Glial-mediated dysregulation of neurodevelopment in Fragile X Syndrome.

Astrocytes are highly involved in a multitude of developmental processes that are known to be dysregulated in Fragile X Syndrome. Here, we examine these processes individually and review the roles astrocytes play in contributing to the pathology of this syndrome. As a growing area of interest in the field, new and exciting insight is continually emerging. Understanding these glial-mediated roles is imperative for elucidating the underlying molecular mechanisms at play, not only in Fragile X Syndrome, but also other ASD-related disorders. Understanding these roles will be central to the future development of effective, clinically-relevant treatments of these disorders.

BCL11B-related disorder in two canadian children: Expanding the clinical phenotype.

The product of the BCL11B (B-Cell Leukemia 11) gene is a bi-functional transcriptional regulator that can act as either a repressor or an activator. It plays an important role in the development of the nervous, immune, and cutaneous systems, and is also involved in dental and craniofacial development. BCL11B-Related Disorder (BCL11BRD) is a novel rare neurodevelopmental disorder associated with mutations in BCL11B. A total of 17 patients have been described in the literature thus far. The main symptoms of BCL11BRD include global developmental delay, speech impairment, dental anomalies, feeding difficulties, refractive errors, dysmorphic features, and immunological abnormalities. In this report, we describe two Canadian girls, with pathogenic de novo BCL11B variants, both diagnosed via exome sequencing. One of the patients had global developmental delay, dental anomalies, dysmorphic features, dyskinesia and hypotonia; the latter two symptoms have not been previously reported in patients with BCL11BRD. She also had dysgenesis of corpus callosum and dilatation of the frontal horns of lateral ventricles, a brain anomaly that has been previously reported in only one other patient. The second patient had developmental delay, dysmorphic features, spasticity in lower limbs and dental anomalies. Our report contributes to the knowledge of the BCL11BRD, expands the clinical phenotype, and can also aid with genetic counseling of newly identified patients.

Large-scale pharmacogenomic study of sulfonylureas and the QT, JT and QRS intervals: CHARGE Pharmacogenomics Working Group.

Sulfonylureas, a commonly used class of medication used to treat type 2 diabetes, have been associated with an increased risk of cardiovascular disease. Their effects on QT interval duration and related electrocardiographic phenotypes are potential mechanisms for this adverse effect. In 11 ethnically diverse cohorts that included 71 857 European, African-American and Hispanic/Latino ancestry individuals with repeated measures of medication use and electrocardiogram (ECG) measurements, we conducted a pharmacogenomic genome-wide association study of sulfonylurea use and three ECG phenotypes: QT, JT and QRS intervals. In ancestry-specific meta-analyses, eight novel pharmacogenomic loci met the threshold for genome-wide significance (P<5 × 10-8), and a pharmacokinetic variant in CYP2C9 (rs1057910) that has been associated with sulfonylurea-related treatment effects and other adverse drug reactions in previous studies was replicated. Additional research is needed to replicate the novel findings and to understand their biological basis.

Pharmacogenomics study of thiazide diuretics and QT interval in multi-ethnic populations: the cohorts for heart and aging research in genomic epidemiology.

Thiazide diuretics, commonly used antihypertensives, may cause QT interval (QT) prolongation, a risk factor for highly fatal and difficult to predict ventricular arrhythmias. We examined whether common single-nucleotide polymorphisms (SNPs) modified the association between thiazide use and QT or its component parts (QRS interval, JT interval) by performing ancestry-specific, trans-ethnic and cross-phenotype genome-wide analyses of European (66%), African American (15%) and Hispanic (19%) populations (N=78 199), leveraging longitudinal data, incorporating corrected standard errors to account for underestimation of interaction estimate variances and evaluating evidence for pathway enrichment. Although no loci achieved genome-wide significance (P<5 × 10-8), we found suggestive evidence (P<5 × 10-6) for SNPs modifying the thiazide-QT association at 22 loci, including ion transport loci (for example, NELL1, KCNQ3). The biologic plausibility of our suggestive results and simulations demonstrating modest power to detect interaction effects at genome-wide significant levels indicate that larger studies and innovative statistical methods are warranted in future efforts evaluating thiazide-SNP interactions.

Biochemical and Hematologic Manifestations of Gastric Intrinsic Factor (GIF) Deficiency: A Treatable Cause of B12 Deficiency in the Old Order Mennonite Population of Southwestern Ontario.

