Evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a B16 mouse melanoma model.

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model. EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically. RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated gene therapy. No significant increases in serum IL-12 levels were detected. Tumor-bearing mice showed an increased number of atypical hematology values when compared with normal naive controls. Statistically significant differences in chemistry and hematology values were observed sporadically in most of the standard chemistry and hematology categories in all groups. The only histopathologic abnormality specific to the animals receiving both plasmid and electroporation was inflammation associated with the kidney at the last time point. CONCLUSIONS: In general, mice that received both plasmid and electroporation showed the least abnormal histopathologic findings and were found to be in the best health, reflecting the reduced burden of disease. No significant toxic effects due to the IL-12 gene therapy were observed.     

Focal Sleep Spindle Deficits Reveal Focal Thalamocortical Dysfunction and Predict Cognitive Deficits in Sleep Activated Developmental Epilepsy

Childhood epilepsy with centrotemporal spikes (CECTS) is the most common focal epilepsy syndrome, yet the cause of this disease remains unknown. Now recognized as a mild epileptic encephalopathy, children exhibit sleep-activated focal epileptiform discharges and cognitive difficulties during the active phase of the disease. The association between the abnormal electrophysiology and sleep suggests disruption to thalamocortical circuits. Thalamocortical circuit dysfunction resulting in pathologic epileptiform activity could hinder the production of sleep spindles, a brain rhythm essential for memory processes. Despite this pathophysiologic connection, the relationship between spindles and cognitive symptoms in epileptic encephalopathies has not been previously evaluated. A significant challenge limiting such work has been the poor performance of available automated spindle detection methods in the setting of sharp activities, such as epileptic spikes. Here, we validate a robust new method to accurately measure sleep spindles in patients with epilepsy. We then apply this detector to a prospective cohort of male and female children with CECTS with combined high-density EEGs during sleep and cognitive testing at varying time points of disease. We show that: (1) children have a transient, focal deficit in spindles during the symptomatic phase of disease; (2) spindle rate anticorrelates with spike rate; and (3) spindle rate, but not spike rate, predicts performance on cognitive tasks. These findings demonstrate focal thalamocortical circuit dysfunction and provide a pathophysiological explanation for the shared seizures and cognitive symptoms in CECTS. Further, this work identifies sleep spindles as a potential treatment target of cognitive dysfunction in this common epileptic encephalopathy.SIGNIFICANCE STATEMENT Childhood epilepsy with centrotemporal spikes is the most common idiopathic focal epilepsy syndrome, characterized by self-limited focal seizures and cognitive symptoms. Here, we provide the first evidence that focal thalamocortical circuit dysfunction underlies the shared seizures and cognitive dysfunction observed. In doing so, we identify sleep spindles as a mechanistic biomarker, and potential treatment target, of cognitive dysfunction in this common developmental epilepsy and provide a novel method to reliably quantify spindles in brain recordings from patients with epilepsy.     

Calculating the Risk Benefit Equation for Aggressive Treatment of Non-convulsive Status Epilepticus

Objective

To address the question: does non-convulsive status epilepticus warrant the same aggressive treatment as convulsive status epilepticus?

Methods

We used a decision model to evaluate the risks and benefits of treating non-convulsive status epilepticus with intravenous anesthetics and ICU-level aggressive care. We investigated how the decision to use aggressive versus non-aggressive management for non-convulsive status epilepticus impacts expected patient outcome for four etiologies: absence epilepsy, discontinued antiepileptic drugs, intraparenchymal hemorrhage, and hypoxic ischemic encephalopathy. Each etiology was defined by distinct values for five key parameters: baseline mortality rate of the inciting etiology; efficacy of non-aggressive treatment in gaining control of seizures; the relative contribution of seizures to overall mortality; the degree of excess disability expected in the case of delayed seizure control; and the mortality risk of aggressive treatment.

Results

Non-aggressive treatment was favored for etiologies with low morbidity and mortality such as absence epilepsy and discontinued antiepileptic drugs. The risk of aggressive treatment was only warranted in etiologies where there was significant risk of seizure-induced neurologic damage. In the case of post-anoxic status epilepticus, expected outcomes were poor regardless of the treatment chosen. The favored strategy in each case was determined by strong interactions of all five model parameters.

Conclusions

Determination of the optimal management approach to non-convulsive status epilepticus is complex and is ultimately determined by the inciting etiology.     

Global infant mortality trends and attributable determinants – an ecological study using data from 192 countries for the period 1990–2011

Background

Infant mortality rate (IMR) is regarded as an important indicator of population health. IMR rates vary substantially with the highest found in sub-Saharan Africa (SSA) compared to the lowest in Europe. Identifying spatial disparities in IMR and quantifying attributable risk factors is essential for policymakers when tailoring time-appropriate interventions at a global, regional, and country level.

Methods

Data for 192 countries were extracted from the World Bank Development Indicator database for the period 1990–2011. Spatial clustering was used to identify significant higher-risk IMR countries. A robust ecological generalized linear negative binomial regression model was used to quantify risk factors and associated decomposition values (Shapley).

