Adaptive Tolerance Research Articles

IL-7Ralow memory CD8+ T cells are clonally anergic cells and defect in aerobic glycolysis pathway (HUM4P.274)

Abstract We have found two subsets of CD8+ T cells expressing IL-7Rahigh and IL-7Ralow with different cell survival responses to IL-7 in peripheral blood of human. Treating IL-7Rαlow memory CD8+ T cells with anti-CD3/CD28 Abs did not produce IL-2, but comparably expressed IFN-γ and TNF-a. But the mechanism for this is unknown. These cells have reduced calcium flux and subsequent inhibited nuclear translocation of NF-AT1 upon TCR stimulation as well as PMA/ionomycin treatment, suggesting that IL-7Rαlow memory CD8+ T cells also have defected in distal-TCR signaling. Importantly, this anergic status was reversed by exogenous IL-2 stimulation, clearly distinct from adaptive tolerance. When TCR stimulation on IL-7Rαlow memory CD8+ T cells exhibited markedly decreased GATA3 and c-myc expression and increased NUAK1, which are key regulators in aerobic glycolysis, compared to those of IL-7Rαhigh memory CD8+ T cells. The expression of GATA3 and c-myc in IL-7Rαlow memory CD8+ T cells were restored after IL-2 stimulation. In conclusion, we firstly show that specific subset of human CD8+ T cells, IL-7Rαlow memory CD8+ T cells, are T-cell clonally anergy in vivo. We also demonstrate that GATA3-c-myc-NUAK1 system may control the maintenance of clonally anergic IL-7Rαlow memory CD8+ T cells in vivo and provides insight into human immunology. Acknowledgement: This research was supported by National Research Foundation of Korea (2014M3A7B4052194) and by the SNUH Research Fund (34-2013-0206)

Adaptation and cross-adaptation of Escherichia coli ATCC 12806 to several food-grade biocides

The objective of this paper was to determine whether Escherichia coli ATCC 12806 can adapt to growth in increasing concentrations of three biocides commonly used in food processing facilities (trisodium phosphate [TSP], sodium nitrite [SNI] or sodium hypochlorite [SHY]), and whether cross-adaptation occurs. Adaptability over time was estimated by the growth kinetic parameters (modified Gompertz equation): lag phase, maximum growth rate and cell density in the stationary phase. Attempts were made to determine whether efflux pumps (in the presence of the efflux inhibitor carbonyl cyanide m-chorophenyl hydrazone [CCCP]) and cell surface hydrophobicity changes (microbial adhesion to solvents [MATS] assay) are associated with this adaptation. After repeated sub-culturing in static broth cultures containing increasing sub-inhibitory concentrations of TSP, SNI or SHY, E. coli exhibited an adaptive tolerance, especially to SNI and SHY. The increased tolerance was stable and remained after 7 successive passages in biocide-free growth media. Cross-adaptation between dissimilar biocides (SNI and SHY) was observed. Both efflux pumps and changes in cell surface hydrophobicity appear to be associated with adaptation to SNI and SHY. It is suggested that sub-lethal exposure to food-grade biocides represents a risk for the development of adaptation and cross-adaptation to such compounds.

Application of Data Mining Using Artificial Neural Network: Survey

The use of neural network is very wide in data mining due to some characteristic like parallel performance, Self-organizing adaptive, robustness and fault tolerance. Data mining models depend on task they accomplish: Association Rules, Clustering, Prediction, and Classification. Neural network is used to find pattern in data. The grouping of neural network model and data mining method can greatly increase the efficiency of data mining methods and it has been broadly used. Different algorithms have been discussed for optimizing the artificial neural network (ANN). ANN combines with other algorithms to find out the high accurate data as compare to traditional algorithm. The role of ANN using data mining techniques is playing an important role in forecasting or prediction about games and weather. This produces high accurate predictions than that of traditional algorithm. Data mining approaches using ANN can also work well. ANN is a highly class algorithm which can be accelerated using neuron. The result of which will produce a high speed up ANN. ANN can also be used for the purpose of extracting rules from trained neural networks.