Intrinsic factor deficiency (OMIM #261000, IFD) is a rare inherited disorder of vitamin B12 metabolism due to mutations in the gastric intrinsic factor (GIF) gene.We report three individuals from an Old Order Mennonite community who presented with B12 deficiency. Two cases are siblings born to consanguineous parents and the third case is not known to be closely related. The older male sib presented at 4 years with gastrointestinal symptoms, listlessness, and pallor. He had pancytopenia with megaloblastic anemia. Serum B12 was 61 (198-615 pmol/L). Methylmalonic aciduria was present. C3 was elevated on acylcarnitine profile. Homocysteine was high at 16.7 (5.0-12.0 umol/L). His asymptomatic female sibling was also found to have B12 deficiency. Genetic testing for methylmalonic aciduria (MMAA), transcobalamin deficiency (TCN2), and Imerslund-Gräsbeck syndrome (AMN) showed no mutation in both siblings. The third patient, a 34-year-old woman, had presented in infancy with a diagnosis of pernicious anemia. Mutation analysis of GIF revealed compound heterozygosity for a c.79+1G>A substitution and a c.973delG deletion in all three individuals. Oral or parenteral vitamin B12 has led to complete recovery of clinical parameters and vitamin B12 levels. Newborn screening samples on the siblings revealed normal methylcitrate, C3, and C3/C2 ratios thus indicating no disruption of propionic or methylmalonic acid metabolism.A high index of suspicion should be maintained if children present with megaloblastic anemia since GIF deficiency is a treatable disorder and newborn screening may not be able to detect this condition.

The clinical effectiveness and cost-effectiveness of cetuximab (mono- or combination chemotherapy), bevacizumab (combination with non-oxaliplatin chemotherapy) and panitumumab (monotherapy) for the treatment of metastatic colorectal cancer after first-line chemotherapy (review of technology appraisal No.150 and part review of technology appraisal No. 118): a systematic review and economic model.

BACKGROUND: Colorectal cancer is the third most commonly diagnosed cancer in the UK after breast and lung cancer. People with metastatic disease who are sufficiently fit are usually treated with active chemotherapy as first- or second-line therapy. Recently, targeted agents have become available including anti-epidermal growth factor receptor (EGFR) agents, for example cetuximab and panitumumab, and anti-vascular endothelial growth factor (VEGF) receptor agents, for example bevacizumab. OBJECTIVE: To investigate the clinical effectiveness and cost-effectiveness of panitumumab monotherapy and cetuximab (mono- or combination chemotherapy) for Kirsten rat sarcoma (KRAS) wild-type (WT) patients, and bevacizumab in combination with non-oxaliplatin chemotherapy, for the treatment of metastatic colorectal cancer after first-line chemotherapy. DATA SOURCES: The assessment comprises a systematic review of clinical effectiveness and cost-effectiveness studies, a review and critique of manufacturer submissions and a de novo cohort-based economic analysis. For the assessment of effectiveness, a literature search was conducted in a range of electronic databases, including MEDLINE, EMBASE and The Cochrane Library, from 2005 to November 2010. REVIEW METHODS: Studies were included if they were randomised controlled trials (RCTs) or systematic reviews of RCTs of cetuximab, bevacizumab or panitumumab in participants with EGFR-expressing metastatic colorectal cancer with KRAS WT status that has progressed after first-line chemotherapy (for cetuximab and panitumumab) or participants with metastatic colorectal cancer that has progressed after first-line chemotherapy (bevacizumab). All steps in the review were performed by one reviewer and checked independently by a second. Synthesis was mainly narrative. An economic model was developed focusing on third-line and subsequent lines of treatment. Costs and benefits were discounted at 3.5% per annum. Probabilistic and univariate deterministic sensitivity analyses were performed. RESULTS: The searches identified 7745 titles and abstracts. Two clinical trials (reported in 12 papers) were included. No data were available for bevacizumab in combination with non-oxaliplatin-based chemotherapy in previously treated patients. Neither of the included studies had KRAS status performed prospectively, but the studies did report retrospective analyses of the results for the KRAS WT subgroups. Third-line treatment with cetuximab plus best supportive care or panitumumab plus best supportive care appears to have statistically significant advantages over treatment with best supportive care alone in patients with KRAS WT status. For the economic evaluation, five studies met the inclusion criteria. The base-case incremental cost-effectiveness ratio (ICER) for KRAS WT patients for cetuximab compared with best supportive care is £98,000 per quality-adjusted life-year (QALY), for panitumumab compared with best supportive care is £150,000 per QALY and for cetuximab plus irinotecan compared with best supportive care is £88,000 per QALY. All ICERs are sensitive to treatment duration. LIMITATIONS: In the specific populations of interest, there is a lack of evidence on bevacizumab, cetuximab and cetuximab plus irinotecan used second line and on bevacizumab and cetuximab plus irinotecan used third line. For cetuximab plus irinotecan treatment for KRAS WT people, there is no direct evidence on progression-free survival, overall survival and duration of treatment. CONCLUSIONS: Although cetuximab and panitumumab appear to be clinically beneficial for KRAS WT patients compared with best supportive care, they are likely to represent poor value for money when judged by cost-effectiveness criteria currently used in the UK. It would be useful to conduct a RCT for patients with KRAS WT status receiving cetuximab plus irinotecan. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Sunitinib for the treatment of gastrointestinal stromal tumours: a critique of the submission from Pfizer.

