The GABAB1b isoform mediates long-lasting inhibition of dendritic Ca2+ spikes in layer 5 somatosensory pyramidal neurons

The apical tuft of layer 5 pyramidal neurons is innervated by a large number of inhibitory inputs with unknown functions. Here, we studied the functional consequences and underlying molecular mechanisms of apical inhibition on dendritic spike activity. Extracellular stimulation of layer 1, during blockade of glutamatergic transmission, inhibited the dendritic Ca2+ spike for up to 400 ms. Activation of metabotropic GABAB receptors was responsible for a gradual and long-lasting inhibitory effect, whereas GABAA receptors mediated a short-lasting (approximately 150 ms) inhibition. Our results suggest that the mechanism underlying the GABAB inhibition of Ca2+ spikes involves direct blockade of dendritic Ca2+ channels. By using knockout mice for the two predominant GABAB1 isoforms, GABAB1a and GABAB1b, we showed that postsynaptic inhibition of Ca2+ spikes is mediated by GABAB1b, whereas presynaptic inhibition of GABA release is mediated by GABAB1a. We conclude that the molecular subtypes of GABAB receptors play strategically different physiological roles in neocortical neurons.

Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats

Calcium influx into the dendritic tufts of layer 5 neocortical pyramidal neurons modifies a number of important cellular mechanisms. It can trigger local synaptic plasticity and switch the firing properties from regular to burst firing. Due to methodological limitations, our knowledge about Ca2+ spikes in the dendritic tuft stems mostly from in vitro experiments. However, it has been speculated that regenerative Ca2+ events in the distal dendrites correlate with distinct behavioral states. Therefore it would be most desirable to be able to record these Ca2+ events in vivo, preferably in the behaving animal. Here, we present a novel approach for recording Ca2+ signals in the dendrites of populations of layer 5 pyramidal neurons in vivo, which ensures that all recorded fluorescence changes are due to intracellular Ca2+ signals in the apical dendrites. The method has two main features: 1) bolus loading of layer 5 with a membrane-permeant Ca2+ dye resulting in specific loading of pyramidal cell dendrites in the upper layers and 2) a fiberoptic cable attached to a gradient index lens and a prism reflecting light horizontally at 90 degrees to the angle of the apical dendrites. We demonstrate that the in vivo signal-to-noise ratio recorded with this relatively inexpensive and easy-to-implement fiberoptic-based device is comparable to conventional camera-based imaging systems used in vitro. In addition, the device is flexible and lightweight and can be used for recording Ca2+ signals in the distal dendritic tuft of freely behaving animals.

How reliable and robust are current biomarkers for copper status?

Cu is an essential nutrient for man, but can be toxic if intakes are too high. In sensitive populations, marginal over- or under-exposure can have detrimental effects. Malnourished children, the elderly, and pregnant or lactating females may be susceptible for Cu deficiency. Cu status and exposure in the population can currently not be easily measured, as neither plasma Cu nor plasma cuproenzymes reflect Cu status precisely. Some blood markers (such as ceruloplasmin) indicate severe Cu depletion, but do not inversely respond to Cu excess, and are not suitable to indicate marginal states. A biomarker of Cu is needed that is sensitive to small changes in Cu status, and that responds to Cu excess as well as deficiency. Such a marker will aid in monitoring Cu status in large populations, and will help to avoid chronic health effects (for example, liver damage in chronic toxicity, osteoporosis, loss of collagen stability, or increased susceptibility to infections in deficiency). The advent of high-throughput technologies has enabled us to screen for potential biomarkers in the whole proteome of a cell, not excluding markers that have no direct link to Cu. Further, this screening allows us to search for a whole group of proteins that, in combination, reflect Cu status. The present review emphasises the need to find sensitive biomarkers for Cu, examines potential markers of Cu status already available, and discusses methods to identify a novel suite of biomarkers.

Sustained virologic response and clinical outcomes in patients with chronic hepatitis C and advanced fibrosis

BACKGROUND: Clinical outcomes of chronic hepatitis C infection in patients with advanced fibrosis include liver failure, hepatocellular carcinoma, and death. OBJECTIVE: To investigate whether sustained virologic response to treatment for hepatitis C is associated with improved clinical outcomes. DESIGN: Retrospective cohort study. SETTING: 5 hepatology units of tertiary care centers in Europe and Canada caring for patients with chronic hepatitis C treated between 1990 and 2003. PATIENTS: Consecutively treated patients with chronic hepatitis C who had biopsy-proven advanced fibrosis or cirrhosis (Ishak score, 4 to 6). MEASUREMENTS: Sustained virologic response, defined as absence of detectable hepatitis C virus RNA at 24 weeks after the end of treatment, and clinical outcomes, defined as death (liver-related or non-liver-related), liver failure, and hepatocellular carcinoma. RESULTS: Of 479 patients, 29.6% had sustained virologic response and 70.3% did not. Median follow-up was 2.1 years (interquartile range, 0.8 to 4.9 years). Four patients with and 83 without sustained virologic response had at least 1 outcome event. Sustained virologic response was associated with a statistically significant reduction in the hazard of events (adjusted hazard ratio, 0.21 [95% CI, 0.07 to 0.58]; P = 0.003). The effect was largely attributable to a reduction in liver failure, which developed in no patients with and 42 patients without sustained virologic response (5-year occurrence, 0% vs. 13.3% [CI, 8.4% to 18.2%]; unadjusted hazard ratio, 0.03 [CI, 0.00 to 0.91]). LIMITATIONS: Because few events occurred in the sustained virologic response group, the study had limited ability to detect differences between groups in individual outcomes. In addition, the study was retrospective; selection and survival biases may therefore influence estimates of effect. CONCLUSION: Sustained virologic response to treatment is associated with improved clinical outcomes, mainly prevention of liver failure, in patients with chronic hepatitis C and advanced fibrosis.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [(3)H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1(-/-) line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4',6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 muM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.

Chemotherapeutic strategies against Trypanosoma brucei: drug targets vs. drug targeting

Trypanosoma brucei rhodesiense and T. b. gambiense are the causative agents of sleeping sickness, a fatal disease that affects 36 countries in sub-Saharan Africa. Nevertheless, only a handful of clinically useful drugs are available. These drugs suffer from severe side-effects. The situation is further aggravated by the alarming incidence of treatment failures in several sleeping sickness foci, apparently indicating the occurrence of drug-resistant trypanosomes. Because of these reasons, and since vaccination does not appear to be feasible due to the trypanosomes' ever changing coat of variable surface glycoproteins (VSGs), new drugs are needed urgently. The entry of Trypanosoma brucei into the post-genomic age raises hopes for the identification of novel kinds of drug targets and in turn new treatments for sleeping sickness. The pragmatic definition of a drug target is, a protein that is essential for the parasite and does not have homologues in the host. Such proteins are identified by comparing the predicted proteomes of T. brucei and Homo sapiens, then validated by large-scale gene disruption or gene silencing experiments in trypanosomes. Once all proteins that are essential and unique to the parasite are identified, inhibitors may be found by high-throughput screening. However powerful, this functional genomics approach is going to miss a number of attractive targets. Several current, successful parasiticides attack proteins that have close homologues in the human proteome. Drugs like DFMO or pyrimethamine inhibit parasite and host enzymes alike--a therapeutic window is opened only by subtle differences in the regulation of the targets, which cannot be recognized in silico. Working against the post-genomic approach is also the fact that essential proteins tend to be more highly conserved between species than non-essential ones. Here we advocate drug targeting, i.e. uptake or activation of a drug via parasite-specific pathways, as a chemotherapeutic strategy to selectively inhibit enzymes that have equally sensitive counterparts in the host. The T. brucei purine salvage machinery offers opportunities for both metabolic and transport-based targeting: unusual nucleoside and nucleobase permeases may be exploited for selective import, salvage enzymes for selective activation of purine antimetabolites.

Recent advances in understanding Marfan syndrome: should we now treat surgical patients with losartan?

OBJECTIVE: Marfan syndrome is a systemic connective tissue disorder caused by mutations in the fibrillin-1 gene. It was originally believed that Marfan syndrome results exclusively from the production of abnormal fibrillin-1 that leads to structurally weaker connective tissue when incorporated into the extracellular matrix. This effect seemed to explain many of the clinical features of Marfan syndrome, including aortic root dilatation and acute aortic dissection, which represent the main causes of morbidity and mortality in Marfan syndrome. METHODS: Recent molecular studies, most based on genetically defined mouse models of Marfan syndrome, have challenged this paradigm. These studies established the critical contribution of fibrillin-1 haploinsufficiency and dysregulated transforming growth factor-beta signaling to disease progression. RESULTS: It seems that many manifestations of Marfan syndrome are less related to a primary structural deficiency of the tissues than to altered morphogenetic and homeostatic programs that are induced by altered transforming growth factor-beta signaling. Most important, transforming growth factor-beta antagonism, through transforming growth factor-beta neutralizing antibodies or losartan (an angiotensin II type 1 receptor antagonist), has been shown to prevent and possibly reverse aortic root dilatation, mitral valve prolapse, lung disease, and skeletal muscle dysfunction in a mouse model of Marfan syndrome. CONCLUSION: There are indicators that losartan, a drug widely used to treat arterial hypertension in humans, offers the first potential for primary prevention of clinical manifestations in Marfan syndrome.