Endophytes from Phragmites for metal removal: evaluating their metal tolerance, adaptive tolerance behaviour and biosorption efficacy

This study determined the potential of fungal endophytes as novel group of biosorbents for metal removal. The endophytic fungi were first isolated from Phragmites, a plant typically used to treat wastewater or leachate, and screened for tolerance and biosorption potential towards various metals. Results revealed that all 21 endophytes demonstrated tolerance to metals tested (Cd2+, Cu2+, Cr3+, Pb2+ and Zn2+), with three isolates (Trichoderma asperellum Iso11, Phomopsis sp. Iso9 and Saccharicola bicolour Iso22) showing the most potential. Of the three, T. asperellum demonstrated better tolerance and adaptive tolerance behaviour to various metals compared to Phomopsis sp. and S. bicolour which were unable to adapt to increasing metal concentrations (up to 2,000 mg L−1). All three isolates showed similar efficacy in removing metals in single-metal solutions. On the contrary, in multi-metal solutions, T. asperellum and S. bicolour showed higher affinity to adsorb Cu2+, followed by Cr3+ and Pb2+, while Phomopsis sp. had affinity towards metals in the following trend: Cu2+ > Pb2+ > Cr3+. This study is the first to document the metal tolerance and sorption efficacy of endophytes from Phragmites.

A novel adaptive tolerance filter for random valued impulse noise suppression in digital grey and color images

During acquisition, transmission, storage and retrieval, quite often digital images become noised and mask the important details of an image and also present themselves with an ugly look. Such images if not processed properly, become useless for subsequent image processing operations like segmentation, classification etc. Hence the quality improvement (through denoising) of such noisy images becomes an important operation in the field of image processing. This paper proposes a two stage detection-estimation based filtering system to suppress random valued impulse noise from digital grey and color images based on neighborhood statistics of pixels of the working window under consideration. An adaptive working window and the adaptive tolerance computed from the pixels belonging to the current filtering window facilitates an appropriate detection of noisy pixels in the first phase, followed by the simple and computationally efficient restoration strategy in the second stage. The Simulation results prove that the proposed scheme exhibits much superior performance in comparison with other state of art image filtering methods in suppressing the random valued impulse noise from the digital grey and color images.

Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights.

BackgroundThe northern elephant seal, Mirounga angustirostris, is a valuable animal model of fasting adaptation and hypoxic stress tolerance. However, no reference sequence is currently available for this and many other marine mammal study systems, hindering molecular understanding of marine adaptations and unique physiology.ResultsWe sequenced a transcriptome of M. angustirostris derived from muscle sampled during an acute stress challenge experiment to identify species-specific markers of stress axis activation and recovery. De novo assembly generated 164,966 contigs and a total of 522,699 transcripts, of which 68.70% were annotated using mouse, human, and domestic dog reference protein sequences. To reduce transcript redundancy, we removed highly similar isoforms in large gene families and produced a filtered assembly containing 336,657 transcripts. We found that a large number of annotated genes are associated with metabolic signaling, immune and stress responses, and muscle function. Preliminary differential expression analysis suggests a limited transcriptional response to acute stress involving alterations in metabolic and immune pathways and muscle tissue maintenance, potentially driven by early response transcription factors such as Cebpd.ConclusionsWe present the first reference sequence for Mirounga angustirostris produced by RNA sequencing of muscle tissue and cloud-based de novo transcriptome assembly. We annotated 395,102 transcripts, some of which may be novel isoforms, and have identified thousands of genes involved in key physiological processes. This resource provides elephant seal-specific gene sequences, complementing existing metabolite and protein expression studies and enabling future work on molecular pathways regulating adaptations such as fasting, hypoxia, and environmental stress responses in marine mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1253-6) contains supplementary material, which is available to authorized users.

Physiological mechanisms of adaptation of Dianthus carthusianorum L. to growth on a Zn-Pb waste deposit - the case of chronic multi-metal and acute Zn stress

This study investigates the response of metallicolous (M) and nonmetallicolous (NM) ecotypes of Dianthus carthusianorum L. to chronic multi-metal and acute Zn stress. Plants were cultivated on the Zn-Pb waste heap substrate and under Zn excess in hydroponics. Growth parameters as well as accumulation of organic acids and thiol peptides were determined as a function of metal accumulation. When grown on the metalliferous substrate, the M plants showed less phytotoxicity symptoms, lower foliar metal (Zn, Pb, Cd) accumulation, higher malate and citrate but lower glutathione content than the NM plants. When exposed to Zn excess in hydroponics, the M ecotype was also more tolerant but accumulated more Zn in comparison with the NM ecotype, accompanied by greater malate and citrate concentrations in the shoots, which were however not affected by increasing Zn doses. No phytochelatins were detected under any experimental conditions. Both constitutive and adaptive tolerance was found in D. carthusianorum. Under chronic metal stress, enhanced tolerance results from restricted metal uptake to the shoots and probably from detoxification by organic acids; however, under acute Zn stress it is not related to diminished metal uptake or organic acids. Glutathione and phytochelatins are not implicated in adaptive metal tolerance.