Results

Significant reductions were observed in IMR for all of the World Health Organization regions for the period 1990–2011 except for SSA, which indicated a reversal of this trend in the 1990s due to HIV. Significant high-risk clustering of IMR is also indicated in SSA countries and parts of Asia. Maternal mortality (survival), lack of water and sanitation and female education were confirmed as prominent and high attributable risk factors for IMR. Distinct temporal changes in the attributability of these factors were observed, as well as significant heterogeneity with regards to the most attributable factor by region and country.

Conclusions

Our study suggests that maternal mortality is the most prominent attributable risk factor for infant mortality, followed by lack of access to sanitation, lack of access to water, and lower female education. Variation exists across regions and countries with regards to the most attributable factor. Our study also suggests significant underestimation of IMR in regions known for poorer data quality. The results will aid policymakers in re-tailoring time-appropriate interventions to more effectively reduce IMR in line with Millennium Development Goal 4.

     

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.Multiple lines of evidence suggest that if achievable, inhibiting V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) signaling may have therapeutic advantages in cancer. Approximately 30% of all human cancers contain activating Ras mutations, making them one of the most common identifiable molecular drivers of cancer (1, 2). K-Ras mutation–positive tumors tend to be less responsive to current therapy than other biological subtypes, and patients with these tumors have worse cancer outcomes (3, 4). We aim to develop and evaluate direct-acting inhibitors of K-Ras as a therapeutic strategy.Ras proteins are GTPase enzymes that transduce extracellular signals when growth factors bind to extracellular receptors, resulting in cellular responses such as proliferation, apoptosis, and differentiation (5). Activating signals are transmitted when Ras is bound to GTP and cease once the bound GTP is hydrolyzed to GDP. Two structurally dynamic loops, so-called “switch I” (residues 25–40 in K-Ras) and “switch II” (residues 57–75 in K-Ras) form a key portion of the binding interface between K-Ras and a number of regulators and downstream effectors, including Raf kinases, PI3 kinases, and RalGDS (6–8). We hypothesize that to be successful, direct-acting K-Ras inhibitors must alter the conformation of switch I and/or switch II such that it becomes incapable of transmitting activating signals. Because the guanine nucleotide (GN)-binding pocket dictates the switch conformation, we postulate that developing compounds binding to this region will have a high likelihood of modulating K-Ras signaling.Development of GN competitive inhibitors of K-Ras is challenging because GTP and GDP bind with subnanomolar affinity and intracellular concentrations of GTP and GDP are high. Recently, our group (9) and Shokat and coworkers (10) independently developed and reported two classes of compounds that have a direct impact on productive nucleotide binding to the GN-binding site. Both target a common activating mutation, G12C, to achieve irreversible binding. K-Ras G12C is present in an estimated 10–20% of Ras-driven cancers and in roughly 50% of Ras-mutated lung adenocarcinomas (11–13). For lung cancer alone, this means that therapeutics targeting the G12C mutation could treat roughly 25,000 people per year in the United States (14). Mutations at codon 12, along with the other most common cancer-causing Ras mutations at codons 13 and 61, decrease intrinsic GTPase activity to some extent and impair interactions with GTPase-activating proteins that modulate (GTP) hydrolysis. Codon 12 is adjacent to the active site, such that the mutation places a solvent-accessible cysteine near the GN terminal phosphate.We previously reported development of SML-8-73-1 (SML), a GDP analog containing an electrophilic warhead extending from the beta-phosphate that undergoes a Michael reaction addition to Cys-12, forming a stable thioether linkage (9). Even in the presence of large excesses of GDP and GTP, quantitative complete irreversible binding was observed by MS. In biochemical assays, SML prevents K-Ras association with the Ras-binding domain of the downstream effector BRaf. Preliminary cellular tests using a cell-permeable caged prodrug version, SML-10-70-1, demonstrated that treatment of a G12C mutant K-Ras cancer cell line with the SML class of compounds, albeit at a high concentration (100 μM), leads to inactivation of K-Ras and down-regulation of Akt and Erk signaling pathways, demonstrating as a proof of concept that the GN-binding pocket is a viable target for inhibitors of Ras signaling.Here, we provide three lines of evidence to support further the concept of targeting the GN-binding pocket of K-Ras. First, we report high-resolution X-ray crystal structures of K-Ras, including a structure containing the irreversible inhibitor, SML, bound to K-Ras G12C. The models are analyzed with the aim of understanding implications for K-Ras interactions with downstream effectors. Second, we address compound selectivity with MS-based in situ profiling demonstrating that SML preferentially interacts with K-Ras G12C over most other cellular GTP-binding proteins. Finally, we provide additional evidence that in a purified system, SML is able to compete for the GN-binding site of K-Ras G12C in the presence of millimolar concentrations of GTP and GDP, similar to those found in a living cell. The prospects for developing GTPase inhibitors are also broadly considered.