The Cape Triage Group (CTG) convened with the intention of producing a triage system for the Western Cape, and eventually South Africa. The group includes in-hospital and prehospital staff from varied backgrounds. The CTG triage protocol is termed the Cape Triage Score (CTG), and has been developed by a multi-disciplinary panel, through best available evidence and expert opinion. The CTS has been validated in several studies, and was launched across the Western Cape on 1 January 2006. The CTG would value feedback from readers of this journal, as part of the ongoing monitoring and evaluation process.

Haskew-Layton et al. (1) reported that subtoxic doses of H2O2 fails to activate nuclear factor erythroid 2-related factor (Nrf2) in astrocytes and triggers Nrf2-independent responses that protect cocultured neurons. Contrary to this, we show that mild oxidative insults, including subtoxic H2O2, strongly activate astrocytic Nrf2/antioxidant response element (ARE)-dependent gene expression, which, moreover, contributes to neuroprotective ischemic preconditioning. In mixed neuron/astrocyte cultures (2, 3), treatment with physiologically relevant H2O2 doses (25–100 μM, similar/less than those recorded postischemia) (4) induced Nrf2-target genes sulfiredoxin ( Srxn1 ) and heme-oxygenase 1 ( Hmox1 ) in wild-type but not Nrf2−/− cultures (Fig. 1). Similarly, exposing cultures to oxygen–glucose deprivation (OGD; an in vitro ischemia model), followed by reoxygenation, also induced Nrf2-target genes. Induction of Hmox1 in mixed cultures was restricted to astrocytes (Fig. 1), and Nrf2-target gene induction was not observed in enriched neuronal cultures (<0.2% astrocytes) (Fig. 1), strongly suggesting that astrocytes are the sole locus for Nrf2 activation by oxidative stress. Furthermore, study of enriched Nrf2+/+ and Nrf2−/− astrocyte cultures showed clear H2O2 (and OGD)-induced Nrf2-dependent gene activation, contrary to that reported in ref. 1. Fig. 1. Subtoxic oxidative insults activate Nrf2-dependent gene expression in astrocytes. ( Upper and Lower Left ) Cells were treated with H2O2 or 5 μM tert -Butylhydroquinone (tBHQ) for 6 h or subjected to a 3-h episode of oxygen–glucose deprivation (OGD), after which they were returned to normoxia for an additional 3 h. Expression of Srxn1 and Hmox1 mRNA was analyzed by quantitative real-time PCR as described (3) and normalized to Gapdh . Cell types treated include WT and Nrf2−/− mixed neuronal/astrocyte cultures [90% neuronal nuclear protein (NeuN+) neurons and 10% GFAP+ astrocytes, made as … 1To whom correspondence should be addressed. E-mail: Giles.Hardingham{at}ed.ac.uk.

Following treatment with a demethylating agent, 5 of 11 renal cell carcinoma (RCC) cell lines showed increased expression of hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2/Bikunin), a Kunitz-type protease inhibitor that regulates HGF activity. As activating mutations in the MET proto-oncogene (the HGF receptor) cause familial RCC, we investigated whether HAI-2/SPINT2 might act as a RCC tumor suppressor gene. We found that transcriptional silencing of HAI-2 in RCC cell lines was associated with promoter region methylation and HAI-2/SPINT2 protein expression was down-regulated in 30% of sporadic RCC. Furthermore, methylation-specific PCR analysis revealed promoter region methylation in 30% (19 of 64) of clear cell RCC and 40% (15 of 38) of papillary RCC, whereas mutation analysis (in 39 RCC cell lines and primary tumors) revealed a missense substitution (P111S) in one RCC cell line. Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, but the P111S mutant had no significant effect. Increased cell motility associated with HAI-2/SPINT2 inactivation was abrogated by treatment with extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phospholipase C-γ inhibitors, but not by an inhibitor of atypical protein kinase C. These findings are consistent with frequent epigenetic inactivation of HAI-2/SPINT2, causing loss of RCC tumor suppressor activity and implicate abnormalities of the MET pathway in clear cell and papillary sporadic RCC. This information provides opportunities to develop novel targeted approaches to the treatment of RCC.

The glial environment is an important determinant of neuronal health in experimental models of neurodegeneration. Specifically, astrocytes have been shown, dependent on context, to be both injurious and protective. Human pluripotent stem cells offer a powerful new system to improve our understanding of the mechanisms underlying astrocyte-mediated neuroprotection. Here, we describe a human embryonic stem cell (HESC)-based system to assess the scope and mechanism of human astrocyte-mediated neuroprotection. We first report the generation of enriched and functional HESC-derived astrocytes, by combining BMP-mediated Smad and LIF-mediated JAK-STAT signalling. These astrocytes promote the protection of HESC-derived neurons against oxidative insults. Moreover, their neuroprotective capacity can be greatly enhanced by treatment with the nuclear factor-erythroid 2-related factor 2 (Nrf2)-activating triterpenoid 1[2-Cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoroethylamide (CDDO^TFEA). Activation of the transcription factor Nrf2 in human astrocytes by CDDO^TFEA treatment induced expression of the glutamate-cysteine ligase (GCL) catalytic subunit, leading to enhanced GCL activity and glutathione production, and strong neuroprotection against H₂O₂. This enhanced neuroprotection was found to be dependent on astrocytic GCL activity, unlike the basal neuroprotection afforded by untreated astrocytes. Direct treatment of HESC-derived neurons with CDDO^TFEA elicited no induction of Nrf2 target genes, nor any neuroprotection. Thus, human astrocytes can mediate neuroprotection through glutathione-dependent and glutathione-independent mechanisms, and represent a therapeutic target for human disorders associated with neuronal oxidative stress.

Thanks to the development of efficient differentiation strategies, human pluripotent stem cells (HPSC) offer the opportunity for modelling neuronal injury and dysfunction in human neurons in vitro. Critically, the effective use of HPSC-derived neural cells in disease-modelling and potentially cell replacement therapies hinges on an understanding of the biology of these cells, specifically their development, subtype specification and responses to neurotoxic signalling mediators. Here, we generated neurons from human embryonic stem cells and characterised the development of vulnerability to glutamate excitotoxicity, a key contributor to neuronal injury in several acute and chronic neurodegenerative disorders. Over two months of differentiation we observed a gradual increase in responsiveness of neurons to glutamate-induced Ca(2+) influx, attributable to NMDA receptor activity. This increase was concomitant with an increase in expression of mRNA encoding NMDA and AMPA receptor subunits. Differentiated neurons were vulnerable to glutamate excitotoxicity in a dose-dependent manner, which was reduced by NMDA receptor antagonists.