Exploring the in situ expression of vascular endothelial growth factor and endoglin in pemphigus foliaceus variants and pemphigus vulgaris.

BACKGROUND: Erythroderma is a severe manifestation of pemphigus foliaceus (PF), a blistering disease mediated by IgG autoantibodies against desmoglein 1. Increasing evidence supports the contribution of angiogenic mediators in the pathogenesis of erythroderma. OBJECTIVE: To evaluate the in situ expression of vascular endothelial growth factor (VEGF) and endoglin in patients with PF with erythroderma. METHODS: Formalin-fixed paraffin-embedded skin samples obtained from patients with erythrodermic PF (n = 19; 12 patients with endemic PF), non-erythrodermic PF (n = 17), pemphigus vulgaris (PV; n = 10), psoriasis (n = 10) and healthy individuals (HI; n = 10) were processed in an automated immunohistochemistry platform utilizing anti-VEGF and anti-endoglin as primary antibodies. Reactivity was evaluated both manually (0 = negative; 1+ = mild; 2+ = intense) and through an automated microvessel analysis algorithm. RESULTS: Vascular endothelial growth factor expression in erythrodermic PF was higher than in non-erythrodermic PF (P = 0.034) and in HI (P = 0.004), and similar to psoriasis (P = 0.667) and PV (P = 0.667). In non-erythrodermic PF, VEGF positivity was similar to HI (P = 0.247), and lower than psoriasis (P = 0.049) and PV (P = 0.049). Both erythrodermic and non-erythrodermic PF presented similar endoglin expression (P = 0.700). In addition, endoglin positivity during erythrodermic PF was similar to psoriasis (P = 0.133) and lower than PV (P = 0.0009). Increased expression of in situVEGF suggests that healing processes are triggered in response to tissue damage led by autoantibodies in PF, especially during erythroderma. Reduced endoglin positivity suggests that an unbalanced angiogenesis may occur during erythrodermic PF. Further studies may help to confirm if the regulation of VEGF and endoglin expression in patients with PF can contribute to control the healing process and enable disease remission. CONCLUSION: Overexpression of VEGF in erythrodermic PF as well as in PV and psoriasis points out a dysregulated repair process in severe forms of these diseases and suggests VEGF and endoglin could act as prognostic markers and future therapeutic targets to enable proper healing in PF.

Increased serum levels of vascular endothelial growth factor in pemphigus foliaceus patients with erythroderma.

BACKGROUND: Erythroderma is a clinical skin syndrome shared by patients with cutaneous disorders of distinct aetiologies as a result of the combined actions of chemokines, adhesion molecules, and cytokines, such as vascular endothelial growth factor (VEGF). OBJECTIVE: To evaluate the profile of serum levels of VEGF and soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) in pemphigus foliaceus (PF) patients with erythroderma. METHODS: We conducted a retrospective study, which included (i) a chart review of all PF patients from the Autoimmune Blistering Clinic, University of Sao Paulo, Brazil, from January 1991 to December 2014, together with an evaluation of demographic variables, hospitalization duration and complications and (ii) analysis of the circulating VEGF and sVEGFR-1 levels in PF patients with erythroderma by ELISA. The controls included patients with pemphigus vulgaris or psoriasis. RESULTS: We observed higher serum VEGF levels in PF patients during erythroderma than during the non-erythrodermic phase. PF patients showed increased serum levels of sVEGFR-1 during the erythrodermic phase in comparison to controls. Interestingly, the sVEGFR-1 and antidesmoglein-1 levels were positively correlated during the non-erythrodermic period. CONCLUSION: Erythroderma, which represents one clinical form of PF, implies more severe outcomes. The circulating levels of VEGF, a potent endothelial activator, are increased in PF patients with erythroderma; this result suggests the contribution of the blood vessel endothelium to the pathogenesis of this clinical syndrome. Interestingly, our findings showed a positive correlation between the sVEGFR-1 and antidesmoglein-1 antibody levels, indicating a suppressive response to VEGF augmentation during the erythrodermic phase of PF.

The effect of imatinib mesylate on the proliferation, invasive ability, and radiosensitivity of retinoblastoma cell lines.

