Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells

IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th(2) cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its "normal" and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.     

The peptaibol alamethicin induces an rRNA-cleavage-associated death in Arabidopsis thaliana

The plant-metabolic response to amphipathic peptides produced by the soil fungi of the genus Trichoderma remains largely unknown. The present investigation was undertaken to examine the death process in alamethicin-treated Arabidopsis thaliana plantlets. The rapid death triggered by alamethicin (at 50 microM) was shown to be associated with protein-synthesis arrest and with specific cleavage of 18S and 25S ribosomal RNA. The use of an inhibitor of nitric oxide (NO) synthases and of an NO scavenger suggested that rRNA cleavage was suppressed by NO. Experiments conducted with a synthetic alamethicin analogue, in which all alpha-aminoisobutyric acid (Aib) residues have been replaced by leucine moieties, showed that the non-coded residues are essential for the ability of the peptaibol to induce rRNA cleavage in Arabidopsis. Our data indicate that further investigations on the mode of action of alamethicin in planta could be of great interest to study the death-signaling pathway associated with rRNA degradation in plants.     

Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

Despite increasing evidence of behavioural manipulation of their vectors by pathogens, the underlying mechanisms causing infected vectors to act in ways that benefit pathogen transmission remain enigmatic in most cases. Here, 2-D DIGE coupled with MS were employed to analyse and compare the head proteome of mosquitoes (Anopheles gambiae sensu stricto (Giles)) infected with the malarial parasite (Plasmodium berghei) with that of uninfected mosquitoes. This approach detected altered levels of 12 protein spots in the head of mosquitoes infected with sporozoites. These proteins were subsequently identified using MS and functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling, and cytoskeletal groups. Our results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapse-associated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins sheds light on potential molecular mechanisms that underlie behavioural modifications and offers new insights into the study of intimate interactions between Plasmodium and its Anopheles vector.     

Differential kinetics of antigen-specific CD4+ and CD8+ T cell responses in the regression of retrovirus-induced sarcomas

Despite the accepted role for CD4+ T cells in immune control, little is known about the development of Ag-specific CD4+ T cell immunity upon primary infection. Here we use MHC class II tetramer technology to directly visualize the Ag-specific CD4+ T cell response upon infection of mice with Moloney murine sarcoma and leukemia virus complex (MoMSV). Significant numbers of Ag-specific CD4+ T cells are detected both in lymphoid organs and in retrovirus-induced lesions early during infection, and they express the 1B11-reactive activation-induced isoform of CD43 that was recently shown to define effector CD8+ T cell populations. Comparison of the kinetics of the MoMSV-specific CD4+ and CD8+ T cell responses reveals a pronounced shift toward CD8+ T cell immunity at the site of MoMSV infection during progression of the immune response. Consistent with an important early role of Ag-specific CD4+ T cell immunity during MoMSV infection, CD4+ T cells contribute to the generation of virus-specific CD8+ T cell immunity within the lymphoid organs and are required to promote an inflammatory environment within the virus-infected tissue.     

Alcohol Consumption in Midlife and Cognitive Performance Assessed 13 Years Later in the SU.VI.MAX 2 Cohort

Background

Associations between alcohol consumption and cognitive function are discordant and data focusing on midlife exposure are scarce.

Objective

To estimate the association between midlife alcohol consumption and cognitive performance assessed 13 y later while accounting for comorbidities and diet.

Methods

3,088 French middle-aged adults included in the SU.VI.MAX (1994) study with available neuropsychological evaluation 13 y later. Data on alcohol consumption were obtained from repeated 24h dietary records collected in 1994–1996. Cognitive performance was assessed in 2007–2009 via a battery of 6 neuropsychological tests. A composite score was built as the mean of the standardized individual test scores (mean = 50, SD = 10). ANCOVA were performed to estimate mean differences in cognitive performance and 95% confidence intervals (CI).

Results

In women, abstainers displayed lower cognitive scores than did low-to-moderate alcohol drinkers (1 to 2 drinks/day) (mean difference = −1.77; 95% CI: −3.29, −0.25). In men, heavy drinkers (>3 drinks/day) had higher cognitive scores than did low-to-moderate (1 to 3 drinks/day) (mean difference = 1.05; 95% CI: 0.10, 1.99). However, a lower composite cognitive score was detected in male drinkers consuming ≥90 g/d (≈8 drinks/d). A higher proportion of alcohol intake from beer was also associated with lower cognitive scores. These associations remained significant after adjustment for diet, comorbidities and sociodemographic factors.

Conclusion

In men, heavy but not extreme drinking was associated with higher global cognitive scores. Given the known harmful effects of alcohol even in low doses regarding risk of cancer, the study does not provide a basis for modifying current public health messages.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00272428","term_id":"NCT00272428"}}NCT00272428     

Revisiting an Old Riddle: What Determines Genetic Diversity Levels within Species?

Understanding why some species have more genetic diversity than others is central to the study of ecology and evolution, and carries potentially important implications for conservation biology. Yet not only does this question remain unresolved, it has largely fallen into disregard. With the rapid decrease in sequencing costs, we argue that it is time to revive it.What evolutionary forces maintain genetic diversity in natural populations? How do diversity levels relate to census population sizes (Box 1)? Do low levels of diversity limit adaptation to novel selective pressures? Efforts to address such questions spurred the rise of modern population genetics and contributed to the development of the neutral theory of molecular evolution—the null hypothesis for much of evolutionary genetics and comparative genomics [1]–[3]. Yet these questions remain wide open and, for close to two decades, have been neglected [4]. Most notably, little progress has been made to resolve a riddle first pointed out 40 years ago on the basis of allozyme data: the mysteriously narrow range of genetic diversity levels seen across taxa that vary markedly in their census population sizes [5]. This gap in our understanding is glaring, and may hamper efforts at conservation (e.g., [6]).