Response of <i>Anopheles gambiae</i> detoxification enzymes to levels of physico-chemical environmental factors from northwest Nigeria

The objective of this study was to investigate the response of Anopheles gambiae detoxification enzymes to levels of various physico-chemical environmental factors present in their breeding sites. Mosquito breeding sites were grouped into three different breeding sites (designated as study zones A, B & C) on the bases of human related activities (intensive agriculture, petrochemical and domestic) taking place within and/or around the breeding sites, followed by sampling of Anopheles gambiae larvae from all the breeding sites across the designated study zones. Some of the sampled larvae were reared to pupae and adult life stages. Levels of 7 physical (pH, temperature, conductivity, transparency, total dissolved solids, dissolved oxygen and biological oxygen demand) and 6 chemical (sulphates, phosphates, nitrites, nitrates, carbon content and oil and grease) environmental factors were determined from these mosquito breeding sites. Activities of the 3 major detoxification enzymes (Cytochrome P450 oxygenase, GST and α & β-esterases) were evaluated in the sampled larvae as well as the pupae and adult samples that ultimately emerged from the larvae. Following statistical analysis, results showed that P450 activities were higher in the petrochemical sites (zone C) and the activities were highly associated with pH, temperature as well as carbon content and oil and grease. The activities of GST and α & β- esterases were higher in the intensive agriculture sites (Zone A) and were highly correlated with all the chemicals environmental factors. A deduced statistical model established all the chemical in combination with some of the physical environmental factors as producing an inductive effect on these three detoxification enzymes. These observations could have a significant impact on the insecticide-based approach to vector control. An. gambiae samples may have developed intrinsic enzymatic machinery to produce an adaptive tolerance to various insecticides used for their control since most of these insecticides and the environmental chemical factors share similar routes of metabolism.Keywords: Cytochrome, Anopheles, Inescts, Northwestern Nigeria

Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

Common and metal-specific proteomic responses to cadmium and zinc in the metal tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn.

Although adaptive metal tolerance may arise in fungal populations in polluted soils, the mechanisms underlying metal-specific tolerance are poorly understood. Comparative proteomics is a powerful tool to identify variation in protein profiles caused by changing environmental conditions, and was used to investigate protein accumulation in a metal tolerant isolate of the ericoid mycorrhizal fungus Oidiodendron maius exposed to zinc and cadmium. Two-dimensional gel electrophoresis and shotgun proteomics followed by mass spectrometry lead to the identification of common and metal-specific proteins and pathways. Proteins selectively induced by cadmium exposure were molecular chaperons of the Hsp90 family, cytoskeletal proteins and components of the translation machinery. Zinc significantly up-regulated metabolic pathways related to energy production and carbohydrates metabolism, likely mirroring zinc adaptation of this fungal isolate. Common proteins induced by the two metal ions were the antioxidant enzyme Cu/Zn superoxide dismutase and ubiquitin. In mycelia exposed to zinc and cadmium, both proteomic techniques also identified agmatinase, an enzyme involved in polyamine biosynthesis. This novel finding suggests that, like plants, polyamines may have important functions in response to abiotic environmental stress in fungi. Genetic evidence also suggests that the biosynthesis of polyamines via an alternative metabolic pathway may be widespread in fungi.

Adaptive 3D Imaging and Tolerance Analysis of Prefabricated Components for Accelerated Construction

Tolerance analysis of prefabricated components poses challenges to effective quality control of accelerated construction projects in urban areas. In busy urban environments, accelerated construction methods quickly assemble prefabricated components to achieve workflows that are more efficient and reduce impacts of construction on urban traffic and business. Accelerated constructions also bring challenges of “fit-up:” misalignments between components can occur due to less detailed tolerance assessments of components. Conventional tolerance checking approaches, such as manual mock-up, cannot provide detailed geometric assessments in a timely manner. This paper proposes the integration of an adaptive 3D imaging and spatial pattern analysis methods to achieve detailed and frequent “fit-up” analysis of prefabricated components. The adaptive 3D imaging methods progressively adjust imaging parameters of a laser scanner according to the geometric complexities of prefabricated components captured in data collected so far. The spatial pattern analysis methods automatically analyze deviations of prefabricated components from as-designed models to derive tolerance networks that capture relationships between tolerances of components and identify risks of misalignments.