PURPOSE: Our aim was to evaluate the potential effect of imatinib mesylate (IM), a small molecule that specifically inhibits the tyrosine quinase receptors, on the proliferation and invasive abilities of two human retinoblastoma (Rb) cell lines. Furthermore, the ability of IM to radiosensitize Rb cells was evaluated. The potential targets of IM (C-kit, PDGRF-α and -ß, and c-Abl) were also investigated in these cell lines. METHODS: Two human Rb cell lines (WERI-RB-1 and Y79) were cultured under normal growth conditions. An MTT-based proliferation assay and a Matrigel invasion assay were performed with and without exposure to 10 µM of IM. The cells were also irradiated with graded dosages of 0, 2, 4, 6, 8, and 10 Gy with and without IM and their proliferations rates were analyzed. Western blot and immunocytochemical analysis of cytospins were performed to evaluate the expression of C-kit, PDGRF-α and -ß, and c-Abl. RESULTS: When IM was added to both cell lines a statistically significant (P<0.05) reduction in proliferation and invasive ability were observed. Exposure to IM also significantly increased the radiosensitivity of both Rb cell lines. The c-Abl expression was strongly positive, PDGRF-α and -ß expression were also positive but the C-kit expression was negative in both cell lines. CONCLUSIONS: These results indicate that Gleevec may be useful as an adjuvant treatment in Rb patients, specially those considered for radiation therapy.

Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis.

The liver is a major site of metastasis for human malignancies, yet the factors that regulate tumor cell survival and growth in this organ remain elusive. Previously, we reported that M-27(IGF-IR) murine lung carcinoma cells with ectopic insulin-like growth factor-1 (IGF-I) receptor overexpression acquired a site-specific, liver-metastasizing potential. Gene expression profiling and subsequent RNA and protein analyses revealed that this was associated with major changes to the expression of extracellular matrix (ECM) protein-encoding genes including type III, IV and XVIII collagen genes, and these changes were also observed in the respective tumors in vivo. Because type IV collagen was the most prominently altered ECM protein in this model, we further analyzed its functional relevance to liver metastasis. M-27 cells stably overexpressing type IV collagen α1 and α2 chains were generated and their growth and metastatic properties investigated. We found that these cells acquired a site-selective growth advantage in the liver and this was associated with cell rescue from anoikis in a collagen IV/α2 integrin/FAK-dependent manner and increased responsiveness to IGF-I. Conversely, collagen IV or focal adhesion kinase (FAK) silencing by small-interfering RNA in highly metastatic tumor cells enhanced anoikis and decreased liver metastases formation. Moreover, analysis of human surgical specimens revealed uniformly high collagen IV expression in 65/65 hepatic metastases analyzed, regardless of tissue of origin, whereas it was variable and generally low in 50/50 primary colorectal carcinoma specimens examined. The results suggest that collagen IV-conveyed signals are essential cues for liver metastasis in diverse tumor types and identify mediators of collagen IV signaling as potential therapeutic targets in the management of hepatic metastases.

[Intraocular tumor diagnosed two years after perforating trauma]. / Tumor intraocular diagnosticado dos años después de un trauma perforante.

CASE REPORT: We describe the unusual diagnosis of a ciliary body medulloepithelioma by histopathology of a subretinal membrane obtained from vitreoretinal surgery of a 10-year-old boy. The patient had a history of perforating trauma OS 2 years earlier, and both fundus exam and B-scan ultrasound revealed only a retinal detachment with a subretinal membrane. No detectable mass was present. DISCUSSION: The membrane removed from underneath the peripheral retina revealed a blue cell tumor confirmed by histopathology and immunohistochemistry to be a primitive neuroectodermal tumor. Currently, the patient has been followed for 5 years with no signs of recurrence.

Expression of the metastasis suppressor gene KISS1 in uveal melanoma.

