Rapid and Continued T-Cell Differentiation into Long-term Effector and Memory Stem Cells in Vaccinated Melanoma Patients.

<b>Purpose:</b> Patients with cancer benefit increasingly from T-cell-based therapies, such as adoptive T-cell transfer, checkpoint blockade, or vaccination. We have previously shown that serial vaccinations with Melan-A <sup>MART-1</sup> <sub>26-35</sub> peptide, CpG-B, and incomplete Freund adjuvant (IFA) generated robust tumor-specific CD8 T-cell responses in patients with melanoma. Here, we describe the detailed kinetics of early- and long-term establishment of T-cell frequency, differentiation (into memory and effector cells), polyfunctionality, and clonotype repertoire induced by vaccination. <b>Experimental Design:</b> Twenty-nine patients with melanoma were treated with multiple monthly subcutaneous vaccinations consisting of CpG-B, and either the native/EAA ( <i>n</i> = 13) or the analogue/ELA ( <i>n</i> = 16) Melan-A <sup>MART-1</sup> <sub>26-35</sub> peptide emulsified in IFA. Phenotypes and functionality of circulating Melan-A-specific CD8 T cells were assessed directly <i>ex vivo</i> by multiparameter flow cytometry, and TCR clonotypes were determined <i>ex vivo</i> by mRNA transcript analyses of individually sorted cells. <b>Results:</b> Our results highlight the determining impact of the initial vaccine injections on the rapid and strong induction of differentiated effector T cells in both patient cohorts. Moreover, long-term polyfunctional effector T-cell responses were associated with expansion of stem cell-like memory T cells over time along vaccination. Dominant TCR clonotypes emerged early and persisted throughout the entire period of observation. Interestingly, one highly dominant clonotype was found shared between memory and effector subsets. <b>Conclusions:</b> Peptide/CpG-B/IFA vaccination induced powerful long-term T-cell responses with robust effector cells and stem cell-like memory cells. These results support the further development of CpG-B-based cancer vaccines, either alone or as specific component of combination therapies. <i>Clin Cancer Res; 23(13); 3285-96. ©2016 AACR</i>

Design of a pulse power supply unit for micro-ECM

Electrochemical micro-machining (μECM) requires a particular pulse power supply unit (PSU) to be developed in order to achieve desired machining performance. This paper summarises the development of a pulse PSU meeting the requirements of μECM. The pulse power supply provides tens of nanosecond pulse duration, positive and negative bias voltages and a polarity switching functionality. It fulfils the needs for tool preparation with reversed pulsed ECM on the machine. Moreover, the PSU is equipped with an ultrafast overcurrent protection which prevents the tool electrode from being damaged in case of short circuits. The developed pulse PSU was used to fabricate micro-tools out of 170 μm WC-Co alloy shafts via micro-electrochemical turning and drill deep holes via μECM in a disk made of 18NiCr6. The electrolyte used for both processes was a mixture of sulphuric acid and NaNO3 aqueous solutions.The research reported in this paper is supported by the European Commission within the project “Minimizing Defects in Micro-Manufacturing Applications (MIDEMMA)” (FP7-2011-NMP-ICT-FoF-285614

Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8(+) and CD4(+) T-cell responses with multiple specificities including a novel DR7-restricted epitope.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8(+) and CD4(+) T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8(+) and NY-ESO-183-99 for CD4(+) T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7(+) patients generated strong CD4(+) T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8(+) and CD4(+) T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach

Vaccination with LAG-3Ig (IMP321) and Peptides Induces Specific CD4 and CD8 T-Cell Responses in Metastatic Melanoma Patients-Report of a Phase I/IIa Clinical Trial.

PURPOSE: Cancer vaccines aim to generate and maintain antitumor immune responses. We designed a phase I/IIa clinical trial to test a vaccine formulation composed of Montanide ISA-51 (Incomplete Freund's Adjuvant), LAG-3Ig (IMP321, a non-Toll like Receptor agonist with adjuvant properties), and five synthetic peptides derived from tumor-associated antigens (four short 9/10-mers targeting CD8 T-cells, and one longer 15-mer targeting CD4 T-cells). Primary endpoints were safety and T-cell responses. EXPERIMENTAL DESIGN: Sixteen metastatic melanoma patients received serial vaccinations. Up to nine injections were subcutaneously administered in three cycles, each with three vaccinations every 3 weeks, with 6 to 14 weeks interval between cycles. Blood samples were collected at baseline, 1-week after the third, sixth and ninth vaccination, and 6 months after the last vaccination. Circulating T-cells were monitored by tetramer staining directly ex vivo, and by combinatorial tetramer and cytokine staining on in vitro stimulated cells. RESULTS: Side effects were mild to moderate, comparable to vaccines with Montanide alone. Specific CD8 T-cell responses to at least one peptide formulated in the vaccine preparation were found in 13 of 16 patients. However, two of the four short peptides of the vaccine formulation did not elicit CD8 T-cell responses. Specific CD4 T-cell responses were found in all 16 patients. CONCLUSIONS: We conclude that vaccination with IMP321 is a promising and safe strategy for inducing sustained immune responses, encouraging further development for cancer vaccines as components of combination therapies. Clin Cancer Res; 22(6); 1330-40. ©2015 AACR

Identification and thermochemical analysis of high-lignin feedstocks for biofuel and biochemical production

Background - Lignin is a highly abundant biopolymer synthesized by plants as a complex component of plant secondary cell walls. Efforts to utilize lignin-based bioproducts are needed. Results - Herein we identify and characterize the composition and pyrolytic deconstruction characteristics of high-lignin feedstocks. Feedstocks displaying the highest levels of lignin were identified as drupe endocarp biomass arising as agricultural waste from horticultural crops. By performing pyrolysis coupled to gas chromatography-mass spectrometry, we characterized lignin-derived deconstruction products from endocarp biomass and compared these with switchgrass. By comparing individual pyrolytic products, we document higher amounts of acetic acid, 1-hydroxy-2-propanone, acetone and furfural in switchgrass compared to endocarp tissue, which is consistent with high holocellulose relative to lignin. By contrast, greater yields of lignin-based pyrolytic products such as phenol, 2-methoxyphenol, 2-methylphenol, 2-methoxy-4-methylphenol and 4-ethyl-2-methoxyphenol arising from drupe endocarp tissue are documented. Conclusions - Differences in product yield, thermal decomposition rates and molecular species distribution among the feedstocks illustrate the potential of high-lignin endocarp feedstocks to generate valuable chemicals by thermochemical deconstruction

Climate driven life histories: the case of the Mediterranean Storm petrel

Seabirds are affected by changes in the marine ecosystem. The influence of climatic factors on marine food webs can be reflected in long-term seabird population changes. We modelled the survival and recruitment of the Mediterranean storm petrel (Hydrobates pelagicus melitensis) using a 21-year mark-recapture dataset involving almost 5000 birds. We demonstrated a strong influence of prebreeding climatic conditions on recruitment age and of rainfall and breeding period conditions on juvenile survival. The results suggest that the juvenile survival rate of the Mediterranean subspecies may not be negatively affected by the predicted features of climate change, i.e., warmer summers and lower rainfall. Based on considerations of winter conditions in different parts of the Mediterranean, we were able to draw inferences about the wintering areas of the species for the first time

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3- fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids

Impact of oil on bacterial community structure in bioturbated sediments

Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions - with tidal cycles and natural seawater - was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g21 wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities