S-adenosyl-methionine-induced apoptosis in PC12 cells

Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.     

To IPD or not to IPD? Advantages and disadvantages of systematic reviews using individual patient data

Systematic reviews and meta-analyses that obtain original research data on individual participants enrolled in trials have been described as the gold standard of review. However, they may take longer and be more resource intensive than other types of review. The authors describe potential advantages and disadvantages of the individual patient data (IPD) approach, including benefits from improved data quality, benefits afforded by the type of analyses that can be done, and advantages in achieving consensus around results and interpretation by an international multidisciplinary team. Disadvantages and barriers relating to resource and expertise, negotiating collaboration, and software requirements are also discussed. At the outset, reviewers should consider the methodological factors likely to influence results in their particular review setting, together with time and resource constraints, so that an active decision can be made about whether to extract data from published reports, collect additional or replacement summary data from trialists, or collect IPD.     

Effect of renal impairment on the pharmacokinetics and tolerability of capecitabine (Xeloda) in cancer patients

PURPOSE: The primary objective of this study was to investigate the influence of renal impairment on the pharmacokinetics of capecitabine and its metabolites in cancer patients. Capecitabine (Xeloda) is an orally administered precursor of 5'-deoxy-5-fluorouridine (5'-DFUR), which is preferentially activated to 5-fluorouracil (5-FU) in tumors. METHODS: A total of 27 patients were enrolled, of whom 24 were evaluable for pharmacokinetics (6 with normal renal function, 8 with mild, 6 with moderate, and 4 with severe renal impairment at baseline). Patients received capecitabine orally at the standard dosing regimen (1250 mg/m(2) capecitabine twice daily for 2 weeks followed by a 1-week rest period). On study days 1 and 14, blood samples were collected to evaluate the pharmacokinetics of capecitabine and its metabolites. The relationship between the area under the plasma concentration-time curve (AUC) and creatinine clearance (CL(CR)) was assessed by log-linear regression analysis. RESULTS: The primary pharmacokinetic parameter with respect to the effect of renal dysfunction was systemic exposure to 5'-DFUR, 5-FU and FBAL determined on study day 14. Renal impairment led to an increase in the systemic exposure to 5'-DFUR and FBAL (23% and 109% increase in AUC, respectively) for a 50% reduction in CL(CR). By contrast, renal impairment may lead to decreased exposure to 5'-DFCR. There was no evidence for an effect of renal impairment on systemic exposure to 5-FU or capecitabine. Renal impairment did not have a major effect on peak concentration (C(max)) or elimination half-life (t(1/2)) of capecitabine, 5'-DFCR, 5'-DFUR, and 5-FU. However, in the case of FBAL, moderate or severe renal impairment caused up to a twofold increase in C(max) and prolongation of t(1/2). All patients with severe renal impairment (four patients) had drug-related grade 3 or 4 adverse-events (AEs) and serious AEs. Patients with moderate renal impairment experienced a similar number of grade 3 or 4 AEs (six of nine patients) but had a higher incidence of serious AEs (three of nine patients) when compared with those with normal renal function (four of six patients and one of six patients, respectively). A similar effect was seen in patients with mild renal dysfunction (grade 3 or 4 AEs in four of eight patients; serious AEs in three of eight patients). The relationship between systemic exposure to capecitabine or its metabolites and safety was investigated using logistic regression. This exploratory analysis showed a strong positive relationship between AUC of 5'-DFUR and treatment-related grade 3 or 4 AEs, whereas there was no relationship with exposure to capecitabine, 5'-DFCR, 5-FU or FBAL. CONCLUSIONS: Renal impairment has no effect on the pharmacokinetics of capecitabine or 5-FU, but leads to an increase in the systemic exposure to 5'-DFUR and FBAL. However, only the AUC of 5'-DFUR is correlated with safety. Based on the safety results in patients with severe renal impairment, a dose modification cannot be recommended for these patients and they should not be treated with capecitabine. Additional data from the clinical safety database and pharmacokinetic results from the present study support the recommendation that patients with moderate renal impairment should be treated with 75% of the recommended standard starting dose to achieve systemic exposure comparable to that in patients with normal renal function.     

Early expression of thyroid hormone deiodinases and receptors in human fetal cerebral cortex

Thyroid hormones are known to be important for optimal development of the human central nervous system. Classically, maternal thyroid hormones have not been thought to have a major role in defining central nervous system development. However, recent epidemiological evidence has indicated that subtle deficiencies in circulating maternal thyroid hormones in the first trimester of pregnancy are associated with adverse neurodevelopment. We have used real time PCR to quantitate the expression of mRNAs encoding the thyroid receptor isoforms (TR alpha1, alpha2, beta1 and beta2) and thyronine deiodinase subtypes (5'-DI, 5'-DII and 5-DIII) in human fetal cerebral cortex from the first and second trimesters of pregnancy. Deiodinase subtype activities have also been determined in these tissues and compared to 'normal' adult human cerebral cortex. Iodothyronine deiodinase mRNAs were expressed in human fetal cerebral cortex from 7 to 8 weeks of gestation. The expression of 5'-DI mRNA was variable in fetal life but increased relative to adult cortex (P<0.05), whereas the activity of this enzyme was below the level of assay detection. 5'-DII mRNA and activity in fetal cerebral cortex was detectable from as early as 7-8 weeks but not significantly different from that in adult life except at 15-16 weeks when mRNA expression increased (P<0.05). Fetal cortex 5-DIII mRNA expression was present from the early first trimester but less abundant than in adult tissue (P<0.01) and 5-DIII activity appeared greater in fetal cortex (P<0.01) as compared to adults. Only TR alpha1 mRNA was more abundantly expressed in fetal cortex than adult tissues (P<0.01). In contrast, the TR isoforms (alpha2 and beta1) were expressed significantly less than in adult tissues (P<0.05). Only 26% of fetal cerebral cortex samples expressed TR beta 1. There is evidence that the developing fetal brain, as early as the first trimester, expresses TRs and exhibits the mechanisms of pre-receptor control of thyroid hormone supply.     

