Gene expression profiles of progestin-induced canine mammary hyperplasia and spontaneous mammary tumors.

Spontaneous mammary tumors are the most prevalent type of neoplasms in women as well as in female dogs. Although ovarian hormones estrogen and progesterone are known to play a key role in mammary tumorigenesis, conflicting reports have been obtained from in vivo and in vitro studies concerning the role of especially progesterone in mammary tumorigenesis. Prolonged exposure to high concentrations of progesterone during the unusually long luteal phase of the estrous cycle is suspected to be the key event in canine mammary tumorigenesis. Accordingly, previous studies have shown the development of mammary hyperplasia in dogs upon prolonged progestin administration. In this study, a dog-specific cDNA microarray was used to identify oncogenic determinants in progestin-induced canine hyperplasia (CMH) and spontaneous mammary tumors (CMC) by comparing expression profiles to those obtained from mammary glands of healthy dogs. The CMH profile showed elevated expression of genes involved in cell proliferation such as PCNA, NPY, RAN and also alterations in expression of transcription factors and cell adhesion molecules. Whereas in CMC, major alterations to the expression of genes involved in cell motility, cytoskeletal organization and extra cellular matrix production was evident besides differential expression of cell proliferation inducing genes. The overall gene expression profile of CMH was related to cell proliferation where as that of CMC was associated with both cell proliferation as well as neoplastic transformation. In conclusion, our findings support a strong cell proliferation inducing potential of progestins in the canine mammary gland. Moreover, deregulated genes identified in CMC are potentially involved in their malignant and may serve as prospective therapeutic targets.

Transcriptomic profile of two canine mammary cancer cell lines with different proliferative and anti-apoptotic potential.

The aim of the study was to identify the genes responsible for the high growth rate and antiapoptotic potential in selected canine mammary cancer cells. cDNA canine microarrays were used to compare the transcriptome in simple carcinoma CMT-U27 and spindle-cell tumor CMT-U309 cell lines. In CMT-U27 cell line the growth rate (shorter cell cycle), anti-apoptotic potential (higher expression of Bcl-2) was higher and spontaneous and induced apoptosis was lower. Comparison of transcriptomes revealed 333 genes which expression differed similarly. We focused on genes involved in cell proliferation, adhesion and apoptosis, and selected 29 of them. The high growth rate and anti-apoptotic potential in CMT-U27 cells was associated with enhanced expression of genes (at the level of transcripts) involved in Ca(2+) signaling pathway (Calmodulin 1, 2, 3 and SPSB2) and growth hormone cellular pathway. The low-proliferative and pro-apoptotic phenotype of CMTU309 cells was more dependent on TGFbeta, neuregulin 1 pathways and adhesion-related molecules.

Expression of heat shock protein 27 and alpha-crystallins in human retinoblastoma after chemoreduction.

AIM: Small heat shock proteins (sHSP) play an important role in the resistance to anticancer drugs. We examined the expression of the sHSP family, HSP27 and alpha-crystallins, in human retinoblastoma with and without preoperative chemotherapy. METHODS: Eighteen enucleated eyes from patients with retinoblastoma were used. Six patients had undergone chemotherapy before enucleation. Formalin-fixed, paraffin-embedded tissue sections were processed for H&E staining and examined by immunohistochemistry using anti-HSP27 and alpha-crystallins antibodies. RESULTS: Eleven of 12 cases with no history of preoperative chemotherapy showed weakly positive or negative staining for HSP27, whereas six and five cases were strongly positive for alphaA and alphaB-crystallin, respectively. In the six cases with a history of chemotherapy, several viable retinoblastoma cells remained. Immunoreactivity for HSP27 and alphaB-crystallin was strongly detected in the cytoplasm of viable retinoblastoma cells, while alphaA-crystallin immunoreactivity was less marked. Immunoreactivity for HSP27 was significantly higher in retinoblastoma cases with preoperative chemotherapy than in those without chemotherapy (p<0.0001). In contrast, immunoreactivity for alphaA-crystallin was significantly lower in cases with chemotherapy than in cases without chemotherapy (p<0.01). CONCLUSIONS: HSP27 and alphaB-crystallin, but not alphaA-crystallin, were highly expressed in viable tumour cells after chemotherapy, suggesting that HSP27 and alphaB-crystallin may protect tumour cells from apoptotic signals produced by anticancer drugs in retinoblastoma.

cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.

Early onset vitreous amyloidosis in familial amyloidotic polyneuropathy with a transthyretin Glu54Gly mutation is associated with elevated vitreous VEGF.

AIM: To report the early vitreous involvement in a rare familial amyloidotic polyneuropathy (FAP) mutation and associated vitreous vascular endothelial growth factor (VEGF) levels. DESIGN: Observational case series. METHODS: Review of clinical, pathological, photographic, and angiographic records of two FAP siblings with severe vitreous involvement. Laboratory ELISA analysis of vitreous samples for VEGF, and DNA sequence analysis of peripheral blood for transthyretin (TTR) mutational analysis. RESULTS: Two patients underwent 25-gauge vitrectomy in three eyes with marked improvement of visual acuity. Neovascularisation seen intraoperatively responded to endolaser. Analysis of vitrectomy samples for VEGF showed raised levels in all three specimens. Mutational analysis revealed an isolated Glu54Gly mutation in the transthyretin gene. CONCLUSIONS: Early involvement of the vitreous occurs in a rare transthyretin mutation of FAP, with increased vitreous levels of VEGF.

Microglial stability and repopulation in the retina.

BACKGROUND/AIMS: Parenchymal central nervous system microglia are repopulated by bone marrow derived monocytes more slowly than any other reticuloendothelial cells. The contribution of bone marrow derived monocytes to the uninflammed retina has not been studied. The present study sought to determine repopulation of retinal microglia in uniflammed retina by bone marrow derived monocytes in bone marrow chimeric rats. METHODS: Chimeric (Y-->X) Lewis rats were constructed by transplanting 5 x 10(7) male bone marrow cells into lethally irradiated female recipient rats. The chimeras were sacrificed 8, 10, 12, 30, and 52 weeks after bone marrow transplant, and retina, brain, lung, and spleen samples were collected. DNA was extracted and quantified. Y positive infiltrating cells in the collected samples were detected by polymerase chain reaction amplification of a Y chromosome specific 104 bp fragment. RESULTS: There was a rapid repopulation of haematopoietic tissues in the spleen (at 8 weeks), confirming the establishment of chimerism, and to a lesser extent, of lung (at 30 weeks). This repopulation was absent in the brain parenchyma and retina until 52 weeks after transplantation. CONCLUSIONS: These data indicate that resident microglia in the retina, much like those in the brain, are stable in number in the retinal compartment (up to 1 year), and repopulation by bone marrow derived cells may be delayed for a year.

The effects of atorvastatin in experimental autoimmune uveitis.

AIM: To investigate the effect of atorvastatin (Lipitor), a commonly used drug for dyslipidaemia in experimental autoimmune uveitis (EAU). METHODS: 48 B10-RIII mice were immunised with human interphotoreceptor retinoid binding protein (IRBP) peptide p161-180. They were divided into three groups of 16 each and treated orally once daily for 14 days; group one received phosphate buffered saline (control group), group two received 1 mg/kg of atorvastatin (low dose group), and group three received 10 mg/kg (high dose). On day 14 lymph nodes, spleens, and right eyes were harvested. RNA was extracted from lymph nodes for RNase protection assay (RPA) to determine proinflammatory (IL-1 alpha and IL-1 beta), Th1 (TNF-alpha, IL-2, IL-12), and Th2 (IL-4, IL-5, and IL-10) cytokine levels. Protein was extracted from spleens for western blot to detect the expression of phosphorylated signal transducer and activator of transcription (STAT) 4 and STAT6. The severity of inflammation in enucleated eyes was graded by a masked observer. Paired t test was performed for the mean difference in histological scoring between treated groups and the immunised control group. RESULTS: Surprisingly, atorvastatin did not modulate the immune response. The proinflammatory cytokines, IL-1 alpha and IL-1 beta, and Th1 cytokines, TNF-alpha and IL-2, were upregulated equally in control and atorvastatin treated groups. IL-12 and Th2 cytokines were not upregulated in all three groups. Western blot analysis showed high levels of phosphorylated STAT4, but not STAT6 protein in the control and atorvastatin treated groups. Mean differences in histological scoring between treated groups and the immunised control group were not statistically significant. CONCLUSIONS: Atorvastatin treatment had no effect on Th1 and Th2 cytokine transcription. Although histological grading suggested mildly decreased inflammation in the high dose treated group, the equivalence of cytokine expression in all groups suggests that the statins may not modulate IRBP induced uveoretinitis.

Necrotising retinopathies simulating acute retinal necrosis syndrome.

AIM: To determine an aetiological diagnosis in patients presenting with necrotising retinopathies that simulate acute retinal necrosis (ARN). METHODS: Retrospective non-comparative case series. The charts of 16 patients presenting with a clinical impression of ARN at Pitie-Salpetriere Hospital, Paris, France, between 1994 and 1999, who required initial antiviral therapy were reviewed. All of the patients had extensive laboratory tests. Anterior chamber paracentesis was performed on 14 patients and evaluated by polymerase chain reaction (PCR) and/or the Witmer-Goldmann coefficient to determine the cause of retinitis. Three of the 14 cases also had diagnostic vitrectomy. Responses to specific treatment, initiated based on laboratory results, and the final outcome were evaluated. RESULTS: Seven of the 16 patients were female and nine were male. The retinitis was bilateral in five patients and unilateral in 11 patients. The average age of the patients at presentation was 53.6 years. 13 patients were immune deficient for various reasons. Upon initial presentation, the patients' visual acuities were less than 20/200 in 68% of the affected eyes. The final diagnoses, based on laboratory data and therapeutic response were toxoplasmic retinochoroiditis (62.5%), syphilitic retinitis (12.5%), aspergillus endophthalmitis (12.5%), Behcet's disease (6.2%), and intraocular lymphoma (6.2%). Visual acuity was stabilised or improved in 12 patients (75%). Two patients with aspergillosis died despite antifungal therapy. CONCLUSIONS: Toxoplasmic retinochoroiditis is the major cause of retinal necrosis that simulates ARN, and PCR analysis of the aqueous humour is helpful in establishing the diagnosis. Such atypical toxoplasma retinochoroiditis may be associated with poor visual outcome.

Metastatic adenocarcinoma with rupture through the Bruch membrane simulating a choroidal melanoma.

PURPOSE: To report a case of adenocarcinoma metastatic to the choroid with rupture through the Bruch membrane, thus, simulating a choroidal melanoma. METHODS: Interventional case report. Evaluation in a university-based clinic, including a complete eye examination, fluorescein angiography, and ultrasonography; oncological evaluation; and eventual enucleation with histopathological study, including immunohistochemical stains. RESULTS: A 62-year-old Hispanic female presented with visual loss, right eye, of short duration because of a choroidal mass with retinal detachment. Ultrasonography showed a dome-shaped lesion with an eccentric collar-button projection and medium internal reflectivity, which suggested a choroidal melanoma. Initial systemic evaluation was negative. Severe pain necessitated enucleation, RE, and histopathology of the choroidal mass demonstrated an adenocarcinoma. Further examination revealed a left, upper lobe, nonsmall cell lung carcinoma. CONCLUSION: Metastatic choroidal tumors may present, although rarely, with collar-button configurations.

Juvenile xanthogranuloma masquerading as pediatric chronic uveitis: a clinicopathologic study.