Orally administered extract from Prunella vulgaris attenuates spontaneous colitis in mdr1a(-/-) mice.

To investigate the ability of a Prunella vulgaris (P. vulgaris) ethanolic extract to attenuate spontaneous typhlocolitis in mdr1a(-/-) mice. Vehicle (5% ethanol) or P. vulgaris ethanolic extract (2.4 mg/d) were administered daily by oral gavage to mdr1a(-/-) or wild type FVB(WT) mice from 6 wk of age up to 20 wk of age. Clinical signs of disease were noted by monitoring weight loss. Mice experiencing weight loss in excess of 15% were removed from the study. At the time mice were removed from the study, blood and colon tissue were collected for analyses that included histological evaluation of lesions, inflammatory cytokine levels, and myeloperoxidase activity. Administration of P. vulgaris extracts to mdr1a(-/-) mice delayed onset of colitis and reduced severity of mucosal inflammation when compared to vehicle-treated mdr1a(-/-) mice. Oral administration of the P. vulgaris extract resulted in reduced (P < 0.05) serum levels of IL-10 (4.6 ± 2 vs 19.4 ± 4), CXCL9 (1319.0 ± 277 vs 3901.0 ± 858), and TNFα (9.9 ± 3 vs 14.8 ± 1) as well as reduced gene expression by more than two-fold for Ccl2, Ccl20, Cxcl1, Cxcl9, IL-1α, Mmp10, VCAM-1, ICAM, IL-2, and TNFα in the colonic mucosa of mdr1a(-/-) mice compared to vehicle-treated mdr1a(-/-) mice. Histologically, several microscopic parameters were reduced (P < 0.05) in P. vulgaris-treated mdr1a(-/-) mice, as was myeloperoxidase activity in the colon (2.49 ± 0.16 vs 3.36 ± 0.06, P < 0.05). The numbers of CD4(+) T cells (2031.9 ± 412.1 vs 5054.5 ± 809.5) and germinal center B cells (2749.6 ± 473.7 vs 4934.0 ± 645.9) observed in the cecal tonsils of P. vulgaris-treated mdr1a(-/-) were significantly reduced (P < 0.05) from vehicle-treated mdr1a(-/-) mice. Vehicle-treated mdr1a(-/-) mice were found to produce serum antibodies to antigens derived from members of the intestinal microbiota, indicative of severe colitis and a loss of adaptive tolerance to the members of the microbiota. These serum antibodies were greatly reduced or absent in P. vulgaris-treated mdr1a(-/-) mice. The anti-inflammatory activity of P. vulgaris ethanolic extract effectively attenuated the severity of intestinal inflammation in mdr1a(-/-) mice.

Constructing MICDS Algorithm for Data Transmission using Maximal Independent Set in Network Graph

Wireless Sensor Networks (WSNs) is a distributed wireless network which consists of a large number of small (sensor) nodes with sensing, computation and wireless communication capabilities. These sensor nodes work together to monitor various applications and they communicate with each other in multiple hops or directly, with the objective of handing over the processed data to the sink node or access point (AP). WSNs have many issues like self-configuration, faulttolerance, adaptation, flexibility, energy efficiency, security, scalability, interference, architectural issues, mobility and delay tolerance but the most critical and important issue is to reduce energy wastage and interference and increasing network lifetime [1,2, 3]. In order to manage these issues, WSNs require some virtual backbone. A Connected Dominating Set (CDS) can be used to create a virtual backbone in WSNs [5,6,7,8, 9,10]. The CDS is a dominating set which induces a connected subgraph. A maximal independent set is an independent set that is not a subset of any other independent set [7,8]. In this paper, proposed a MICDS (Maximal Independent Connected Dominating Set) algorithm for the WSNs, which are based on the construction of the maximal independent set (MIS) of the representing network graph. There are two different steps in the implementation of MICDS algorithm. In the first phase, the maximal independent set of the network is constructed. In second phase, the dominating set and connectors for the dominating set are constructed using MIS. Using the dominating set and connectors, all the data is transmitted to the sink node or access point (AP). The simulation results indicate that the MICDS algorithm reduce the interference and energy wastage in the network graph, further it also increases the lifetime of the network.