Increased risk of hepatocellular carcinoma among patients with hepatitis C cirrhosis and diabetes mellitus

Recent studies suggest that diabetes mellitus increases the risk of developing hepatocellular carcinoma (HCC). The aim of this study is to quantify the risk of HCC among patients with both diabetes mellitus and hepatitis C in a large cohort of patients with chronic hepatitis C and advanced fibrosis. We included 541 patients of whom 85 (16%) had diabetes mellitus. The median age at inclusion was 50 years. The prevalence of diabetes mellitus was 10.5% for patients with Ishak fibrosis score 4, 12.5% for Ishak score 5, and 19.1% for Ishak score 6. Multiple logistic regression analysis showed an increased risk of diabetes mellitus for patients with an elevated body mass index (BMI) (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.00-1.11; P = 0.060) and a decreased risk of diabetes mellitus for patients with higher serum albumin levels (OR, 0.81; 95% CI, 0.63-1.04; P = 0.095). During a median follow-up of 4.0 years (interquartile range, 2.0-6.7), 11 patients (13%) with diabetes mellitus versus 27 patients (5.9%) without diabetes mellitus developed HCC, the 5-year occurrence of HCC being 11.4% (95% CI, 3.0-19.8) and 5.0% (95% CI, 2.2-7.8), respectively (P = 0.013). Multivariate Cox regression analysis of patients with Ishak 6 cirrhosis showed that diabetes mellitus was independently associated with the development of HCC (hazard ratio, 3.28; 95% CI, 1.35-7.97; P = 0.009). CONCLUSION: For patients with chronic hepatitis C and advanced cirrhosis, diabetes mellitus increases the risk of developing HCC.

Biomechanical comparison of different surface modifications for dental implants

Purpose: A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is also critically important. Biomechanical and microcomputerized tomographic evaluation of implant osseointegration was performed to compare alternative structural, chemical and biochemical, and/or pharmaceutical surface treatments applied to an identical established implant design. Materials and Methods: Dental implants with the same geometry but with 6 different surface treatments were tested in vivo in a sheep model (pelvis). Peri-implant bone density and removal torque were compared at 2, 4, and 8 weeks after implantation. Implant surfaces tested were: sandblasted and acid-etched titanium (Ti), sandblasted and etched zirconia, Ti coated with calcium phosphate (CaP), Ti modified via anodic plasma-chemical treatment (APC), bisphosphonate-coated Ti (Ti + Bisphos), and Ti coated with collagen containing chondroitin sulfate (CS). Results: All dental implants were well integrated at the time of sacrifice. There were no significant differences observed in peri-implant bone density between implant groups. After 8 weeks of healing, removal torque values for Ti, Ti + CaP, Ti + Bisphos, and Ti + collagen + CS were significantly higher than those for zirconia and Ti + APC. Conclusions: Whereas the sandblasted/acid-etched Ti implant can still be considered the reference standard surface for dental implants, functional surface modifications such as bisphosphonate or collagen coating seem to enhance early peri-implant bone formation and should be studied further.

Outcomes of the South African National Antiretroviral Treatment Programme for children: the IeDEA Southern Africa collaboration

OBJECTIVES: To assess paediatric antiretroviral treatment (ART) outcomes and their associations from a collaborative cohort representing 20% of the South African national treatment programme. DESIGN AND SETTING: Multi-cohort study of 7 public sector paediatric ART programmes in Gauteng, Western Cape and KwaZulu-Natal provinces. SUBJECTS: ART-naive children (< or = 16 years) who commenced treatment with > or = 3 antiretroviral drugs before March 2008. OUTCOME MEASURES: Time to death or loss to follow-up were assessed using the Kaplan-Meier method. Associations between baseline characteristics and mortality were assessed with Cox proportional hazards models stratified by site. Immune status, virological suppression and growth were described in relation to duration of ART. RESULTS: The median (interquartile range) age of 6 078 children with 9 368 child-years of follow-up was 43 (15 - 83) months, with 29% being < 18 months. Most were severely ill at ART initiation. More than 75% of children were appropriately monitored at 6-monthly intervals with viral load suppression (< 400 copies/ml) being 80% or above throughout 36 months of treatment. Mortality and retention in care at 3 years were 7.7% (95% confidence interval 7.0 - 8.6%) and 81.4% (80.1 - 82.6%), respectively. Together with young age, all markers of disease severity (low weight-for-age z-score, high viral load, severe immune suppression, stage 3/4 disease and anaemia) were independently associated with mortality. CONCLUSIONS: Dramatic clinical benefit for children accessing the national ART programme is demonstrated. Higher mortality in infants and those with advanced disease highlights the need for early diagnosis of HIV infection and commencement of ART.