision. The surgery site healed without complication, and the pinnal cartilage became fully incorporated into the globe. Twelve months postoperatively, the patient remains visual with a normal intraocular and fundic examination. The pinnal harvest site on the right ear healed without complication. To the authors’ knowledge, this is the first reported case of corneoscleral grafting using autologous pinnal cartilage. This may represent a viable alternative to other corneoscleral grafting procedures for large defects and is an attractive treatment option due to lack of host rejection, readily available source of donor cartilage, and provision of tectonic support to the globe.     

Chronic lameness caused by vascular compression of the iliac artery and vein by a malignant melanoma in the pelvis of a grey Spanish gelding

An 11-year-old grey Spanish gelding was presented with chronic lameness of the right hindlimb, with abduction of the limb during the swing phase. Based on a comprehensive lameness examination, the problem was localised to the pelvic region. Rectal and ultrasonographic examinations revealed a mass in the pelvic region in close association with the iliac artery and vein, and ultrasonographic indications of vascular compression. Considering the clinical presentation of a grey middle-aged horse with multifocal melanoma-like nodules at the level of the perineum, tail base, preputium and penis, a malignant melanoma with secondary vascular or neurological compression was suspected. Post-mortem and histological examination confirmed the diagnosis of malignant melanoma with vascular compression of the iliac artery and vein and infiltrative growth in the iliac vein.     

Tumour‐infiltrating lymphocytes in canine melanocytic tumours: An investigation on the prognostic role of CD3+ and CD20+ lymphocytic populations

The study of the immune response in several types of tumours has been rapidly increasing in recent years with the dual aim of understanding the interactions between neoplastic and immune cells and their importance in cancer pathogenesis and progression, as well as identifying targets for cancer immunotherapy. Despite being considered one of the most immunogenic tumour types, melanoma can progress in the presence of abundant lymphocytic infiltration, therefore suggesting that the immune response is not able to efficiently control tumour growth. The purpose of this study was to investigate whether the density, distribution and grade of tumour‐infiltrating lymphocytes (TILs) in 97 canine melanocytic tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in the dog. As a further step in the characterization of the immune response in melanocytic tumours, an immunohistochemical investigation was performed to evaluate the two main populations of TILs, T‐lymphocytes (CD3⁺) and B‐lymphocytes (CD20⁺). The results of our study show that TILs are present in a large proportion of canine melanocytic tumours, especially in oral melanomas, and that the infiltrate is usually mild. The quantity of CD20⁺ TILs was significantly associated with some histologic prognostic factors, such as the mitotic count, the cellular pleomorphism and the percentage of pigmented cells. Remarkably, a high infiltration of CD20⁺ TILs was associated with tumour‐related death, presence of metastasis/recurrence, shorter overall and disease‐free survival, increased hazard of death and of developing recurrence/metastasis, hence representing a potential new negative prognostic factor in canine melanocytic tumours.     

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow‐up of 6 months. Epidemiological, clinical and histological data were collected and quantitative‐PCR were performed on formalin‐fixed paraffin‐embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.     

Surgical treatment of epibulbar melanocytomas by complete excision and homologous corneoscleral grafting in dogs: 11 cases

Objective The aim of the study was to evaluate the efficacy, outcome, and complications following penetrating sclerokeratoplasty and frozen homologous corneoscleral grafting for the management of extensive canine epibulbar melanocytomas. Methods Medical records of canine patients treated at three different veterinary hospitals between 1999 and 2010 were reviewed. Signalment, location and extent of melanocytomas, recurrence rate, and early and late complications were reported. Patients were re‐examined postoperatively to provide follow‐up information. Results Patients included one intact male, three castrated males, six intact females, and one spayed female, with a median age of 5 years (range, 3–9). German Shepherds were overrepresented. Tumors extended from 2 to 4 clock hours at the limbus and up to 17 mm from clear cornea to globe equator. One case showed iridocorneal angle invasion; corneal involvement was present in all cases, and lipid keratopathy was present in four cases. In two cases, there was incomplete resection owing to tumor extent. Follow‐up time ranged from 3 to 72 months (median, 17 months), with one case of intraocular tumor progression. Early complications included anterior uveitis (11/11), intracameral fibrin (5/11), hyphema (4/11), corneal edema (4/11), exuberant corneal granulation tissue (2/11), focal retinal edema (1/11), dyscoria (1/11), and partial suture dehiscence (1/11). Late complications included corneal fibrosis and/or pigmentation (11/11), faint anterior cortical cataracts (3/11), and lipid keratopathy (1/11). Vision was retained in all cases. Conclusions This technique offers a surgically challenging but effective treatment for extensive epibulbar melanocytomas. In this case series, complications were mild and transient, with preservation of ocular anatomy and function.