TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th₁ polarized CD4⁺ and CD8⁺ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8⁺ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.     

A Farm to Fork Risk Assessment for the Use of Wastewater in Agriculture in Accra,Ghana

The need to minimise consumer risk, especially for food that can be consumed uncooked, is a continuing public health concern, particularly in places where safe sanitation and hygienic practices are absent. The use of wastewater in agriculture has been associated with disease risks, though its relative significance in disease transmission remains unclear. This study aimed at identifying key risk factors for produce contamination at different entry points of the food chain. Over 500 produce and ready-to-eat salad samples were collected from fields, markets, and kitchens during the dry and wet seasons in Accra, Ghana, and over 300 soil and irrigation water samples were collected. All samples were analysed for E. coli, human adenovirus and norovirus using standard microbiological procedures, and real time RT-PCR. Finally, critical exposures associated with microbial quality of produce were assessed through observations and interviews. The study found that over 80% of produce samples were contaminated with E. coli, with median concentrations ranging from 0.64 to 3.84 Log E. coli/g produce. Prepared salad from street food vendors was found to be the most contaminated (4.23 Log E. coli/g), and that consumption of salad exceeded acceptable health limits. Key risk factors identified for produce contamination were irrigation water and soil at the farm level. Storage duration and temperature of produce had a significant influence on the quality of produce sold at markets, while observations revealed that the washed water used to rinse produce before sale was dirty. The source of produce and operating with a hygiene permit were found to influence salad microbial quality at kitchens. This study argues for a need to manage produce risk factors at all domains along the food chain, though it would be more effective to prioritise at markets and kitchens due to cost, ease of implementation and public health significance.     

Impact of herbivory on performance of <Emphasis Type="Italic">Vincetoxicum</Emphasis> spp., invasive weeds in North America

The alien invasive vines Vincetoxicum rossicum and Vincetoxicum nigrum (swallow-wort) are of major concern in eastern North America, where both species invade forested landscapes and threaten faunal and plant diversity. Among the few native natural enemies reported in Eurasia, the specialist chrysomelid, Chrysochus (Eumolpus) asclepiadeus (Coleoptera; Chrysomelidae), feeds on Vincetoxicum both above ground (as adults) and below ground (as larvae). The goal of our study was to assess the potential for using this beetle to manage invasive Vincetoxicum spp. in North America by quantifying the impact of herbivory by C. asclepiadeus on Vincetoxicum and determining whether this effect was influenced by plant density (“Allee effect”). Experimental work was carried out using a split plot design in the field in southern France. Pots of V. nigrum and V. hirundinaria, a substitute for V. rossicum, were planted at high (255 plants/m²), medium (127 plants/m²), and low (32 plants/m²) plant densities, and received treatments of 0 (control), 2 or 4 C. asclepiadeus adult beetles/pot. Leaf damage, root and shoot biomass, and quantity of seeds were measured after 4 weeks of adult feeding. Densities of 2 and 4 beetles/pot caused similar damage, with significant reductions in plant biomass at low plant density. While V. hirundinaria increased allocation of resources to roots in response to herbivory, V. nigrum did not. Seed production was greatest for both species grown at low plant densities, but only V. nigrum produced fewer seeds in response to herbivory. Our results, based on the effects of herbivory by C. asclepiadeus adults, suggest that if this beetle were to be introduced into North America for the management of Vincetoxicum spp. such as V. rossicum, reductions in plant biomass and spread would be greatest if beetles were released on edges or in newly-established satellite populations at low plant densities. In the case of V. nigrum, beetles could be released irrespective of plant density as reproductive output and seed dispersal would be reduced similarly.     

The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.     

Amyloid-beta42 interacts mainly with insoluble prion protein in the Alzheimer brain

The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-β (Aβ42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aβ42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aβ42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aβ from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aβ binding sites on huPrP are identified in vitro. One specifically binds to Aβ42 and the other binds to both Aβ42 and Aβ40. Notably, Aβ42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aβ40 and Aβ42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aβ42 in vivo. Although this work indicated the interaction of Aβ42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aβ42 interaction remains to be established.