The submission's evidence for the clinical effectiveness and cost-effectiveness of sunitinib for the treatment of gastrointestinal stromal tumours (GISTs) is based on a randomised controlled trial (RCT) comparing sunitinib with placebo for people with unresectable and/or metastatic GIST after failure of imatinib and with Eastern Cooperative Oncology Group (ECOG) progression status 0-1, and an ongoing, non-comparative cohort study of a similar population but with ECOG progression status 0-4. The searches are appropriate and include all relevant studies and the RCT is of high quality. In the RCT sunitinib arm overall survival was 73 median weeks [95% confidence interval (CI) 61 to 83] versus 75 median weeks (95% CI 68 to 84) for the cohort study. However, time to tumour progression in the cohort study was different from that in the RCT sunitinib arm [41 (95% CI 36 to 47) versus 29 (95% CI 22 to 41) median weeks respectively]. Median progression-free survival with sunitinib was 24.6 weeks (95% CI 12.1 to 28.4) versus 6.4 weeks (95% CI 4.4 to 10.0) on placebo (hazard ratio 0.333, 95% CI 0.238 to 0.467, p < 0.001). The manufacturer used a three-state Markov model to model the cost-effectiveness of sunitinib compared with best supportive care for GIST patients; the modelling approach and sources and justification of estimates are reasonable. The base-case incremental cost-effectiveness ratio (ICER) was 27,365 pounds per quality-adjusted life-year (QALY) with the first cycle of sunitinib treatment not costed; when we included the cost of the first treatment cycle we estimated a base-case ICER of 32,636 pounds per QALY. Pfizer's sensitivity analysis produced a range of ICERs from 15,536 pounds per QALY to 59,002 pounds per QALY. Weaknesses of the manufacturer's submission include that the evidence is based on only one published RCT; that 84% of the RCT control population crossed over to the intervention group, giving rise to the use of unusual rank preserved structural failure time (RPSFT) analysis to correct for possible bias; and that a number of errors and omissions were made in the probabilistic sensitivity analysis, meaning that it is not possible to come to firm conclusions about the cost-effectiveness of sunitinib for GIST in this patient population. In conclusion, during the blinded phase of the RCT, overall survival was significantly longer in the sunitinib arm than in the placebo arm (hazard ratio 0.491, 95% CI 0.290 to 0.831, p <0.007). However, intention-to-treat analysis of the entire study showed no statistically significant difference in overall survival for those who received sunitinib (73 weeks) versus those who received placebo (65 weeks) (hazard ratio 0.876, 95% CI 0.679 to 1.129, p = 0.306).

Late onset antibody-mediated rejection and endothelial localization of vascular endothelial growth factor are associated with development of cardiac allograft vasculopathy.

BACKGROUND: Improvements in cardiac transplant practice and immunosuppressive treatment have done much to curb the incidence of acute cellular rejection (ACR); however, antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) remain prevalent. Recent studies have shown that allograft rejection is governed by both allogeneic and nonallogeneic factors such as inflammation. Initial studies have suggested that vascular endothelial growth factor (VEGF), a leukocyte mitogen produced by activated endothelial cells and leukocytes, may play a specific role in not only leukocyte trafficking, but also in the augmentation of ACR and development of CAV. METHODS: We investigated the localization of VEGF protein using immunohistochemistry in a cohort of 76 heart transplant patients during periods of ACR and AMR and assessed the development of CAV. RESULTS: We showed a significant correlation between lymphocytic localization of VEGF protein and severe ACR (P<0.001). Antibody-mediated rejection positive biopsies taken at 12 months posttransplantation showed significantly greater endothelial localization of VEGF than time-matched AMR negative biopsies (P=0.006). Diffuse endothelial expression of VEGF was also associated with a 2.5-fold increase in the risk of developing CAV (P=0.001). CONCLUSIONS: These results show that localization of VEGF protein to the vascular endothelium during AMR is significantly increased in patients who develop CAV. This study also highlights the potential pathogenic role of the endothelial cell in late onset AMR and the development of CAV.

Increased vascular endothelial growth factor mRNA in endomyocardial biopsies from allografts demonstrating severe acute rejection: a longitudinal study.

Investigation into the contribution of the immune system and inflammatory cascade to acute rejection (AR) and cardiac allograft vasculopathy (CAV) has implicated vascular endothelial growth factor (VEGF). The endomyocardial biopsy (EB) has proved invaluable in the diagnosis of AR, and in providing information concerning the biological processes occurring following transplantation. The association between VEGF and AR and the development of CAV was examined in endomyocardial biopsies (EBs) from a cohort of 76 heart transplant recipients. VEGF mRNA levels were quantified through real time RT-PCR in 712 EBs, obtained at routine intervals during post-operative monitoring. VEGF and leukocyte and endothelial markers were assessed in a subset of biopsies through immunohistochemistry. The results of generalised linear modelling, adjusting for covariates, revealed VEGF mRNA expression was 19% greater during severe AR as compared to no rejection (p=0.007). Immunohistochemical results supported these findings. Mean VEGF mRNA levels were not significant predictors for the development of CAV (p=0.554). However the risk of cardiac related death increased 9-fold for a 1 unit increase in mean VEGF expression (p=0.006). Similarly, a single unit increase in mean AR severity equated to a 10-fold increase in the risk of cardiac related death (p<0.005). Our data suggest that increased VEGF expression is strongly associated with severe AR and cardiac related death.

Predictors of decline in renal function after lung transplantation.

BACKGROUND: Survival after lung transplantation has improved, but with the consequence that long-term toxicities of treatment are of growing importance. In particular, renal impairment is common, has many causes, and carries with it increased morbidity and mortality. METHODS: We retrospectively analyzed clinical and laboratory data of 136 patients who underwent lung and heart-lung transplantation at our institution between 1990 and 2004 inclusive. Using multivariate analysis we considered the impact of age, gender, pulmonary diagnosis, transplant type (single lung, double lung, heart-lung), hypertension, diabetes mellitus, cigarette smoking, current immunosuppression, duration of calcineurin inhibitor (CNI) exposure and pre-existing renal impairment on renal function. RESULTS: At transplantation, creatinine clearance (CrCl) for the patient population was 108 +/- 3.28 (mean +/- SEM) ml/min/1.73 m(2). At end of follow-up (6 +/- 0.32 years) there was a significant decline in glomerular filtration rate (GFR) to 56.7 +/- 1.78 ml/min/1.73 m(2) (p < 0.001). Five of 136 patients (3.7%) developed end-stage renal failure (ESRF). On multivariate analysis, factors most strongly associated with this decline included (in order of significance): CrCl at transplantation; pack-years of cigarette smoking; exposure to sirolimus (SLM); CNI exposure; and age at transplantation. The rate of decline in GFR was linked to CrCl and age at the time of transplantation. CONCLUSIONS: This analysis has demonstrated that patients with a lower baseline CrCl, older age at transplantation, and a smoking history are at high risk for rapid loss of renal function after transplantation. To best preserve kidney function, these patients should be targeted for aggressive risk factor modification as well as minimization of CNI exposure wherever possible.

Beyond operational tolerance: effect of ischemic injury on development of chronic damage in renal grafts.

BACKGROUND: The induction of operational tolerance is the holy grail of clinical transplantation. However, in animal models with operational tolerance, long- term grafts still develop chronic damage. The elucidation of the impact of allogenic versus nonallogeneic factors in such a model is important. This study examined the effect of a clinically relevant combination of warm ischemia and cold preservation in the absence of allogeneic response (isografts) and in the context of operational tolerance. METHODS: Dark Agouti (DA) rat kidneys were transplanted into DA recipients (isografts) or Albino Surgery recipients (allografts) tolerized by two transfusions of DA blood, under cover of cyclosporin A. Grafts were subjected to minimal cold preservation or to 30 mins warm ischemia followed by 24 hrs cold preservation. RESULTS: After an initial peak of renal dysfunction, serum creatinine concentration returned to normal in isografts and nonischemic allografts, but remained significantly elevated in ischemic allografts (P<0.0002) throughout 6 months follow-up. Both allograft groups developed proteinuria. At 6 months, ischemic isografts and nonischemic allografts demonstrated very mild tubular atrophy and interstitial fibrosis. Tubulointerstitial injury was significantly more severe in ischemic allografts (P<0.01 vs. nonischemic allografts) and was associated with increased infiltrating monocyte/macrophages and NK cells (P<0.05). Moderate glomerulosclerosis was a feature of both allograft groups (P<0.05). CONCLUSIONS: The modified allogeneic response in operationally tolerant recipients acts in synergy with ischemia/reperfusion injury in the development of chronic damage. Strategies to limit or modify the initial ischemia/reperfusion injury may ameliorate chronic tubulointerstitial damage. Progressive glomerular damage and proteinuria in allografts may require other pharmacological intervention.

Increased expression of urotensin II and urotensin II receptor in human diabetic nephropathy.

BACKGROUND: Urotensin II (UII) is an 11-amino acid vasoactive peptide, recently identified as the ligand for a novel G protein-coupled receptor, GPR-14 (renamed urotensin receptor [UT]). In addition to its potent vasoconstrictive actions, UII also has trophic and profibrotic effects, leading to its implication in the pathogenesis of heart failure. However, elevated plasma UII levels also were reported in association with renal impairment and diabetes. Accordingly, the present study sought to examine the expression and localization of UII and its receptor in kidney tissue from patients with diabetic nephropathy. METHODS: We quantified UII and UT gene expression in renal biopsy tissue samples from patients with diabetic nephropathy by using quantitative real-time polymerase chain reaction and determined the intrarenal distribution of their peptides by means of immunohistochemistry. RESULTS: In human diabetic tissue, gene expression of UII and UT were increased 45- and almost 2,000-fold in comparison to control nephrectomy tissue, respectively (P < 0.0001). Immunohistochemical studies showed intense UII peptide staining in diabetic tissue localized predominantly to tubular epithelial cells, and fluorescein-labeled ligand binding studies showed a similar tubular pattern of distribution. CONCLUSION: In the context of its known biological actions, the dramatic overexpression of UII and its receptor implicate this vasoactive peptide as a possible novel factor in the pathogenesis of diabetic nephropathy.

Lessons from the Clinical Support Systems Program: facilitating better practice through leadership and team building.

The increasing array of strategies and models for improving clinical practice and patient outcomes can be confusing for clinicians. The Clinical Support Systems (CSS) model has proved to be effective in local environments because it demystifies the design and implementation of evidence-based practice improvement projects. The CSS model is simple and has a wide scope. It provides a broad framework with minimalist specifications, enabling clinicians to design their own systems of care that cut across fragmented organisational structures. Implementing simple rules can be an effective strategy for change in complex care systems. These rules do not impose solutions on clinicians, but rather, help them to find creative solutions that have meaning for them and are contextually relevant.

A phase I (tumour site-specific) study of carboplatin and temozolomide in patients with advanced melanoma.

Temozolomide is an oral alkylating agent that readily crosses the blood-brain barrier and has activity in patients with advanced melanoma. Carboplatin is a convenient outpatient treatment that also has activity in patients with melanoma. The purpose of this study was to assess the safety of a combination of temozolomide and carboplatin, and provide preliminary evidence of efficacy. In all, 30 patients were treated in two stages. In stage 1, patients received temozolomide 750 mg x m(-2), with escalating doses of carboplatin AUC 3-6. In stage 2, patients received temozolomide 1000 mg x m(-2), with increasing doses of carboplatin until dose-limiting toxicity (DLT) was experienced. In stage 1, 12 patients received 33 cycles of treatment. No grade 3/4 haematological toxicity was experienced up to carboplatin AUC 6. In stage 2, 18 patients received 55 cycles of treatment. The DLT was haematological with grade 4 myelosuppression seen with carboplatin AUC 5. In all, 11 patients were treated with carboplatin AUC 4 to gain further information on toxicity. Myelosuppression remained significant and common with grade 4 thrombocytopenia experienced in 50% of cycles. Two of 28 patients (7%) assessable for efficacy achieved a partial response. None of the 11 patients with brain metastases responded to treatment. The addition of carboplatin to temozolomide 1000 mg x m(-2) significantly adds to toxicity with frequent grade 3/4 myelosuppression. Preliminary information on efficacy demonstrates that it is unlikely that the combination can be given in doses sufficient to improve on the efficacy of temozolomide alone.

Over-expression of platelet-derived growth factor in human diabetic nephropathy.

BACKGROUND: The pathogenetic mechanisms responsible for progressive renal impairment of diabetic nephropathy are still poorly understood, despite its growing incidence. Increasing evidence suggests that growth factors may contribute to the initiation and progressive fibrosis of diabetic nephropathy. In this study, the gene expression and protein distribution of platelet-derived growth factor-A and -B (PDGF-A and PDGF-B) in human diabetic nephropathy were examined. METHODS: PDGF-A and PDGF-B mRNA levels in surplus renal biopsy tissue from seven patients with overt diabetic nephropathy and six nephrectomy samples were examined using quantitative reverse transcription-polymerase chain reaction (RT-PCR). In addition, each sample was also examined immunohistochemically to quantify and localize peptide expression of each PDGF isoform. RESULTS: Gene expression of PDGF-A and PDGF-B mRNA were increased 22- and 6-fold, respectively, in biopsies from patients with diabetic nephropathy compared with control tissue. Immunostaining also demonstrated increased peptide expression of both PDGF-A and PDGF-B in diabetic nephropathy, with each isoform showing a specific pattern of tissue distribution. CONCLUSIONS: The findings of increased gene and protein expression of PDGF in renal biopsies from patients with diabetic nephropathy imply a potential role for this prosclerotic growth factor in the development of the progressive fibrosis that characterizes human diabetic kidney disease.

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study.

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab' cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab' maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab')(2). Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab' maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab' maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab')(2) used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab' maleimide over murine versions of this antibody suggesting that humanised divalent-Fab' maleimide should be a useful vehicle for repeated therapies.

Electrochemical sensor for detection of unmodified nucleic acids.

We have developed a nucleic acid (NA) sensor based on mediated electrochemical oxidation of guanine residues. In this method, oligonucleotide probes are bound to a tin-doped indium oxide (ITO) electrode through a self-assembled phosphonate monolayer. The end carboxyl moiety of the monolayer is activated with carbodiimide and reacted with the amine group of a C6 alkyl linker which has been added to the 5'-end of the oligonucleotide probe. Upon hybridization of the complementary target NA, the hybrid is detected using a redox-active mediator, tris(2,2'-bipyridyl) ruthenium(II). We speculate that the monolayer does not impede electron-transfer since it contains many defect sites when assembled on a polycrystalline ITO surface. These defect sites are accessible to the mediator, but not to NA or proteins. The electrocatalytic current was a linear function of the amount of guanine bound at the electrode surface, with a detection limit of 120 amoles of guanine cm(-2) at 0.28 cm(2) ITO electrodes.

Novel chemotherapeutic agents in colorectal cancer.

Colorectal cancer remains the second most common cause of cancer death in Europe. A number of new agents, with varying mechanisms of action, have been developed and are being evaluated, both as single agents and in combination. Irinotecan, a topisomerase I inhibitor, and oxaliplatin, a new platinum compound, have proven efficacy in advanced colorectal cancer resistant to 5-fluorouracil. Capecitabine and other oral fluoro-pyrimidines are also emerging as potentially effective drugs. Raltitrexed and other inhibitors of thymidylate synthase (TS) are entering clinical practice although their role has yet to be determined. The new fluoro-pyrimidines and TS inhibitors are important classes of drug which have been designed to take advantage of the knowledge of folate metabolism gained from basic clinical research. Their design features could perhaps reduce the toxicity seen with the first generation cytotoxic agents. This review will focus on these new chemotherapeutic agents in colorectal cancer with respect to their mechanisms of action, current clinical activity and their potential use both in the adjuvant and palliative settings.