Evaluation of rapamycin-induced cell death.

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase that integrates signals from nutrients and growth factors for the coordinate regulation of many cellular processes, including proliferation and cell death. Constitutive mTOR signaling characterizes multiple human malignancies, and pharmacological inhibitors of mTOR such as the immunosuppressant rapamycin and some of its nonimmunosuppressive derivatives not only have been ascribed with promising anticancer properties in vitro and in vivo but are also being extensively evaluated in clinical trials. mTOR inhibition rapidly leads to the activation of autophagy, which most often exerts prosurvival effects, although in some cases it accompanies cell death. Thus, depending on the specific experimental setting (cell type, concentration, stimulation time, and presence of concurrent stimuli), rapamycin can activate/favor a wide spectrum of cellular responses/phenotypes, ranging from adaptation to stress and survival to cell death. The (at least partial) overlap among the biochemical and morphological responses triggered by rapamycin considerably complicates the study of cell death-associated variables. Moreover, rapamycin presumably triggers acute cell death mainly via off-target mechanisms. Here, we describe a set of assays that can be employed for the routine quantification of rapamycin-induced cell death in vitro, as well as a set of guidelines that should be applied for their correct interpretation.

IKK connects autophagy to major stress pathways.

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

The IKK complex contributes to the induction of autophagy.

In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

p53 represses the polyploidization of primary mammary epithelial cells by activating apoptosis.

Tetraploidy may constitute a metastable state leading to numeric and structural chromosome abnormalities that are associated with cancer. Here, we show that cultured primary p53(-/-) (but not wild type, WT) mouse mammary epithelial cells (MMECs) accumulate a tetraploid sub-population in vitro. This occurs spontaneously, yet can be exacerbated by the addition of microtubule inhibitors as well as of inhibitors of cytokinesis. As compared to WT cells, tetraploid p53(-/-) MMECs contain supernumerary centrosomes and exhibit a reduced propensity to initiate the mitochondrial pathway of apoptosis. Moreover, tetraploid p53(-/-) MMECs are more resistant against anthracyclin-induced cell killing than their diploid counterparts. Altogether, these data indicate that p53 normally suppresses the generation of tetraploid cells, presumably by activating the intrinsic pathway of apoptosis. In the absence of p53, tetraploid cells accumulate as a result of inhibited apoptosis, which contributes to the acquisition of chemotherapy resistance.

Minimal chemical modification of reductive end of dextran to produce an amphiphilic polysaccharide able to incorporate onto lipid nanocapsules.

In order to graft an amphiphilic polysaccharide to lipid nanocapsules, we present here a new method of dextran lipidation. The lipidation strategy is based on the formation of an oxime linkage between the amphiphilic hydroxylamine C16E20ONH2 and the reductive end of a 40 kDa dextran. This chemoselective reaction allows us to control the lipidation site and the number of lipid introduced on the dextran molecule. This new amphiphilic dextran was used to coat the surface of lipid nanocapsules. The coating efficiency was followed by dynamic light scattering and the presence of the polysaccharide was confirmed by (1)H NMR and observed by electronic microscopy.

Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough.

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.

High-resolution magic angle spinning NMR of the neuronal tau protein integrated in Alzheimer's-like paired helical fragments.

HRMAS NMR of tau paired helical fragments assembled with heparin show an intensity decrease for those amino acids that are incorporated into the rigid core region, whereas the N-terminal amino acids maintain their full mobility.

Human eosinophils express and release IL-13 following CD28-dependent activation.

Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)-4, a key cytokine in the pathogenesis of allergic inflammation, and that IL-4 and IL-13 share common biological functions, we investigated the possibility that IL-13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL-13. Granule localization was demonstrated after subcellular fractionation, and IL-13 immunoreactivity was localized to crystalloid, granule-enriched fractions. Furthermore, electron microscopic analyses specifically localized IL-13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL-13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL-13 secretion. Furthermore, eosinophil-derived IL-13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL-13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.