DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

The patient presenting with decompensated cirrhosis.

The rates of liver disease in the UK are rising and hence more patients than ever are presenting to acute medical units with potentially life threatening sequelae. Early recognition and treatment of sepsis, kidney injury, bleeding and alcoholic hepatitis can significantly improve outcomes, but requires a comprehensive approach to assessment. This patient cohort often suffers from a perceived uniform poor prognosis, especially in alcohol related disease, but evidence for this is changing and reassessment of prognosis after 48 hours of organ support may be more accurate than that made 'at the front door'. This article summarises the most important complications of decompensated cirrhosis, their early management, and presents a targeted system of care: 'RING Liver'--Renal failure, Infection, Nutrition, Gastrointestinal bleeding and transit, Liver dysfunction/transplantation. Factors favouring transfer to tertiary units are also explored.

Review article: towards a considered and ethical approach to organ support in critically-ill patients with cirrhosis.

BACKGROUND: Increasing numbers of patients are being admitted to hospital with decompensated chronic liver disease in the UK. A significant proportion will develop complicating extra-hepatic organ dysfunction, but the selection of those who should be admitted to intensive care is complex and challenging. Alcohol-related liver disease also presents complex ethical dilemmas. AIM: To review recent survival analyses and explore differences in secondary and tertiary care; to highlight strengths and weaknesses of prognostic models, therapeutic advances and shifts in prognostic expectation. We also aim to explore the ethical challenges presented by addiction and self-injury in an area of limited resource. METHODS: We searched PubMed for articles discussing 'cirrhosis', 'prognosis', 'critical illness', 'organ failure', 'renal failure', 'alcohol', 'ethics' and 'addiction'. We also explored particular ethical dilemmas encountered by the authors and colleagues. RESULTS: Prognosis has improved in many cirrhotic complications and historically poor outcomes in tertiary care may reflect a more complex patient cohort. Previously 'untreatable' complications are now being managed successfully. Estimates of survival are more accurate after a 48-h period of supportive care. Physicians are not best placed to make judgments with regard to deservingness, moral responsibility, rationing and access to organ support in cases of acute deterioration related to alcoholism, and the case for denying support must be made on purely medical grounds. CONCLUSIONS: An early, aggressive approach to organ support is justified. Further discussions between hepatologists and critical care physicians are required to determine acceptable burden-to-benefit ratios for prolonged intensive care support in young alcoholic patients.

High-power, widely-tunable Cr(2+):ZnSemaster oscillator power amplifier systems.

We demonstrate high-power Cr(2+):ZnSe master oscillator power amplifier (MOPA) pure continuous wave (CW) laser systems with output power of 14 W and amplifier gain greater than 2X. In addition, we develop a theoretical model for this type of amplification and show single-knob tunability at high powers over 400 nm.

Admission levels and early changes in serum interleukin-10 are predictive of poor outcome in acute liver failure and decompensated cirrhosis.

BACKGROUND & AIM: Immunoparesis contributes to prognosis in acute liver failure (ALF) and decompensated cirrhosis, a phenomenon thought to be mediated by the anti-inflammatory cytokine interleukin (IL)-10. We investigated the prognostic value of admission IL-10 levels and their evolution during the early phase of treatment in intensive care, in comparison to the pro-inflammatory cytokines IL-6 and tumour necrosis factor (TNF)-alpha. METHODS: We measured these cytokines within 48 h of admission in 51 ALF and 39 decompensated cirrhosis patients admitted to intensive care, and obtained follow-up measurement a median of 2 days later in 35 patients. RESULTS: Levels of all cytokines were higher in those with a poor outcome. IL-10 performed as well as TNF-alpha and IL-6 in the whole cohort (area under receiver operator curve 0.73 vs 0.66 and 0.72). However IL-10 outperfomed pro-inflammatory cytokines in the subgroups with ALF (0.80 vs 0.63 and 0.70) and acetaminophen-induced ALF (0.92 vs 0.67 and 0.81). Levels of all cytokines rose significantly in non-surviving patients (n=15); IL-10 by a factor of 2, TNF-alpha by 2.6 and IL-6 by 1.13. No significant changes were seen in the surviving patients. In ALF, IL-10 was an independent predictor of outcome in multivariate analysis. CONCLUSION: The magnitude of the compensatory anti-inflammatory response at admission, and its development during the early phase of treatment, predicts outcome as well as the pro-inflammatory response in acute hepatic syndromes and supports a vital role for this immunological phenomenon in the outcome of these patients.

Multiple organ failure and severe bone marrow dysfunction in two 18 year-old Caucasian patients: Epstein-Barr virus and the haemophagocytic syndrome.

Haemophagocytic lymphohistiocytosis secondary to viral infection is an unusual but well recognised cause of bone marrow dysfunction and multiple organ failure in young patients. Two 18 year-old patients were admitted to a tertiary liver unit with features of acute liver failure, cardio-respiratory collapse and pancytopenia. Serological tests and bone marrow examination with in-situ hybridisation revealed severe acquired haemophagocytic lymphohistiocytosis secondary to acute Epstein-Barr virus infection. Both patients died despite full supportive therapy; the first due to pulmonary haemorrhage, the second due to acute respiratory distress syndrome refractory to high frequency oscillatory ventilation. The clinical spectrum, diagnostic features and current evidence based recommendations for treatment of this condition are explored. The diagnosis of haemophagocytic lymphohistiocytosis should be considered in young patients with marked bone marrow dysfunction and multiple organ failure. Further research into appropriate therapy for patients with acute severe forms of the disease who require intensive organ support is required.

Vastus medialis cross-sectional area is associated with patella cartilage defects and bone volume in healthy women.

OBJECTIVES: Although vastus medialis and vastus lateralis are important muscular determinants of patellofemoral joint function, it is unclear how these muscles relate to the structure of the patellofemoral joint. The aim of this cross-sectional study was to determine the relationship between the vasti muscles and patella cartilage volume and defects and patella bone volume. METHODS: One hundred and seventy-five women, aged 40-67 years, with no knee pain or clinical lower-limb disease had magnetic resonance imaging (MRI) of their dominant knee. The cross-sectional areas of the distal vastus medialis and lateralis were measured 37.5mm superior to the quadriceps tendon insertion at the proximal pole of the patella. Patella cartilage volume and defects and patella bone volume were measured from these images using validated methods. RESULTS: There was no significant association between the distal vastus medialis cross-sectional area and patella cartilage volume. For every 1mm(2) increase in the distal vastus medialis cross-sectional area, there was an associated increased risk of patella cartilage defects [odds ratio (OR): 1.2; 95% confidence interval (CI) 1.004, 1.5; P=0.05], and an associated increase in patella bone volume (OR: 3.9; 95% CI 2.0, 5.8; P<0.001) after adjustment for potential confounders. There was no significant relationship between vastus lateralis cross-sectional area and measures of patella cartilage or bone. CONCLUSION: An increased cross-sectional area of the distal portion of the vastus medialis muscle is associated with an increased risk of patella cartilage defects, and an increase in patella bone volume among healthy women. Although these results need to be confirmed in longitudinal studies, they suggest that an increase in the distal vastus medialis cross-sectional area is associated with structural change at the patellofemoral joint.

Hepatitis A vaccine associated with autoimmune hepatitis.

To describe a case of probable relapsing autoimmune hepatitis associated with vaccination against hepatitis A virus (HAV). A case report and review of literature were written concerning autoimmune hepatitis in association with hepatitis A and other hepatotropic viruses. Soon after the administration of formalin-inactivated hepatitis A vaccine, a man who had recently recovered from an uncharacterized but self-limiting hepatitic illness, experienced a severe deterioration (AST 1687 U/L, INR 1.4). Anti-nuclear antibodies were detectable, and liver biopsy was compatible with autoimmune hepatitis. The observation supports the role of HAV as a trigger of autoimmune hepatitis. Studies in helper T-cell activity and antibody expression against hepatic proteins in the context of hepatitis A infection are summarized, and the concept of molecular mimicry with regard to other forms of viral hepatitis and autoimmunity is briefly explored.

Decreased wild-type full-length Et-A and -B receptors in neuroblastoma and Ewing sarcoma cells.

BACKGROUND: Endothelins and their receptors, Et-A and Et-B, play an essential role in differentiation and migration of neural crest cells. Expression of endothelin receptors has been examined in neuroblastoma and Ewing sarcoma cell lines. PROCEDURE: RNA was amplified for Et-A and Et-B by RT-PCR. Amplified products were cloned into the expression vector pLNCX, which was used to transfect CHO cells. Binding characteristics of transfected CHO cells were examined. RESULTS: Full-length Et-A mRNA was identified in all cell lines, in addition to a truncated Et-A product. CHO cells expressing full-length Et-A bound to endothelin, but cells expressing truncated Et-A did not. Full length Et-B mRNA was not detected, but two smaller molecular weight products were amplified. These are as yet uncharacterised. CONCLUSIONS: These results suggest that endothelins and their receptors may be important in the development and biology of neuroblastoma and Ewing sarcoma.

Truncated human endothelin receptor A produced by alternative splicing and its expression in melanoma.

