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.     

Evolutionary and Functional Analysis of Celiac Risk Loci Reveals SH2B3 as a Protective Factor against Bacterial Infection

Celiac disease (CD) is an intolerance to dietary proteins of wheat, barley, and rye. CD may have substantial morbidity, yet it is quite common with a prevalence of 1%–2% in Western populations. It is not clear why the CD phenotype is so prevalent despite its negative effects on human health, especially because appropriate treatment in the form of a gluten-free diet has only been available since the 1950s, when dietary gluten was discovered to be the triggering factor. The high prevalence of CD might suggest that genes underlying this disease may have been favored by the process of natural selection. We assessed signatures of selection for ten confirmed CD-associated loci in several genome-wide data sets, comprising 8154 controls from four European populations and 195 individuals from a North African population, by studying haplotype lengths via the integrated haplotype score (iHS) method. Consistent signs of positive selection for CD-associated derived alleles were observed in three loci: IL12A, IL18RAP, and SH2B3. For the SH2B3 risk allele, we also show a difference in allele frequency distribution (F_st) between HapMap phase II populations. Functional investigation of the effect of the SH2B3 genotype in response to lipopolysaccharide and muramyl dipeptide revealed that carriers of the SH2B3 rs3184504^∗A risk allele showed stronger activation of the NOD2 recognition pathway. This suggests that SH2B3 plays a role in protection against bacteria infection, and it provides a possible explanation for the selective sweep on SH2B3, which occurred sometime between 1200 and 1700 years ago.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Potential celiac patients: a model of celiac disease pathogenesis

Background and Aim

Potential celiacs have the ‘celiac type’ HLA, positive anti-transglutaminase antibodies but no damage at small intestinal mucosa. Only a minority of them develops mucosal lesion. More than 40 genes were associated to Celiac Disease (CD) but we still do not know how those pathways transform a genetically predisposed individual into an affected person. The aim of the study is to explore the genetic features of Potential CD individuals.

Methods

127 ‘potential’ CD patients entered the study because of positive anti-tissue transglutaminase and no mucosal lesions; about 30% of those followed for four years become frankly celiac. They were genotyped for 13 polymorphisms of ‘candidate genes’ and compared to controls and celiacs. Moreover, 60 biopsy specimens were used for expression studies.

Results

Potential CD bear a lighter HLA-related risk, compared to celiac (χ² = 48.42; p value = 1×10⁻⁸). They share most of the polymorphisms of the celiacs, but the frequency of c-REL* G allele was suggestive for a difference compared to celiac (χ² = 5.42; p value = 0.02). One marker of the KIAA1109/IL-2/IL-21 candidate region differentiated potentials from celiac (rs4374642: χ2 = 7.17, p value = 0.01). The expression of IL-21 was completely suppressed in potentials compared to celiacs (p value = 0.02) and to controls (p value = 0.02), in contrast IL-2, KIAA1109 and c-REL expression were over-expressed.

Conclusions

Potential CD show genetic features slightly different from celiacs. Genetic and expression markers help to differentiate this condition. Potential CD is a precious biological model of the pathways leading to the small intestinal mucosal damage in genetically predisposed individuals.     

The early postnatal nonhuman primate neocortex contains self-renewing multipotent neural progenitor cells

The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine.     

Bleomycin-induced pulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2.

The importance of HER2/HER3 signaling in decreasing the effects of lung injury was recently demonstrated. Transgenic mice unable to signal through HER2/HER3 had significantly less bleomycin-induced pulmonary fibrosis and showed a survival benefit. Based on these data, we hypothesized that pharmacological blockade of HER2/HER3 in vivo in wild-type mice would have the same beneficial effects. We tested this hypothesis in a bleomycin lung injury model using 2C4, a monoclonal antibody directed against HER2 that blocks HER2/HER3 signaling. The administration of 2C4 before injury decreased the effects of bleomycin at days 15 and 21 after injury. HER2/HER3 blockade resulted in less collagen deposition (362.8 +/- 37.9 compared with 610.5 +/- 27.1 microg/mg; P = 0.03) and less lung morphological changes (injury score of 1.99 +/- 1.55 vs. 3.90 +/- 0.76; P < 0.04). In addition, HER2/HER3 blockade resulted in a significant survival advantage with 50% vs. 25% survival at 30 days (P = 0.04). These results confirm that HER2 signaling can be pharmacologically targeted to reduce lung fibrosis and remodeling after injury.     

Expression of mutant human epidermal receptor 3 attenuates lung fibrosis and improves survival in mice.

Neuregulin-1 (NRG-1), binding to the human epidermal growth factor receptor HER2/HER3, plays a role in pulmonary epithelial cell proliferation and recovery from injury in vitro. We hypothesized that activation of HER2/HER3 by NRG-1 would also play a role in recovery from in vivo lung injury. We tested this hypothesis using bleomycin lung injury of transgenic mice incapable of signaling through HER2/HER3 due to lung-specific dominant-negative HER3 (DNHER3) expression. In animals expressing DNHER3, protein leak, cell infiltration, and NRG-1 levels in bronchoalveolar lavage fluid increased after injury, similar to that in nontransgenic littermate control animals. However, HER2/HER3 was not activated, and DNHER3 animals displayed fewer lung morphological changes at 10 and 21 days after injury (P = 0.01). In addition, they contained 51% less collagen in injured lungs (P = 0.04). Transforming growth factor-beta1 did not increase in bronchoalveolar lavage fluid from DNHER3 mice compared with nontransgenic littermate mice (P = 0.001), suggesting that a mechanism for the decreased fibrosis was lack of transforming growth factor-beta1 induction in DNHER3 mice. Severe lung injury (0.08 units bleomycin) resulted in 80% mortality of nontransgenic mice, but only 35% mortality of DNHER3 transgenic mice (P = 0.04). Thus inhibition of HER2/HER3 signaling protects against pulmonary fibrosis and improves survival.