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.

Preference for rizatriptan 10-mg wafer vs. eletriptan 40-mg tablet for acute treatment of migraine.

Preference is a composite, patient-oriented endpoint incorporating efficacy, tolerability, formulation, and convenience of medications. The objective of this study was to compare patient preference for rizatriptan 10-mg wafer vs. eletriptan 40-mg tablet for acute treatment of migraine. In this multicentre, open-label, two-period, crossover study, out-patients were randomly assigned to treat the first of two moderate to severe migraines with rizatriptan or eletriptan and the second with the alternate therapy. Patients completed diary assessments at baseline and up to 24 h after taking study medication. At the last visit, patients completed a psychometrically validated preference questionnaire. A total of 372 patients (mean age 38 years, 85% female) treated two migraine attacks, and 342 patients (92%) expressed a preference for treatment. Significantly more (P < or = 0.001) patients preferred rizatriptan 10-mg wafer [61.1%; 95% confidence interval (CI) 55.7, 66.3] to eletriptan 40-mg tablet (38.9%; 95% CI 33.7, 44.3). The most common reason given for preference of either treatment was speed of headache relief. At 2 h, 80% and 69% of patients reported that rizatriptan and eletriptan, respectively, was convenient or very convenient to take (mean convenience score 1.99 vs. 2.31, respectively; P < or = 0.001). Both triptans were well tolerated. In this head-to-head study designed to evaluate global patient preference, significantly more patients preferred the rizatriptan 10-mg wafer to the eletriptan 40-mg tablet for acute treatment of migraine. The single most important reason for preference was speed of relief, consistent with results from previous preference studies.

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.

Prevalence of pulmonary tuberculosis in Karachi central prison.

OBJECTIVE: To determine the extent of pulmonary tuberculosis among prisoners in Karachi central jail, so that strategy for targeted intervention can be planned. METHOD: This prospective observational study was done at Karachi central prison from 7-2-2002 to 14-2-2002. A team of doctors, laboratory and x-ray technicians visited the central prison. Patients who had symptoms suggestive of pulmonary tuberculosis were included in the study. Their chest x-ray was taken in the jail and three "spot specimens" of sputum were collected for three consecutive days. The sputum specimens were processed at the laboratory of Ojha Institute of Chest Diseases, Karachi. RESULTS: Out of 4870 prisoners, 79 (1.62%) were pulmonary tuberculosis suspects. All were male and their mean age was 32 (22-75) years. Sixteen suspects already diagnosed were on anti-tuberculosis treatment (ATT) while 11 suspects gave history of ATT in the past for incomplete duration varying from 3-4 weeks to 3 months either regularly or irregularly. Twenty-two (28%) suspects were not expectorating while fifty-seven (72%) submitted the sputum for AFB (Acid Fast Bacilli), of which only one was smear positive. Radiologically, 39 (49%) chest x-rays including those of 22 who were not expectorating were normal. Eight (10%) showed healed lesion. Thirty-two (40.5%) chest x-rays were suggestive of active pulmonary tuberculosis, so clinically and radiologically 32 prisoners were suffering from active pulmonary tuberculosis. The prevalence of active pulmonary tuberculosis in jail population determined by using the formula, number of persons with active TB in jail divided by the total number of persons booked into jail was 657 per 100,000, which is 3.75 times higher than general population. CONCLUSION: Pulmonary tuberculosis is 3.75 times more common than general population in Karachi central prison and concrete efforts are needed to eradicate tuberculosis from this segment of population. The integration of provincial TB control program with that of jail health services is urgently required.

Polymerase chain reaction analysis of aqueous humour samples in necrotising retinitis.

AIM: To evaluate the diagnostic value of polymerase chain reaction (PCR) performed on aqueous humour for the detection of viral DNA in patients with necrotising herpetic retinitis. METHODS: The clinical features and laboratory results of 22 patients (29 eyes) presenting with necrotising herpetic retinitis between March 1999 and June 2001 were reviewed retrospectively. Aqueous humour was obtained after anterior chamber paracentesis and PCR was performed in all cases. RESULTS: Viral DNA was detected in the aqueous humour of 19 patients (86.4%). Epstein-Barr virus (EBV) seroconversion was evidenced in one additional patient. In the acute retinal necrosis (ARN) group (n = 19), varicella zoster virus (VZV) DNA was identified in six patients, herpes simplex virus 1 (HSV-1) DNA in two patients, herpes simplex virus 2 (HSV-2) DNA in four patients, and cytomegalovirus (CMV) genome in four patients. In the progressive outer retinal necrosis (PORN) group (n = 3), VZV DNA was detected in all patients. No sample was positive for more than one virus. CONCLUSIONS: PCR analysis of aqueous humour in patients with clinical features of necrotising viral retinitis can provide specific aetiological orientation and the method appears to be safe and highly sensitive.