X-ray microtomography study of otic capsule deficiencies: three-dimensional modelling of the fissula ante fenestram.

BACKGROUND: The postulated sites of perilymph fistulae involve otic capsule deficiencies, in particular, at the fissula ante fenestram. Histological studies have revealed this to be a channel extending from the middle ear, and becoming continuous with the inner ear medial to the anterior limit of the oval window. The relationship between a patent fissula and symptoms of perilymph fistula is contentious. OBJECTIVE: The understanding of the anatomy of the fissula ante fenestram is incomplete. Histopathology is inherently destructive to the delicate ultrastructure of the middle and inner ear. Conversely, X-ray microtomography allows non-destructive examination of the otic capsule. In this study, we used X-ray microtomography to characterise the fissula ante fenestram. MATERIALS AND METHODS: We imaged cadaveric temporal bones with X-ray microtomography. We used the Avizo Fire (Visualization Science Group, Merignac Cedex, France) software to perform post-processing and image analysis. RESULTS: Three-dimensional modelling of the fissula ante fenestram allowed stratification into four forms: rudimentary pit; partial fissula; complete occluded fissula; and complete patent fissula. CONCLUSION: X-ray microtomography showed that the fissula ante fenestram is present in various forms from rudimentary pit to complete deficiency of the otic capsule. This understanding may have implications for otologic surgery and clinical diagnosis of perilymph fistula.

Endolymphatic hydrops is prevalent in the first weeks following cochlear implantation.

AIM: To explore morphological or electrophysiological evidence for the presence of endolymphatic hydrops (EH) in guinea pig cochleae in the first 3 months after cochlear implantation. METHODS: Dummy silastic electrodes were implanted atraumatically into the basal turn of scala tympani via a cochleostomy. Round window electrocochleography (ECochG) was undertaken prior to and after implantation. Animals survived for 1, 7, 28 or 72 days prior to a terminal experiment, when ECochG was repeated. The cochleae were imaged using micro-CT after post-fixing with osmium tetroxide to reveal the inner ear soft tissue structure. EH was assessed by visual inspection at a series of frequency specific places along the length of the cochlea, and the extent to which Reissner's membrane departed from its neutral position was quantified. Tissue response volumes were calculated. Using ECochG, the ratio of the summating potential to the action potential (SP/AP ratio) was calculated in response to frequencies between 2 and 32 kHz. RESULTS: There was minimal evidence of electrode trauma from cochlear implantation on micro-CT imaging. Tissue response volumes did not change over time. EH was most prevalent 7 days after surgery in implanted ears, as determined by visual inspection. Scala media areas were increased, as expected in cases of EH, over the first month after cochlear implantation. SP/AP ratios decreased immediately after surgery, but were elevated 1 and 7 days after implantation. CONCLUSIONS: EH is prevalent in the first weeks after implant surgery, even in the absence of significant electrode insertion trauma.

[Cancer incidence among petrochemical workers in the Porto Torres industrial area, 1990-2006]. / Incidenza di patologie neoplastiche nei lavoratori del complesso petrolchimico di Porto Torres, 1990-2006.

BACKGROUND: Various epidemiological studies explored cancer mortality and incidence among petrochemical workers. We followed up cancer incidence in a cohort of 5350 male petrochemical workers in the industrial area of Porto Torres (Sardinia, Italy). MATERIAL AND METHODS: The follow-up covered the period from 01/01/1990, when completeness of the cohort was certain and reference rates by the local Cancer Registry became available, up to 31/12/2006. Cohort members were subjects employed for six months or more in one of the chemical plants of the industrial area, alive as at 01/01/1990. Overall, a total of 81,392 person-years at risk were accumulated. The standardized incidence ratio (sir), as the ratio of observed to expected events, and its 95% confidence interval (CI) were calculated for all cancers and selected cancer sites, in the total cohort and in sub-cohorts of workers in plants where exposure to chemical agents evaluated in the IARC Monographs might have occurred. RESULTS: An increase in risk for all cancers was observed in the total cohort (596 cases; sir = 1.09; 95% CI 1.00-1.18), and it was highest for non-Hodgkin lymphoma (NHL, 26 cases: sir = 1.78; 95% CI 1.22-2.62). Risk for haemolymphatic cancer was highest in the sub-cohort of workers employed for 10 years or more, with a latency period of 20 years or longer, and among those employed in the manufacture and polymerization of vinyl chloride (VCM; all cancers, 51 cases: sir = 1.43; 95% CI 1.08-1.88; NHL, 4 cases: sir=4.06; 95% CI 1.64-10.0). Risk of haemolymphatic cancer was not significantly elevated in the sub-cohort potentially exposed to benzene. An excess risk of bladder cancer (RR = 1.46; 95% CI 1.09-1.96), but not of pleural cancer, was observed in the sub-cohort potentially exposed to asbestos. No significant increase in cancer risk was observed among workers potentially exposed to acrylonitrile, butadiene, or styrene. CONCLUSIONS: Our follow-up study of petrochemical workers showed an increase in risk for all cancers, and particularly NHL, apparently concentrated among workers potentially exposed to VCM

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy.

Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N- and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1(W437X), interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1(G309D), a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process.

Immunogenicity of allo-vesicle carrying ERBB2 tumor antigen for dendritic cell-based anti-tumor immunotherapy.

Dendritic cells (DCs) are able to orchestrate innate and acquired immunity and can activate and sustain a long-lasting anti-tumor immune response in vivo when used as anti-tumor cell therapy. The selection of the antigen and the choice of its formulation are key points in designing anti-cancer DC-based vaccines. Cell released vesicles/exosomes have been shown to transfer antigens, HLAI/peptide complexes and co-stimulatory molecules to recipient cells. In this study we describe the generation of an allogenic microvesicle cell factory in which the expression of a specific tumor antigen was combined to the expression of co-stimulatory and allogeneic molecules. The DG75 lymphoblastoid cell line was selected as microvesicle producer and transfected with ErbB2, as tumor antigen prototype. The shed microvesicles transferred antigenic components to recipient DCs, increasing their immunogenicity. DC pulsing resulted in cross-presentation of ErbB2 both in HLAI and HLAII compartments, and ErbB2-specific CD8+ T cells from cancer patients were activated by DCs pulsed with vesicle-bound ErbB2. The microvesicle cell factory proposed may represent a source of cell free immunogen to be used for DC-based cancer therapy.

Lipido-sterolic extract of Serenoa repens (LSESr, Permixon) treatment affects human prostate cancer cell membrane organization.

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.

Type-3 metabotropic glutamate receptors negatively modulate bone morphogenetic protein receptor signaling and support the tumourigenic potential of glioma-initiating cells.

Targeted-therapies enhancing differentiation of glioma-initiating cells (GICs) are potential innovative approaches to the treatment of malignant gliomas. These cells support tumour growth and recurrence and are resistant to radiotherapy and chemotherapy. We have found that GICs express mGlu3 metabotropic glutamate receptors. Activation of these receptors sustained the undifferentiated state of GICs in culture by negatively modulating the action of bone morphogenetic proteins, which physiologically signal through the phosphorylation of the transcription factors, Smads. The cross-talk between mGlu3 receptors and BMP receptors was mediated by the activation of the mitogen-activated protein kinase pathway. Remarkably, pharmacological blockade of mGlu3 receptors stimulated the differentiation of cultured GICs into astrocytes, an effect that appeared to be long lasting, independent of the growth conditions, and irreversible. In in vivo experiments, a 3-month treatment with the brain-permeant mGlu receptor antagonist, LY341495 limited the growth of infiltrating brain tumours originating from GICs implanted into the brain parenchyma of nude mice. While clusters of tumour cells were consistently found in the brain of control mice, they were virtually absent in a large proportion of mice treated with LY341495. These findings pave the way to a new non-cytotoxic treatment of malignant gliomas based on the use of mGlu3 receptor antagonists.

Effects of long-term treatment with the anti-androgen bicalutamide on human testis: an ultrastructural and morphometric study.

AIMS: To assess the effects of more than 4 years' treatment with the anti-androgen bicalutamide on human testis by clinical, ultrastructural and morphometric analysis. METHODS AND RESULTS: Two patients (aged 74 and 69 years) with prostate cancer were treated for more than 4 years with bicalutamide 50 mg daily. Clinical characterization and follow-up included luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and prostate-specific antigen (PSA) measurements and clinical response of the tumours. Due to progression of the disease, patients underwent surgical orchidectomy as a further androgen withdrawal therapy. Testis biopsies were studied by light and electron microscopy and analysed by morphometry. Control samples were obtained from the normal testis of two patients with testicular cancer who underwent orchidectomy. Clinical follow-up showed a good response in the control of tumour growth and serum PSA decreased to < 4 ng/mL; concentrations of serum LH, FSH and testosterone were within the normal range. Testicular morphology of treated patients was unexpectedly well preserved; the organization of seminiferous tubules was normal with all the germ line elements and mature spermatozoa present. In some areas, a net increase of peritubular connective tissue was evident which may be a consequence of the age of the patients. CONCLUSIONS: Long-term bicalutamide (50 mg) treatment appears to have very little impact on testis ultrastructure and sperm maturation, while it is effective in the control of androgen-dependent prostatic tumours.

The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression.

Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties.

Heat shock protein-90, IL-6 and IL-10 in bladder cancer.

The progression of transitional-cell carcinomas of the bladder is associated with changes in general and local immune status. To understand the factors involved in the progression of transitional cell carcinoma and in the maintenance of an efficient anti-tumoural response, in this study we investigated by immunohistochemistry expression of HSP-90, IL-6 and IL-10 proteins in 56 surgical specimens obtained from superficial and deeply invasive bladder carcinomas. Of the 56 bladder carcinoma 52 (92.9%) expressed HSP-90, 48 (85.7%) IL-6 and 45 (80.3%) IL-10. High-grade and muscle-invasive tumours contained significantly higher levels of HSP-90 and IL-6 antibodies than low-grade and superficial tumours (p < 0.05). Linear regression showed a significant correlation between HSP-90 and IL-10 (p = 0.022) but not between HSP-90 and IL-6, or IL-6 and IL-10 expression. The variable quantities of HSP-90, IL-6 and IL-10 in the high-grade bladder carcinomas studied suggest that these proteins have independent functional roles and may be the immunogenic targets for an anti-tumoural response.

Nitric oxide synthases in normal and benign hyperplastic human prostate: immunohistochemistry and molecular biology.

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The inducible NOS (iNOS or NOS-2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co-expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium.

Use and costs of incontinence pads in female study volunteers. Continence Program for Women Research Group.

OBJECTIVES: We examined the use and cost of incontinence pads and the relationship to factors such as age, duration of incontinence, diurnal frequency, incontinence severity indices, urodynamic diagnosis, and quality of life. SUBJECTS AND SETTING: Three hundred fifteen women with urinary incontinence who volunteered to participate in 1 of 3 incontinence studies (behavioral intervention, estrogen supplementation, or surgery) were analyzed. Subjects were community-dwelling women aged 45 years and older living in 3 cities in the southeastern United States. METHODS: Pad use was recorded on a daily diary. The type of pads used was reported on the history. Average price of pad types was assessed at local stores and reported in 1995 dollars. Statistical comparisons used nonparametric methods. MAIN OUTCOME MEASURES: The number of pads used per week and annual cost of pads in 1995 dollars. RESULTS: Seventy-seven percent of subjects used pads at baseline. Median cost per year for the entire cohort was $46 (interquartile range $3-$138). For pad users, median annual cost was $76 (interquartile range $36-$177), with costs being greater for women with detrusor instability than those with pure genuine stress incontinence (median $135-$138 versus $63). This increased cost was likely associated with the greater use of special incontinence products among women with detrusor instability. For the entire cohort, cost and usage did not differ by urodynamic diagnosis. Cost and pad usage were significantly associated with number of incontinent episodes and quality of life, but not with age, pad weight, or duration of incontinence. CONCLUSIONS: The majority of incontinent women who sought treatment used absorbent pads at least once per week, with menstrual pads being the most common type of pad. The annual cost of pad usage was not as high as in previous estimates.

Hepatocellular carcinoma associated with oral contraceptive use and pregnancy.

Numerous undesirable side effects have been attributed to oral contraception, from mild breast discomfort to thromboembolism. The authors present a case report of hepatocellular carcinoma associated with oral contraceptive use and pregnancy and discuss the potentially fatal association of malignant liver tumors with usage of oral contraception.

PIP: The possible association of hepatocellular carcinoma with oral contraceptive (OC) use is supported by the case of a 33-year old black female, gravida 5, para 4. She presented in April 1978 with right upper quadrant pain, nausea, vomiting, and fatty food intolerance. The case had been taking norethindrone, 1 mg with mestranol 0.05, for 2 years. There was no history of liver disease, alcohol abuse, or exposure to chemical toxins. The preoperative diagnosis was subacute cholecystitis; however, an unresectable primary liver tumor of both lobes was detected on surgery. OC use was discontinued, and the case refused chemotherapy. On December 1, 1978, she presented with a 9-week pregnancy which was aborted. Physical examination revealed an enlarged liver and mass in the upper right quadrant. The patient was readmitted December 11 with intractable pain and discharged. She died December 28, 1978. At autopsy the liver tumor appeared as a moderate to poorly differentiated hepatoma with irregular hyperchromatic nuclei. There was no evidence of coexistent benign lesions. The rapid progression of the disease following pregnancy suggests that hepatic growth was stimulated by the high estrogen levels of pregnancy. Earlier diagnosis and improved management are required in such cases. Ultrasonography can be used to confirm the presence of a mass, and liver scan or hepatic angiogram may be useful. Liver biopsy is required for definitive diagnosis. Treatment involves discontinuation of OC use and complete excision of the tumor where possible. If tumors have progressed beyond the stage of resectability, as in this case, the prognosis is poor.