Cold-Hardiness Mechanisms in Third Instar Larvae ofSpodoptera exiguaHübner (Lepidoptera: Noctuidae)

The mechanisms of cold tolerance in insects which are exposed to suboptimal temperatures for survival include accumulation of cryoprotectants and expression of heat shock protein genes (hsps). Spodoptera exigua is a freeze avoidance insect, which can overwinter as third instar larvae in China. Our previous study illustrated that seasonal and diel fluctuating temperatures enhanced its cold hardening. The survival mechanism for this stage, however, is poorly understood. We hypothesized that the survival mechanisms in third instar larvae of S. exigua were correlated with the induction of cryoprotectants and/or expression of hsps. Results showed that trehalose as the main cryoprotectant was accumulated during the cold acclimation (CA), and the level of accumulation fell off sharply when the CA treatment was discontinued (discontinued cold acclimation, DCA). Furthermore, inducible hsp90 was down-regulated in all treatments compared with the control, and hsp70 was up-regulated under conditions of CA and RCH (rapid cold hardening), which induced 1.23- and 44.00-fold increases compared with the control. Therefore, we considered that both trehalose (as the main cryoprotectant) and hsps (i.e. hsp90 and hsp70) were involved in the regulation of physiological activity of third instar larvae of S. exigua to resist low temperatures during winter. These results are helpful for us to understand the mechanisms underlying this species' ecological adaptability and stress tolerance.

Tolerância ao calor em bovinos das raças Nelore branco, Nelore vermelho e Pantaneira

A identificação de parâmetros/ métricas associados à adaptação ambiental é uma das principais tendências e desafios da pecuária mundial. Desta forma, o objetivo deste trabalho foi avaliar as características de tolerância ao calor de bovinos da raça Nelore e Pantaneiro no ecossistema do Pantanal matogrossense no pico de calor da região. Estes animais foram mantidos por duas horas à sombra (8:00h às 10:00h) e em seguida uma hora ao sol (10:00h às 11:00h). Após a exposição foram analisados parâmetros de frequência cardíaca (FC), frequência respiratória (FR), temperatura retal (TR), temperatura superficial da costela (TSC), temperatura superficial do pescoço (TSP) e temperatura superficial da garupa (TSG). Também foram avaliados a taxa de sudação (TXS), comprimento e densidade de pelos e calculados o índice de tolerância ao calor (ITC). No período estudado, a temperatura ambiental variou de 29,0 a 40,0ºC e a média para umidade relativa foi de 75%. O ambiente teve influência marcante sobre as variáveis fisiológicas, com exceção da FR. A TR e a TXS diferiram significativamente entre os grupos genéticos, sendo da raça Nelore mais elevada em relação à Pantaneira, mas todas dentro dos limites fisiológicos. A análise multivariada mostrou a importância de variáveis como taxa de sudação e comprimento de pelos para adaptação de bovinos na região. Apesar do Nelore vermelho estar separado dos outros dois grupos, foi possível concluir que ambas as raças analisadas apresentaram capacidade de adaptação e tolerância ao calor às condições climáticas do Pantanal e podem ser produzidas de modo sustentável na região.

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

QTL mapping of freezing tolerance: links to fitness and adaptive trade-offs.

Local adaptation, defined as higher fitness of local vs. nonlocal genotypes, is commonly identified in reciprocal transplant experiments. Reciprocally adapted populations display fitness trade-offs across environments, but little is known about the traits and genes underlying fitness trade-offs in reciprocally adapted populations. We investigated the genetic basis and adaptive significance of freezing tolerance using locally adapted populations of Arabidopsis thaliana from Italy and Sweden. Previous reciprocal transplant studies of these populations indicated that subfreezing temperature is a major selective agent in Sweden. We used quantitative trait locus (QTL) mapping to identify the contribution of freezing tolerance to previously demonstrated local adaptation and genetic trade-offs. First, we compared the genomic locations of freezing tolerance QTL to those for previously published QTL for survival in Sweden, and overall fitness in the field. Then, we estimated the contributions to survival and fitness across both field sites of genotypes at locally adaptive freezing tolerance QTL. In growth chamber studies, we found seven QTL for freezing tolerance, and the Swedish genotype increased freezing tolerance for five of these QTL. Three of these colocalized with locally adaptive survival QTL in Sweden and with trade-off QTL for overall fitness. Two freezing tolerance QTL contribute to genetic trade-offs across environments for both survival and overall fitness. A major regulator of freezing tolerance, CBF2, is implicated as a candidate gene for one of the trade-off freezing tolerance QTL. Our study provides some of the first evidence of a trait and gene that mediate a fitness trade-off in nature.

