Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.     

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

BackgroundFunctional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras.ResultsA recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~ 35% tumor progression in vivo in already established tumors.ConclusionsSelective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.     

Effects of subinhibitory concentrations of menthol on adaptation, morphological, and gene expression changes in enterohemorrhagic Escherichia coli

Menthol (C(10)H(20)O) possesses antibacterial activity; nevertheless, bacterial adaptation to this compound has never been studied. Here we report that precultivation of enterohemorrhagic Escherichia coli (EHEC) strains in increasing subinhibitory (SI) concentrations of menthol significantly elevates (4- to 16-fold) their resistance to menthol. Concomitant morphological alterations included the appearance of mucoid colonies and reduced biofilm production. Scanning electron microscopy (SEM) examination revealed suppressed curli formation in menthol-adapted cells. Expression of the gene cpsB10 (encoding one of the enzymes responsible for colanic acid production) was elevated in response to SI concentrations of menthol in a laboratory E. coli strain, whereas expression in an rcsC null mutant was reduced, implicating a partial role for the Rcs phosphorelay system in mediating the menthol signal. Adaptation to menthol also reduced expression of the locus of enterocyte effacement-encoded regulator (Ler). This reduction, together with reduced curli and biofilm formation and elevated mucoidity, suggests a general reduction in bacterial virulence following adaptation to menthol. Our results thus suggest menthol as a potential lead in the recently emerging alternative strategy of targeting bacterial virulence factors to develop new types of anti-infective agents.     

TCR zeta down-regulation under chronic inflammation is mediated by myeloid suppressor cells differentially distributed between various lymphatic organs

T cell AgR zeta chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; zeta chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1(+)Mac-1(+) myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their zeta expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer.     

Complementary medicine in the primary care setting: Results of a survey of gender and cultural patterns in Israel

Objective: The purpose of this study was to examine the use of complementary and alternative medicine (CAM) in a primary care practice in Israel to determine prevalence and patterns of use.Methods: Trained research assistants invited all patients attending the administrative, medical, pharmaceutical, or nursing services of 7 clinics in urban and rural areas of northern Israel over a 16-month period, from April 1, 2005, through August 1, 2006, to complete a 13-item written questionnaire about CAM use and beliefs about CAM safety and efficacy. CAM was defined as therapies often referred to as alternative, complementary, natural, or folk/traditional medicine, and which are not usually offered as part of the medical treatment in the clinic, including herbal medicine, Chinese medicine (including acupuncture), homeopathy, folk and traditional remedies, dietary/nutritional therapy (including nutritional supplements), chiropractic, movement/manual healing therapies (including massage, reflexology, yoga, and Alexander and Feldenkrais techniques), mind-body techniques (including meditation, guided imagery, and relaxation), energy and healing therapies, and other naturopathic therapies. The Pearson χ² test and multivariate logistic regression were used to assess univariate associations with the odds ratios of CAM use among Arab and Jewish women. A t test was performed to determine whether there were any differences in the continuous variables between the 2 groups.Results: Of 3972 consecutive patients who received the questionnaire, 3447 responded; 2139 respondents (62%) were women. Of the female respondents, 2121 reported their religion (1238 respondents [58%] self-identified as being Arab, and 883 [41.6%] as being Jewish). Compared with men, more women used CAM during the previous year (46.4% vs 39.4%; P < 0.001). Women were more likely to use CAM and to be interested in receiving CAM at primary care clinics. Arab women reported less CAM use than Jewish women but were more interested in experiencing CAM, had a higher degree of confidence in CAM efficacy and safety, and more frequently supported the integration of CAM practitioners in primary care clinics.Conclusions: In this study, women visiting primary care clinics in northern Israel used CAM more often than men did. Arab women reported less use of CAM than did Jewish women but also reported greater confidence in CAM efficacy and safety.     

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane “sleeve”, split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities’ organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as “ready to go” prefabricated presynaptic terminals. We suggest that the varicosities’ motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse. Electronic Supplementary Material Supplementary material is available in the online version of this article at These authors contributed equally to the paper.