Activated mesothelial cells produce heparin-binding growth factors: implications for tumour metastases

Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121 and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bg(c)) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum.     

Pituitary tumour transforming gene (PTTG) induces genetic instability in thyroid cells

Cancer reflects the progressive accumulation of genetic alterations and subsequent genetic instability of cells. Cytogenetic studies have demonstrated the importance of aneuploidy in differentiated thyroid cancer development. The pituitary tumour transforming gene (PTTG), also known as securin, is a mitotic checkpoint protein which inhibits sister chromatid separation during mitosis. PTTG is highly expressed in many cancers and overexpression of PTTG induces aneuploidy in vitro. Using fluorescent intersimple sequence repeat PCR (FISSR-PCR), we investigated the relationship between PTTG expression and the degree of genetic instability in normal and tumorous thyroid samples. The genomic instability index (GI index) was 6.7-72.7% higher in cancers than normal thyroid tissues. Follicular thyroid tumours exhibited greater genetic instability than papillary tumours (27.6% (n=9) versus 14.5% (n=10), P=0.03). We also demonstrated a strong relationship between PTTG expression and the degree of genetic instability in thyroid cancers (R2=0.80, P=0.007). To further investigate PTTG's role in genetic instability, we transfected FTC133 thyroid follicular cells and observed increased genetic instability in cells overexpressing PTTG compared with vector-only-transfected controls (n=3, GI Index VO=29.7+/-5.2 versus PTTG=63.7+/-6.4, P=0.013). Further, we observed a dose response in genetic instability and PTTG expression (GI Index low dose (0.5 microg DNA/ six-well plate) PTTG=15.3%+/-1.7 versus high dose (3 microg DNA) PTTG=50.8%+/-3.3, P=0.006). Overall, we describe the first use of FISSR-PCR in human cancers, and demonstrate that PTTG expression correlates with genetic instability in vivo, and induces genetic instability in vitro. We conclude that PTTG may be an important gene in the mutator phenotype development in thyroid cancer.     

Participation in health impact assessment: objectives, methods and core values

Health impact assessment (HIA) is a multidisciplinary aid to decision-making that assesses the impact of policy on public health and on health inequalities. Its purpose is to assist decision-makers to maximize health gains and to reduce inequalities. The 1999 Gothenburg Consensus Paper (GCP) provides researchers with a rationale for establishing community participation as a core value of HIA. According to the GCP, participation in HIA empowers people within the decision-making process and redresses the democratic deficit between government and society. Participation in HIA generates a sense that health and decision-making is community-owned, and the personal experiences of citizens become integral to the formulation of policy. However, the participatory and empowering dimensions of HIA may prove difficult to operationalize. In this review of the participation strategies adopted in key applications of HIA in the United Kingdom, we found that HIA's aim of influencing decision-making creates tension between its participatory and knowledge-gathering dimensions. Accordingly, researchers have decreased the participatory dimension of HIA by reducing the importance attached to the community's experience of empowerment, ownership and democracy, while enlarging its knowledge-gathering dimension by giving pre-eminence to "expert" and "research-generated" evidence. Recent applications of HIA offer a serviceable rationale for participation as a means of information gathering and it is no longer tenable to uphold HIA as a means of empowering communities and advancing the aims of participatory democracy.     

Visually evoked eye movements: Effects of labyrinthectomy

Nystagmus responses of cats to both optokinetic and flicker stimuli were studied before and after unilateral and bilateral labyrinthectomy. Destruction of the labyrinths served to reduce significantly the number of eye movements to each type of the visual stimuli. The opposite-direction after-nystagmus elicited by the optokinetic stimulus were abolished by bilateral labyrinthectomy. No changes occurred in the frequency of the flash nystagmus response that could be ascribed to body position. Thus, the data confirm previous work by others, but suggest species differences with regard to several properties of visually evoked nystagmus.     

Fibrin glue for fistula–in–ano: the evidence reviewed

Fibrin glue is increasingly used in the treatment of anal fistulae. This review aims to establish its longterm efficacy and clarify its role in this setting. A search of Medline and Pubmed databases was performed from 1966 to 2004. Data were collated regarding the type of study, fistula aetiology and complexity, technical aspects of glue application, and short– and long–term healing rates. The majority of studies comprised prospective series with fistulae of mixed aetiology. The overall healing rate was 53% with a wide variation between studies (10%–78%). The only factor that could account for this diversity was fistula complexity, with series including a high proportion of complex fistulae reporting worse outcomes. The quality of data to assess the efficacy of fibrin glue in the treatment of anal fistulae is poor and further clinical trials are needed. Fistula complexity is the main factor that adversely influences long–term healing rates.