Evaluation of bacterial internalization using qRT-PCR.

Bacterial resistance to antibiotics currently represents a topic of high interest. Unfortunately, studies addressing their adaptation and multidrug tolerance latency1 are not sufficient for a comprehensive description of the pathogen-host molecular interaction mechanisms (interactome) and the bacterial internalization into human cells, which leads to modified human genes. Bacterial persisters2 are antibiotic-tolerant cells and their resistance can be better understood by exploring their growth status, signals and pathways leading to their formation in infected tissues. Some bacteria, as intracellular pathogens, directly interfere with transcription, translation, and DNA repair. Since metagenomic techniques were used to characterize the human microbiome,3 as in Human Microbiome Project4 and MetaHIT,5 both conducted between 2008-2012, several research projects emerged and all bacterial genomes detected in the human body can be consulted in the Genomes OnLine Database.6 Although culturing techniques still represent the gold standard for bacterial identification, PCR is a valuable tool for detecting bacterial pathogenic agents, especially when they die or lyse easily due to inappropriate storage conditions or prior antibiotic treatment, as it does not require live or intact cells.7 In clinical applications, real-time PCR for broad-range amplification of bacterial DNA offers a rapid turnaround time and a decreased risk of PCR carryover contamination, of outmost importance when using small and qualitative-poor DNA samples. Furthermore, quantitative real-time reverse-transcription PCR (qRT-PCR) can be used to evaluate transcription8 and to measure gene expression levels in different mutant strains,9 offering a complete and realistic picture of the bacterial internalization.

Improved GAA activity in tregitope-treated pompe disease: preclinical studies (TECH1P.864)

Abstract “Tregitopes” are immunoglobulin-derived regulatory T-cell (Treg) epitopes that lead to suppression of inflammation and, when administered with a target antigen, adaptive tolerance. One potential application of Tregitopes is mitigating immune responses to therapeutic proteins, manifest when a protein appears foreign either because it is derived from a different species or because patients express a genetic deletion or modification. In Pompe disease, genetic deficiencies in acid alpha-glucosidase (GAA) result in lysosomal glycogen accumulation throughout the body. Life-saving treatment with fully human recombinant GAA can trigger high titer anti-drug-antibodies (ADA) that interfere with drug efficacy. We report here our efforts to apply Tregitopes to provide GAA-specific tolerance induction. Therapeutic GAA protein was expressed in GAA-deficient mice by administering AAV-GAA under conditions known to induce ADA +/- Tregitopes, also delivered by AAV expression vectors. Tregitope administration inhibited ADA formation compared to vehicle-treated controls. In addition, a significant increase in GAA activity was observed for Tregitope, consistent with Tregitope inhibition of glycogen buildup in muscle. Current experiments are designed to refine the Tregitope treatment regimen to achieve clinically-relevant ADA inhibition and to evaluate cellular immune responses towards the goal of providing effective and lasting Tregitope-mediated tolerance to GAA for Pompe patients.

Flt3L Dependence Helps Define an Uncharacterized Subset of Murine Cutaneous Dendritic Cells

Skin-derived dendritic cells (DC) are potent antigen presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DC carry antigens and constitutively migrate to the skin draining lymph nodes (LN). In mice, Langerin-CD11b− dermal DC are a low-frequency, heterogeneous, migratory DC subset that traffic to LN (Langerin-CD11b-migDC). Here, we build on the observation that Langerin-CD11b− migDC are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice which accumulate migDC, demonstrate these cells are cutaneous residents. Langerin-CD11b-Flt3L responsive DC are largely CD24(+) and CX3CR1low and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11bmigDC present antigen with equal efficiency to other DC subsets ex vivo including classical CD8α cDC and Langerin+CD103+ dermal DC. Finally, transcriptome analysis suggests a close relationship to other skin DC, and a lineage relationship to other classical DC. This work demonstrates that Langerin- CD11b− dermal DC, a previously overlooked cell subset, may be an important player in the cutaneous immune environment.