Juvenile xanthogranuloma (JXG) is a rare, pediatric histiocytic skin disorder that may affect the eye. It can present with protean ocular manifestations, including masquerade uveitis, heterochromia, hyphema, or glaucoma. It very rarely involves the retina and posterior segment; indeed, posterior involvement has been documented histopathologically in only one case. We present the case of a 2-year-old child with ocular JXG presenting as chronic, refractive uveitis, without skin or systemic findings. The blind, painful eye was enucleated and found to harbor a diffuse histiocytic process that involved both the anterior and posterior segments, including the retina and subretinal space. Histological, immunohistochemical, and electron microscopic studies confirmed the diagnosis of JXG. The pathologic classification and differential diagnosis of systemic histiocytic disorders are discussed. Since JXG can present as masquerade pediatric uveitis, this entity should be considered in children with atypical uveitis. In rare instances, JXG may involve the posterior segment and the retina, leading to retinal detachment and blindness.

Endogenous mycotic endophthalmitis: variations in clinical and histopathologic changes in candidiasis compared with aspergillosis.

PURPOSE: To describe clinical and/or histopathologic features that could help distinguish endogenous Candida endophthalmitis from endogenous Aspergillus intraocular inflammation and to provide histologic documentation of intraocular spread of these agents. METHODS: Twenty-five patients who underwent enucleation, 13 with morphologic features and/or positive culture for Aspergillus and 12 with histologic evidence and/or positive culture for Candida were included in the study. Clinical information was sought from each case. Patients with AIDS were excluded. The enucleated globes were analyzed to detect location of the fungi, vascular invasion by these agents, and inflammatory response. RESULTS: Candida endophthalmitis was noted in patients with a history of gastrointestinal surgery, hyperalimentation, or diabetes mellitus, whereas aspergillosis was present in patients who had undergone organ transplantation or cardiac surgery. Histopathologically, the vitreous was the primary focus of infection for Candida, whereas subretinal/subretinal pigment epithelium infection was noted in eyes with aspergillosis. Retinal and choroidal vessel wall invasion by fungal elements was noted in cases of aspergillosis but not in cases with candidiasis. Both infectious agents induced suppurative nongranulomatous inflammation. CONCLUSIONS: Unlike Candida endophthalmitis, aspergillosis clinically presents with extensive areas of deep retinitis/choroiditis, and vitreous biopsy may not yield positive results. Histopathologically, it appears that Aspergillus grows preferentially along subretinal pigment epithelium and subretinal space. This intraocular infection is usually associated with a high rate of mortality caused by cerebral and cardiac complications.

An outbreak of trematode-induced granulomas of the conjunctiva.

PURPOSE: To describe the epidemiologic, clinical, and histopathologic features of trematode granulomas of the conjunctiva, eyelid, and anterior chamber in pediatric patients. DESIGN: Prospective noncomparative case series. PARTICIPANTS: Forty-one children from a southern Indian village with conjunctival granulomas. METHODS: The village of Sellananthal was selected for a field visit after analysis of earlier hospital-based allergic conjunctival granuloma cases. Children with ocular diseases were examined, and histories of exposure to assumed risk factors and clinical findings were evaluated. Selected patients were brought to the base hospital for excisional biopsy. Serial sections obtained from the excised nodules were examined for the presence of a parasite. MAIN OUTCOME MEASURES: Histopathologic examination of excised conjunctival lesions or response of lesions to local medical therapy. RESULTS: In this year-long prospective study, 41 children (16 years or younger; 38 boys and 3 girls) with clinical features of allergic conjunctival granulomas were examined. Thirty-four patients were from a single village located in the southern Indian state of Tamil Nadu; the remaining 7 were from various parts of the same state. All children swam in their village's freshwater pond. Twenty patients with nodules less than 5 mm in diameter received medical treatment; 13 with larger nodules underwent surgical excision of the lesions. Nine of these 13 cases revealed a zonal granulomatous inflammation admixed with eosinophilic leukocytes; 4 of these 9 displayed fragments of the tegument and internal structures of a trematode and Splendore-Hoeppli phenomenon. The remaining 4 of the 13 cases revealed nongranulomatous inflammation made up of lymphocytes, histiocytes, and eosinophils. Eight patients refused surgical treatment. CONCLUSIONS: In southern India, one cause of allergic conjunctival granulomas in children seems to be trematode infection. The clustering of cases in a single village and exposure to a village freshwater pond indicate the need for an epidemiologic investigation and study of the parasite's life cycle. Sporadic cases from other parts of the state with similar histories of exposure to their local pond or river water suggest a widespread distribution of the etiologic agent.

Depigmented atrophic lesions in sunset glow fundi of Vogt-Koyanagi-Harada disease.

PURPOSE: Although the depigmented, small, round to oval lesions seen in the sunset glow fundi of Vogt-Koyanagi-Harada disease are considered to represent Dalén-Fuchs nodules, there is no histopathologic evidence to support such a consideration. An attempt is made herein to clarify the nature of the atrophic lesions and distinguish them from Dalén-Fuchs nodules seen in eyes with Vogt-Koyanagi-Harada disease. METHODS: Eyes from five individuals with clinical diagnoses of Vogt-Koyanagi-Harada disease were subjected to histopathologic examination. The retinal pigment epithelial changes from early active to convalescent and late chronic recurrent stages were evaluated. Particular attention was paid to Dalén-Fuchs nodules, depigmented lesions in the sunset glow fundi, and hyperpigmentation of the chronic recurrent stage. RESULTS: Eyes of two individuals, one in the active stage of Vogt-Koyanagi-Harada disease and the other in the convalescent stage, showed the presence of Dalén-Fuchs nodules. The depigmented small retinal pigment epithelial lesions were seen in two individuals, both of whom exhibited the sunset glow fundus of the convalescent stage. The retinal pigment epithelial lesions represented damage or disappearance of retinal pigment epithelial cells, and the sunset glow fundus appearance was from the loss of choroidal melanocytes. The heavy pigmentation seen in fundi with the chronic recurrent stage was the result of the proliferation of retinal pigment epithelial cells. CONCLUSION: The Dalén-Fuchs nodule is a specific histologic change observed at the level of retinal pigment epithelium in patients with Vogt-Koyanagi-Harada disease. There is no histologic confirmation that the depigmented small atrophic lesions seen in the sunset glow fundi of Vogt-Koyanagi-Harada disease are Dalén-Fuchs nodules. The depigmented lesions represent localized damage or disappearance of retinal pigment epithelial cells.

Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: report of an international committee on nomenclature.

PURPOSE: To present revised criteria for the diagnosis of Vogt-Koyanagi-Harada disease, a chronic, bilateral, granulomatous ocular and multisystem inflammatory condition of unknown cause. METHODS: Diagnostic criteria and nomenclature were subjects of discussion at the First International Workshop on Vogt-Koyanagi-Harada Disease on October 19-21, 1999, at the University of California, Los Angeles, Conference Center, Lake Arrowhead, California. A committee appointed by the workshop participants was charged with drafting revised criteria for Vogt-Koyanagi-Harada disease, based on discussions held during the conference. This article is the consensus committee report. RESULTS: New criteria, taking into account the multisystem nature of Vogt-Koyanagi-Harada disease, with allowance for the different ocular findings present in the early and late stages of the disease, were formulated and agreed upon by the committee. These criteria are based on additional knowledge and experience of experts in the field and are believed to reflect disease features more fully than previously published criteria. CONCLUSIONS: The revised definition of Vogt-Koyanagi-Harada disease, with expanded diagnostic criteria, will facilitate performance of studies involving homogeneous populations of patients, at various stages of disease, that address unanswered questions regarding treatment and disease mechanisms.