Synergistic Antitumor Effect between Gefitinib and Fractionated Irradiation in Anaplastic Oligodendrogliomas Cannot Be Predicted by the Egfr Signaling Activity

In high-grade gliomas, the identification of patients that could benefit from EGFR inhibitors remains a challenge, hindering the use of these agents. Using xenografts models, we evaluated the antitumor effect of the combined treatment “gefitinib + radiotherapy” and aimed to identify the profile of responsive tumors. Expression of phosphorylated proteins involved in the EGFR-dependent signaling pathways was analyzed in 10 glioma models. We focused on three models of anaplastic oligodendrogliomas (TCG2, TCG3 and TCG4) harboring high levels of phospho-EGFR, phospho-AKT and phospho-MEK1. They were treated with gefitinib (GEF 75 mg/kg/day x 5 days/week, for 2 weeks) and/or fractionated radiotherapy (RT: 5x2Gy/week for 2 weeks). Our results showed that GEF and/or RT induced significant tumor growth delays. However, only the TCG3 xenografts were highly responsive to the combination GEF+RT, with ∼50% of tumor cure. Phosphoproteins analysis five days after treatment onset demonstrated in TCG3 xenografts, but not in TCG2 model, that the EGFR-dependent pathways were inhibited after GEF treatment. Moreover, TCG3-bearing mice receiving GEF monotherapy exhibited a transient beneficial therapeutic response, rapidly followed by tumor regrowth, along with a major vascular remodeling. Taken together, our data evoked an “EGFR-addictive” behavior for TCG3 tumors. This study confirms that combination of gefitinib with fractionated irradiation could be a potent therapeutic strategy for anaplastic oligodendrogliomas harboring EGFR abnormalities but this treatment seems mainly beneficial for “EGFR-addictive” tumors. Unfortunately, neither the usual molecular markers (EGFR amplification, PTEN loss) nor the basal overexpression of phosphoproteins were useful to distinguish this responsive tumor. Evaluating the impact of TKIs on the EGFR-dependent pathways during the treatment might be more relevant, and requires further validation.     

Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

Canine Adenovirus Type 2 Vector Generation via I-Sce1-Mediated Intracellular Genome Release

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.     

Improved Diagnosis in Children with Partial Epilepsy Using a Multivariable Prediction Model Based on EEG Network Characteristics

Background

Electroencephalogram (EEG) acquisition is routinely performed to support an epileptic origin of paroxysmal events in patients referred with a possible diagnosis of epilepsy. However, in children with partial epilepsies the interictal EEGs are often normal. We aimed to develop a multivariable diagnostic prediction model based on electroencephalogram functional network characteristics.

Methodology/Principal Findings

Routinely performed interictal EEG recordings at first presentation of 35 children diagnosed with partial epilepsies, and of 35 children in whom the diagnosis epilepsy was excluded (control group), were used to develop the prediction model. Children with partial epilepsy were individually matched on age and gender with children from the control group. Periods of resting-state EEG, free of abnormal slowing or epileptiform activity, were selected to construct functional networks of correlated activity. We calculated multiple network characteristics previously used in functional network epilepsy studies and used these measures to build a robust, decision tree based, prediction model. Based on epileptiform EEG activity only, EEG results supported the diagnosis of with a sensitivity and specificity of 0.77 and 0.91 respectively. In contrast, the prediction model had a sensitivity of 0.96 [95% confidence interval: 0.78–1.00] and specificity of 0.95 [95% confidence interval: 0.76–1.00] in correctly differentiating patients from controls. The overall discriminative power, quantified as the area under the receiver operating characteristic curve, was 0.89, defined as an excellent model performance. The need of a multivariable network analysis to improve diagnostic accuracy was emphasized by the lack of discriminatory power using single network characteristics or EEG''s power spectral density.

Conclusions/Significance

Diagnostic accuracy in children with partial epilepsy is substantially improved with a model combining functional network characteristics derived from multi-channel electroencephalogram recordings. Early and accurate diagnosis is important to start necessary treatment as soon as possible and inform patients and parents on possible risks and psychosocial aspects in relation to the diagnosis.     

Richness and Composition of Niche-Assembled Viral Pathogen Communities

The pathogen and parasite community that inhabits every free-living organism can control host vital rates including lifespan and reproductive output. To date, however, there have been few experiments examining pathogen community assembly replicated at large-enough spatial scales to inform our understanding of pathogen dynamics in natural systems. Pathogen community assembly may be driven by neutral stochastic colonization and extinction events or by niche differentiation that constrains pathogen distributions to particular environmental conditions, hosts, or vectors.Here, we present results from a regionally-replicated experiment investigating the community of barley and cereal yellow dwarf viruses (B/CYDV''s) in over 5000 experimentally planted individuals of six grass species along a 700 km latitudinal gradient along the Pacific coast of North America (USA) in response to experimentally manipulated nitrogen and phosphorus supplies. The composition of the virus community varied predictably among hosts and across nutrient-addition treatments, indicating niche differentiation among virus species. There were some concordant responses among the viral species. For example, the prevalence of most viral species increased consistently with perennial grass cover, leading to a 60% increase in the richness of the viral community within individual hosts (i.e., coinfection) in perennial-dominated plots. Furthermore, infection rates of the six host species in the field were highly correlated with vector preferences assessed in laboratory trials. Our results reveal the importance of niche differentiation in structuring virus assemblages. Virus species distributions reflected a combination of local host community composition, host species-specific vector preferences, and virus responses to host nutrition. In addition, our results suggest that heterogeneity among host species in their capacity to attract vectors or support pathogens between growing seasons can lead to positive covariation among virus species.     

Effectiveness of Patient Adherence Groups as a Model of Care for Stable Patients on Antiretroviral Therapy in Khayelitsha,Cape Town,South Africa

Background

Innovative models of care are required to cope with the ever-increasing number of patients on antiretroviral therapy in the most affected countries. This study, in Khayelitsha, South Africa, evaluates the effectiveness of a group-based model of care run predominantly by non-clinical staff in retaining patients in care and maintaining adherence.

Methods and Findings

Participation in “adherence clubs” was offered to adults who had been on ART for at least 18 months, had a current CD4 count >200 cells/ml and were virologically suppressed. Embedded in an ongoing cohort study, we compared loss to care and virologic rebound in patients receiving the intervention with patients attending routine nurse-led care from November 2007 to February 2011. We used inverse probability weighting to estimate the intention-to-treat effect of adherence club participation, adjusted for measured baseline and time-varying confounders. The principal outcome was the combination of death or loss to follow-up. The secondary outcome was virologic rebound in patients who were virologically suppressed at study entry. Of 2829 patients on ART for >18 months with a CD4 count above 200 cells/µl, 502 accepted club participation. At the end of the study, 97% of club patients remained in care compared with 85% of other patients. In adjusted analyses club participation reduced loss-to-care by 57% (hazard ratio [HR] 0.43, 95% CI = 0.21–0.91) and virologic rebound in patients who were initially suppressed by 67% (HR 0.33, 95% CI = 0.16–0.67).

Discussion

Patient adherence groups were found to be an effective model for improving retention and documented virologic suppression for stable patients in long term ART care. Out-of-clinic group-based models facilitated by non-clinical staff are a promising approach to assist in the long-term management of people on ART in high burden low or middle-income settings.