In this study, reverse transcriptase polymerase chain reaction was used to amplify human endothelin receptor A (ETA) and ETB receptor mRNA. A truncated ETA receptor transcript with exons 3 and 4 skipped was found. The skipping of these two exons results in 109 amino acids being deleted from the receptor. The truncated receptor was expressed in all tissues and cells examined, but the level of expression varied. In melanoma cell lines and melanoma tissues, the truncated receptor gene was the major species, whereas the wild-type ETA was predominant in other tissues. A 1.9-kb ETA transcript was identified in melanoma cell lines by Northern blot, which was much smaller than the transcript in heart and in other tissues reported previously (4.3 kb). The cDNA coding regions of the truncated and wild-type ETA receptors were stably transfected into Chinese hamster ovary (CHO) cells. The truncated ETA receptor-transfected CHO cells did not show binding affinity to endothelin 1 (ET-1) or endothelin 3 (ET-3). The function and biological significance of this truncated ETA receptor is not clear, but it may have regulatory roles for cell responses to ETs.

Contrasting levels of p21ras activation and expression of neurofibromin in peripheral primitive neuroectodermal tumour and neuroblastoma cells, and their response to retinoic acid.

Ras protooncogenes encode small guanine nucleotide binding proteins (p21ras) activated by phosphorylation. Phosphorylation of p21ras is predominantly regulated by the GTPase activating proteins type 1 GAP120 and neurofibromin. Increased levels of p21ras-GTP (active) have been associated with increased cell growth and malignant transformation. In this study the relationship between p21ras, type 1 GAP120 and neurofibromin with growth and differentiation has been examined in neuroblastoma and peripheral primitive neuroectodermal tumour (pPNET) cell lines. The level of p21ras protein in neuroblastoma and pPNET cells was the same. However, the amount of p21ras-GTP bound was higher in pPNET than in neuroblastoma cells. This most likely reflects the absence of neurofibromin. Retinoic acid (RA)-induced differentiation and growth inhibition of neuroblastoma cells was associated with an increase in type 1 GAP120 and neurofibromin mRNA, and a decrease in p21ras-GTP. In pPNET cells levels of type 1 GAP120 but not neurofibromin mRNA were increased to similar levels to those in neuroblastoma cells. This was not associated with decreased p21ras-GTP, modulation of growth or change in morphology. In summary, constitutive activation of p21ras may have a role in the biology of pPNET cells. This may reflect abnormalities in neurofibromin expression, and could inpart explain why RA did not induce morphological differentiation and growth inhibition in pPNETs.

Activation of p21ras by nerve growth factor in neuroblastoma cells.

Nerve growth factor (NGF) is essential for the differentiation and survival of sympathetic and sensory neurones and is thought to play a role in the differentiation of neuroblastoma. In this study we have shown NGF decreased the mRNA level of the two GTPase activating proteins neurofibromin (containing the NF1-GRD) and type 1 GAP120 in two neuroblastoma cell lines, IMR-32 and SK-N-SH. This effect was seen within 15 min exposure to NGF and was maintained up to 2 h after the addition of NGF. Treatment with NGF increased the amount of GTP bound p21ras 3-fold, within 20 min exposure. Western blot analysis showed SK-N-SH and IMR-32 cells to contain equal amounts of p21ras protein and these levels were unchanged by NGF treatment. However, NGF induced an increase in the level of neurofilament L protein, which was accompanied by an increase in neurite extension. These effects of NGF occurred in the absence of growth inhibition. In conclusion, our results demonstrate a decrease in GTPase activating proteins and activation of p21ras by NGF in IMR-32 and SK-N-SH cells, thus implicating p21ras in NGF signal transduction in neuroblastoma.

Effect of retinoic acid on p21ras and regulators of its activity in neuroblastoma.

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. This protein is important in the regulation of cell growth and differentiation in a number of different cell types. Therefore, the aim of the present study was to examine the role of p21ras and regulators of its activity in the differentiation of neuroblastoma cells induced by retinoic acid (RA). Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and neurofibromin. RA-induced differentiation of the two neuroblastoma cell lines SK-N-SH and IMR-32 was closely related to growth inhibition. Differentiation induced by RA resulted in an increase in both type I GAP120 and neurofibromin mRNAs. This increase was accompanied by a decrease in the activation of p21ras. These results suggest that, in neuroblastoma, activation of p21ras is not associated with RA-induced differentiation. However, the GTPase activating proteins type I GAP120 and neurofibromin may have effector functions in RA-induced differentiation of neuroblastoma.

Changing expression of GTPase activating proteins with differentiation in neuroblastoma.

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. Like other guanine nucleotide-binding proteins p21ras is active when GTP bound and inactive when GDP bound. Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and NF1-GRD. In this study we have identified type I GAP120 and two NF1-GRD mRNAs in three neuroblastoma cell lines, IMR-32, SK-N-SH and SK-N-MC. NF1-GRD mRNA was expressed in all cell lines at a similar level but type I GAP120 mRNA was more abundant in the IMR-32 cell line. Retinoic acid induced differentiation of all three cell lines, this effect was most marked in the SK-N-SH line. This differentiation was accompanied by an increase in both type I GAP120 and NF1-GRD mRNAs. Retinoic acid induced differentiation had no effect on the ratio of type I to type II NF1-GRD mRNA. In seven patient tumour samples examined type I GAP120 and NF1-GRD were coexpressed, type I GAP120 at a higher level than NF1-GRD in all tumour stages. Type I was the predominant NF1-GRD mRNA. The expression of type I GAP120 was similar in all tumour stages but the total level of NF1-GRD was higher in stage 2 and 3 tumours than in stage 4 tumours. In summary, these results suggest increased type I GAP120 and NF1-GRD mRNA are associated with differentiation in neuroblastoma cells.

Distribution of dopamine-containing neurons and fibres in the feline medulla oblongata: a comparative study using catecholamine-synthesizing enzyme and dopamine immunohistochemistry.

The distribution of dopamine-immunoreactive neurons and fibres in the feline medulla oblongata was examined by immunocytochemistry with antisera to the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase, and with antisera to the catecholamines dopamine and L-dihydroxyphenylalanine. Neurons immunoreactive for the catecholamine-synthesizing enzymes were found in two regions of the medulla, the ventrolateral A1 region and the dorsomedial A2 region. Double-staining studies with antisera to the enzymes indicated that a population of neurons within both regions were immunoreactive for tyrosine hydroxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase, implying that they synthesize dopamine. Studies using the dopamine antisera demonstrated the presence of dopamine-immunoreactive neurons in both the ventrolateral and dorsomedial regions of the medulla; in the dorsomedial region, they were found in the area postrema, nucleus tractus solitarius and dorsal motor vagal nucleus, mainly at levels caudal to the obex. Dopamine-immunoreactive fibres were found in several areas of the medulla including the nucleus tractus solitarius, inferior olive, dorsal motor vagal, spinal trigeminal, hypoglossal, cuneate, gracile, and raphe nuclei. Double-staining studies with antisera to dopamine and dopamine-beta-hydroxylase revealed a population of cells immunoreactive for dopamine alone. The presence of some double-stained neurons, however, implies some cross-reactivity of the dopamine antiserum with noradrenaline or adrenaline and/or recognition of dopamine present as a metabolic intermediary in some noradrenergic neurons. No L-dihydroxyphenylalanine-immunoreactive neurons were found in the medulla, although fibres were seen. These data provide evidence for the existence of catecholamine neurons which utilize dopamine as a final synthetic product within the medulla oblongata.

Immunolocalization of catecholamine enzymes, serotonin, dopamine and L-dopa in the brain of Dicentrarchus labrax (Teleostei).

Antisera to serotonin (5-HT), dopamine, and L-dopa, and to the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT), were used to localize monoamine containing neurones in the brain of Dicentrarchus labrax (sea bass). In the brain stem, 5-HT-immunoreactive (ir) neurones were recognized in the ventrolateral medulla, vagal motor area, medullary, and mesencephalic raphe nuclei and in the dorsolateral isthmal tegmentum. In the hypothalamus, liquor-contacting 5-HT neurones were seen in various regions of the paraventricular organ. Virtually all regions of the brain contained a dense innervation by 5-HT fibres and terminals. DBH-ir neurones were restricted to three brain stem areas: the locus coeruleus, the area postrema, and the reticular formation of the lower medulla. Neurones in these three groups also displayed TH-ir, and in the latter area, PNMT-ir in addition. In the locus coeruleus and area postrema, TH-ir neurones outnumbered DBH-ir neurones, an observation substantiated by the presence of dopamine-ir neurones. In the forebrain, dopamine- and TH-ir neurones were found in the olfactory bulb, ventral/central telencephalon, periventricular preoptic, and suprachiasmatic areas, dorsolateral and ventromedial thalamus, and posterior tuberal nucleus. In the paraventricular organ, the distribution and morphology of dopamine-ir neurones was similar to that observed with anti-5-HT, but the vast majority of cells were not TH-ir, suggesting accumulation of dopamine by uptake from the ventricle, rather than by synthesis. L-dopa-ir neurones were found only in the central telencephalon, preoptic recess, and dorsolateral thalamus. Fibres and terminals immunoreactive for dopamine, TH, and DBH showed a broadly similar distribution. The results are discussed in relation to the monoaminergic systems previously reported in other teleostean species and the mammalian brain.