PURPOSE: Uveal melanoma (UM) is the most common primary malignant intraocular tumour in adults. Forty-five percent of UM patients develop metastasis within 15 years of initial diagnosis. KISS1, a human metastasis suppressor gene, has been reported to play a role in various human malignancies. The purpose of this study was to investigate the expression of KISS1 in UM and its potential value as a prognostic marker. METHODS: Thirty-seven cases of paraffin-embedded human UM specimens were immunostained with a KISS1 antibody. Clinical-pathological data were obtained. The relationship between the clinical-pathological data and the expression of KISS1 was evaluated. Moreover, the survival rates of the patients were also assessed. Five UM cell lines (92.1, OCM-1, MKTBR, UW1 and SP6.5) were assayed for KISS1 expression. In addition, real-time PCR was used to determine mRNA levels of KISS1and its receptor GPR54in these cell lines. RESULTS: The immunohistochemical results of KISS1 expression displayed cytoplasmic staining in 84% of UM specimens. Low KISS1 expression was associated with a higher risk of metastatic disease (P<0.05). Furthermore, we found that KISS1 was expressed in all five UM cells lines. Real-time PCR analysis confirmed the presence of both KISS1and its receptor GPR54in all five human UM cell lines. CONCLUSIONS: To the best of our knowledge, this is the first time that KISS1has been characterized in UM. The correlation between KISS1 expression and UM survival rate suggests an important role for KISS1as a prognostic marker in this particular tumour.

Amfenac increases the radiosensitivity of uveal melanoma cell lines.

PURPOSE: To evaluate the proliferation rates of five human uveal melanoma (UM) cell lines after treatment with amfenac, a cyclooxygenase (COX)-2 inhibitor, and subsequent radiation exposure. METHODS: Five human UM cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1) and one human fibroblast cell line (BJ) were incubated with amfenac. Treated and non-treated cell lines were then exposed to various doses of gammaradiation: 0, 2, 4, 6, and 8 Gy. Sulphorhodamine-B assay was used to assess proliferation rates 48 h post-radiation. RESULTS: Treatment of UM cell lines with amfenac prior to radiation led to a marked reduction in proliferation rates. This difference was statistically significant in all cell lines at every radiation dose (P<0.005), with the exception of 92.1 at 2 Gy (P=0.157). Fibroblasts treated with amfenac showed significantly higher proliferation rates after 2 and 8 Gy, with no significant differences at 0, 4, and 6 Gy. CONCLUSIONS: The radiosensitivity of UM cell lines was increased by the administration of amfenac, the active metabolite of nepafenac. There appears to be a radioprotective effect of amfenac on human fibroblasts. The topical administration of nepafenac may decrease tumour recurrence and radiation-induced complications while broadening the indications for radiotherapy by treating larger tumours.

Current and emerging concepts in the management of neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. The more severe form of the disease, known as neovascular AMD, is characterized by aberrant growth of blood vessels from the choroid into the subretinal space. This pathologic choroidal neovascularization can have drastic consequences, often seriously impairing vision in affected individuals. Current treatment approaches focus on combination therapies that include photodynamic therapy in conjunction with numerous forms of antiangiogenic or anti-inflammatory drug intervention. To date, however, no adequate treatment is available for the majority of affected individuals. The threat of a rapidly aging population provides the impetus for aggressive efforts to control the prevalence and progression of this disease. This review will outline the currently available pharmacotherapies, discussing the justification for their use as well as their shortcomings. Furthermore, drugs that are currently under investigation as monotherapies and adjuncts will be highlighted. The potential for alternate targets will also be examined, with a focus on the most promising candidates.

Identification of circulating malignant cells and its correlation with prognostic factors and treatment in uveal melanoma. A prospective longitudinal study.

PURPOSE: Most uveal melanoma patients (UMP) do not show evidence of metastases upon diagnosis. However, despite local tumour control, approximately 50% of them will develop metastases. These findings suggest that malignant cells may have already disseminated by the time of initial diagnosis. The purpose of the study was to detect circulating malignant cells (CMCs) in UMP and to correlate them with prognostic factors and therapy. METHODS: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect CMCs. In each UMP, blood was collected every 3 months. In each visit, 20 RT-PCR tests were performed. The date of diagnosis, largest tumour dimension, type, and date of treatment were obtained. RESULTS: A total of 30 UMP were enrolled. Five patients were enrolled at the time of diagnosis and 25 patients between 1 and 17 years following diagnosis. No UMP showed clinical evidence of metastasis. A total of 136 visits were registered, 1360 samples collected, and 2720 RT-PCRs performed. CMCs were identified in 29 patients in 119 visits (87.5%). However, in each visit, a low number of positive tests were recorded. CMCs were found in newly diagnosed, irradiated, enucleated, and observed patients regardless of tumour size and time period following treatment. CONCLUSIONS: Uveal melanoma (UM) is not a localized ocular disease. CMCs were recorded at initial diagnosis confirming the early metastatic nature of UM. CMCs were present following treatment, including enucleation, demonstrating that CMCs are capable of disseminating and surviving, possibly as micrometastasis, which would contribute to the pool of CMCs at a later stage. Systemic therapy should be evaluated.