Box 1. Glossary

Allozymes: Allelic variants of a protein, often detected by differences in gel electrophoresis. Balancing selection: Natural selection that maintains variation longer than expected from genetic drift alone. Census population size: The actual number of individuals in a population; methods to estimate this number vary depending on the species and may involve aerial, transect, or capture/recapture counts. Diversity levels: The measure used here is the probability that a pair of randomly chosen haplotypes differ at a site. Effective population size: The size of an idealized population with some of the same properties as the actual one, e.g., the same rate of genetic drift. Under simplifying assumptions, notably a constant population size and no population structure, this parameter can be estimated from observed diversity levels, given an independent estimate of the mutation rate. Fluctuating selection: When the fitness of an allele changes over time or over space. Genetic draft: A dramatic loss of genetic variation due to strong, frequent selection at nearby sites [8]. Genetic drift: In a finite population, the loss of genetic variation due to the random sampling of gametes at each generation. Local adaptation: Adaptation to a particular environment that is not shared by the entire species. Nearly neutral theory of molecular evolution: A modification of the neutral theory, in which many mutations are slightly deleterious, rather than strictly neutral or strongly deleterious [75]. Neutral theory of molecular evolution: The theory that most genetic variation seen within populations and between species is neutral, and most mutations are either neutral or strongly deleterious [11]. Panmixia: Random mating among individuals, and hence no population structure. Phylogenetically independent contrasts: A statistical method that allows one to compare properties of species controlling for their evolutionary relationship. Purifying (negative) selection: Natural selection that favors the common, fitter allele against rare, deleterious alleles. Selection at linked sites: Selection at sites linked to the locus under consideration, which can affect the population dynamics of alleles at that locus. Silent sites: A general term for synonymous, intronic, and intergenic sites—all sites at which mutations do not change an amino acid. Variation-reducing selection: Selection that leads to a decrease in diversity at linked sites.With the recent technological revolution in sequencing, the data needed to address questions about the determinants of genetic diversity levels are now within reach. As a first step towards reviving these questions, we compile existing estimates of nuclear sequence diversity. These data are highly preliminary, but they underscore how little is known about the narrow span of diversity levels across species or why some species maintain more genetic variation than others [5],[7],[8], and they offer a glimpse of trends that may be worth pursuing.     

TNF inhibited the apoptosis by activation of Akt serine/threonine kinase in the human head and neck squamous cell carcinoma

Tumour necrosis factor (TNF) is known to induce apoptosis, but recently, TNF was shown to promote cell survival, a process regulated by phosphatidylinositol-3-OH kinase (PI3K) and the NFkappaB pathway. In this study, we investigated the relationship between the molecules implicated in regulating TNF-induced cell survival and apoptosis induced by TNF in a human head and neck squamous cell carcinoma cell line (SAS), with special reference to the Akt pathway, one of the pathways related to cell survival. In SAS cells, TNF induced the phosphorylation of Akt at both Ser473 and Thr308, causing the activation of Akt, and also induced the phosphorylation and degradation of IkappaB (inhibitor of NFkappaB). This phosphorylation and degradation was inhibited by pretreating the cells with the PI3K inhibitors, wortmannin or LY294002. The apoptosis of SAS cells induced by TNF was dependent on the concentration: a high concentration of TNF, but not a low concentration, induced apoptosis within 30 h. However, a low concentration of TNF in the presence of wortmannin or LY294002 induced apoptosis. Furthermore, expression of the kinase-negative form of Akt, IKKalpha or IKKbeta, and the undegradable mutant of IkappaB, also induced apoptosis at low concentrations of TNF. When the SAS cells expressed constitutively activated Akt, apoptosis was not induced, even by high concentrations of TNF. These observations suggest that, in the SAS cell line, the PI3K-NFkappaB pathway contributes to TNF-induced cell survival and that inhibition of this pathway accelerates apoptosis.     

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.     

CD4 T cell-dependent autoimmunity against a melanocyte neoantigen induces spontaneous vitiligo and depends upon Fas-Fas ligand interactions

Better understanding of tolerance and autoimmunity toward melanocyte-specific Ags is needed to develop effective treatment for vitiligo and malignant melanoma; yet, a systematic assessment of these mechanisms has been hampered by the difficulty in tracking autoreactive T cells. To address this issue, we have generated transgenic mice that express hen egg lysozyme as a melanocyte-specific neoantigen. By crossing these animals to a hen egg lysozyme-specific CD4 TCR transgenic line we have been able to track autoreactive CD4+ T cells from their development in the thymus to their involvement in spontaneous autoimmune disease with striking similarity to human vitiligo vulgaris and Vogt-Koyanagi-Harada syndrome. Our findings show that CD4-dependent destruction of melanocytes is partially inhibited by blocking Fas-Fas ligand interactions and also highlights the importance of local control of autoimmunity, as vitiligo remains patchy and never proceeds to confluence even when Ag and autoreactive CD4+ T cells are abundant. Immune therapy to enhance or suppress melanocyte-specific T cells can be directed at a series of semiredundant pathways involving tolerance and cell death.     

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.