TGFBI gene mutations in Brazilian patients with corneal dystrophy.

PURPOSE: To investigate the transforming growth factor beta-induced gene (TGFBI) mutations in Brazilian patients with corneal dystrophy and to evaluate the phenotype-genotype correlation in these patients. METHODS: A total of 11 unrelated families were studied. The diagnosis of corneal dystrophy was based on clinical and histopathological findings. Genomic DNA was extracted from peripheral blood leucocytes, and exons 4 and 12 of the TGFBIgene were amplified by polymerase chain reaction followed by direct sequencing on both strands. RESULTS: Five different mutations in the TGFBIgene were found in the probands. We identified the following mutations: lattice corneal dystrophy--R124C and A546T; Reis-Bücklers corneal dystrophy--R555Q and R124L; granular corneal dystrophy--R555W and Avellino dystrophy--R555W. In three of the 11 studied families there was no mutation in exons 4 and 12. CONCLUSIONS: This is the first report of mutations in the TGFBIgene in a series of Brazilian patients with corneal dystrophy. The findings indicate that TGFBIgene screening should be considered in the diagnosis of corneal dystrophy.

The effect of a selective cyclooxygenase-2 (COX-2) inhibitor on the proliferation rate of retinoblastoma cell lines.

PURPOSE: To examine the effect of nepafenac, a selective cyclooxygenase-2 (COX-2) inhibitor, on the proliferation rate of two human retinoblastoma (Rb)cell lines. METHODS: Two human Rb cell lines (WERI-RB and Y79) were cultured. COX-2 expression in these cell lines was verified by immunocytochemical analysis of cytospin sections and Western blotting. An MTT-based proliferation assay was used to compare Rb cell growth with and without amfenac, the active metabolite of nepafenac. The averaged results per condition were recorded. The Student's t-test was used to compare results from the cells cultured with and without amfenac. RESULT: The Y79 cell line showed a higher proliferative rate than the WERI-RB cell line. Both cell lines were negative for COX-2 expression by immunocytochemical analysis; however, both cell lines were positive for COX-2 expression by Western blot. When amfenac was added to both of the cell lines, a statistically significant reduction in proliferation was observed in both cell lines. The two Rb cell lines were positive for COX-2 only in the Western blot, indicating that they probably express low levels of COX-2, which was undetectable by immunocytochemical analysis. CONCLUSION: The selective, anti-COX-2 molecule amfenac inhibited proliferation of both tested Rb cell lines. Further trials should be undertaken to study the effect of selective COX-2 inhibitors on Rb.

The role of ultrasound biomicroscopy in predicting the result of temporal artery biopsy in temporal arteritis patients: a preliminary study.

PURPOSE: Temporal artery biopsy is considered the gold standard for the diagnosis of temporal arteritis (TA). However, complications following this procedure may occur. The goal of this study is to evaluate if ultrasound biomicroscope (UBM) findings are useful in predicting the result (positive or negative) of temporal artery biopsy in patients with TA. METHODS: Twenty-six consecutive patients with clinical diagnosis of TA seen at the Department of Ophthalmology, Royal Victoria Hospital, Montreal, Canada, were involved in this study. All patients were submitted to UBM before temporal artery biopsy. Eight patients presented histopathologic findings consistent with the diagnosis of TA. Thus, UBM findings of these patients were compared with those from 18 patients with negative biopsy. On UBM we searched for the presence of a hypoechoic effect surrounding the walls of the temporal arteries, the so-called halo sign, as well as an intra-arterial middle reflexive filling, the so-called intra-arterial filling. RESULTS: The halo sign and/or the intra-arterial filling were found in 8 (100%) of 8 patients with biopsy-proven TA. However, 10 (55.5%) of 18 patients with a negative biopsy presented one or both of these two UBM findings. On the other hand, the absence of these two parameters on the UBM of a patient with TA strongly suggests that the temporal artery biopsy will be negative (negative predictive value=100%). CONCLUSIONS: This preliminary work suggests that UBM may play a role in predicting a negative result of the temporal artery biopsy in patients with TA. In the present series approximately 30% of the patients could be spared this surgical procedure and its possible complications.

Cost considerations of the new fixed combinations for glaucoma medical therapy.

OBJECTIVE: To compare the costs of the new fixed combinations for glaucoma medical therapy. METHODS: The studied drugs were: Cosopt (5-mL bottle), Combigan (5-mL bottle) and Xalacom (2.5-mL bottle). Five bottles of each drug were obtained from pharmacies, and the medications lot numbers were recorded. To calculate the drop volume, 10 drops and 1 mL of each bottle were weighed with a digital precision scale. Drop volume was calculated by the relation between volume and weight. The cost of each bottle of medication was determined from the average retail price in Canada. The prices were obtained in Canadian dollars (dollars). RESULTS: The drops of Cosopt (39.60 +/- 0.45 microL) were considerably larger than the drops of Combigan (33.75 +/- 0.60 microL) and Xalacom (30.87 +/- 0.37 microL). The average number of drops per millilitre varied from 25.25 +/- 0.29 (Cosopt) to 32.40 +/- 0.39 microL (Xalacom). Combigan presented the lowest daily cost (dollars 0.87 +/- 0.02) followed by Xalacom (dollars 1.09 +/- 0.01) and Cosopt (dollars 1.22 +/- 0.01). The average cost by year varied from dollars 316.75 +/- 5.59 (Combigan) to dollars 445.96 +/- 5.16 (Cosopt), with a total difference of dollars 129.21 per year of treatment. CONCLUSIONS: There was a statistically significant difference in average drop size and cost among the three studied drugs. Combigan presented the lowest daily cost followed by Xalacom and Cosopt.

Basal cell carcinoma of the eyelid in children: a report of three cases.

Basal cell carcinoma is the most common eyelid malignancy, accounting for approximately 90% of malignant eyelid tumours. Despite its prevalence in adults, it is extremely rare in children, and usually occurs in the setting of a known genetic defect or following radiotherapy treatment. We report three cases of de novo basal cell carcinoma in children. These children had no known genetic syndromes and had not undergone radiotherapy.

The role of ultrasound biomicroscopy in predicting the result of temporal artery biopsy in temporal arteritis patients: A preliminary study.

PURPOSE: Temporal artery biopsy is considered the gold standard for the diagnosis of temporal arteritis (TA). However, complications following this procedure may occur. The goal of this study is to evaluate if ultrasound biomicroscope (UBM) findings are useful in predicting the result (positive or negative) of temporal artery biopsy in patients with TA. METHODS: Twenty-six consecutive patients with clinical diagnosis of TA seen at the Department of Ophthalmology, Royal Victoria Hospital, Montreal, Canada, were involved in this study. All patients were submitted to UBM before temporal artery biopsy. Eight patients presented histopathologic findings consistent with the diagnosis of TA. Thus, UBM findings of these patients were compared with those from 18 patients with negative biopsy. On UBM we searched for the presence of a hypoechoic effect surrounding the walls of the temporal arteries, the so-called halo sign, as well as an intra-arterial middle reflexive filling, the so-called intra-arterial filling. RESULTS: The halo sign and/or the intra-arterial filling were found in 8 (100%) of 8 patients with biopsy-proven TA. However, 10 (55.5%) of 18 patients with a negative biopsy presented one or both of these two UBM findings. On the other hand, the absence of these two parameters on the UBM of a patient with TA strongly suggests that the temporal artery biopsy will be negative (negative predictive value=100%). CONCLUSIONS: This preliminary work suggests that UBM may play a role in predicting a negative result of the temporal artery biopsy in patients with TA. In the present series approximately 30% of the patients could be spared this surgical procedure and its possible complications.