Specific Tolerance Research Articles

Characterizing and Augmenting Peripheral Tolerance in in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). Central deletion, however, is incomplete, and donor-reactive host T cells remain following allogeneic IUHCT. Furthermore, central deletion alone cannot explain the absence of GVHD either following IUHCT using non-T cell depleted bone marrow (BM) or in chimeric animals that undergo postnatal donor lymphocyte infusion (DLI) with subsequent conversion to >90% allogeneic donor cell engraftment (Hayashi et al., Blood 2002;100:804-12). Studies in both mice and humans have demonstrated the importance of regulatory T cells, including traditional CD25+FoxP3+ Tregs and CD49b+LAG-3+ Type 1 regulatory T (Tr1) cells, in maintaining tolerance following postnatal allogeneic transplantation. We hypothesized that peripheral regulatory T cells are increased in chimeric animals following IUHCT and that they contribute to the establishment of DST and prevention of GVHD.Methods: 10x106 BM cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP mice (H2Kb, CD45.2+) were injected intravenously (IV) into gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd, CD45.2+). Mice were sacrificed at 2, 4, 8, and 24 weeks of age. Splenocytes were analyzed for the expression of CD4, H2Kb, H2Kd, CD49b, LAG-3, CD25, and FoxP3 to determine the percentage of traditional Tregs and Tr1 cells within the CD4+ population as well as for the expression of intracellular IL-10 - a key mediator of regulatory T cell function. A GVHD model was developed in which 10x106 B6 BM cells were injected IV into day of life 0 (P0) naive Balb/c mice. The ability of IUHCT-induced regulatory T cells to suppress GVHD was assessed in two groups: 1) 10x106 B6 BM cells injected into P0 mice that had undergone IUHCT and 2) 10x106 B6 BM cells co-injected with 5x106 CD4+ splenocytes harvested from 2 week old chimeric mice into P0 naive Balb/c mice. To evaluate the role of regulatory T cells following DLI in chimeric mice, 30x106 B6CD45.1 (H2Kb, CD45.1+) splenocytes were injected IV into 4 week old chimeric recipients. Mice were sacrificed at 48 hours and 2 weeks post-DLI, and regulatory T cells were measured as a percentage of CD45.2+CD4+ cells in the spleen.Results: IUHCT was associated with a significant increase in Tr1 cells, predominantly of donor origin, compared to naive controls (Figure 1). There was a corresponding increase in intracellular IL-10 expression in donor CD4+ splenocytes in chimeric animals compared to naive B6 FoxP3GFP controls at 2 weeks of age (MFI 7.07 vs. 3.26, p=0.0017). Elevated levels of regulatory T cells following IUHCT also had a functional effect in the GVHD model (Figure 2). Mice that had undergone IUHCT (group 1) demonstrated a trend toward improved survival compared to naive mice when injected with B6 BM at P0. Naive mice injected at P0 with B6 BM plus CD4+ splenocytes from chimeric mice (group 2) demonstrated significant improvement in survival compared to naive recipients of B6 BM alone (p=0.049). Although we detected no increase in traditional Tregs after IUHCT alone, we noted a 3-fold increase in traditional Tregs of host origin in chimeric animals at 48 hours post-DLI (25.5% vs. 8.3%, p<0.001).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. We demonstrate an increase in nontraditional regulatory T cells following IUHCT which can minimize GVHD as well as an increase in traditional Tregs following DLI in an established model of complete allogeneic engraftment. These studies suggest an important role for regulatory T cell populations in the induction of tolerance following IUHCT. They are significant in light of a potential obstacle to the clinical translation of IUHCT: the window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges. Augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a technically feasible point later in gestation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Asexual Recombinants of Plasmopara halstedii Pathotypes from Dual Infection of Sunflower.

Genetically homogenous strains of Plasmopara halstedii differing in host specificity and fungicide tolerance were used to test the hypothesis that asexual genetic recombination occurs and may account for the high genotype diversity of this homothallic reproducing oomycete, which causes downy mildew in sunflower. Dual inoculation of sunflower seedlings with single zoospore strains of complementary infection characteristics caused sporulation under conditions where inoculation with each strain alone failed to infect. PCR-based investigation with strain-specific primers proved the presence of genetic traits from both progenitors in single sporangia collected from sporangiophores of such infections. Sister zoospores released from these sporangia revealed the genotype of the one or the other parental strain thus indicating heterokaryology of sporangia. Moreover, some zoospores showed amplification products of both parents, which suggests that the generally mononucleic spores derived from genetic recombination. The possibility of parasexual genetic exchange in the host-independent stage of infection and the evolutionary consequences are discussed.

Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.

Specific oral tolerance induction in childhood.

Food allergy continues to be a significant public health concern for which there are no approved treatments and management strategies primarily include allergen avoidance and pharmacological measures for accidental exposures. Food allergy is thought to result from either a failure to establish oral tolerance or the breakdown of existing oral tolerance, and therefore, experimental preventative and treatment strategies are now aimed at inducing specific oral tolerance. This may occur in infancy prior to the development of food allergy through the optimal timing of dietary exposure (primary oral tolerance induction) or as a treatment for established food allergy through oral immunotherapy (secondary oral tolerance induction). Trials examining the effectiveness of early dietary allergen exposure to prevent food allergy have yielded promising results for peanut allergy but not so for other allergens, although the results of several trials are yet to be published. Although infant feeding guidelines no longer advise to avoid allergenic foods and exposure to food allergens orally is an important step in inducing food tolerance by the immune system, evidence regarding the optimal timing, dose and form of these foods into the infant's diet is lacking. Likewise, oral immunotherapy trials appear promising for inducing desensitization; however, the long-term efficacy in achieving sustained desensitization and optimal protocols to achieve this is unknown. More research is needed in this emerging field.

Expression of a phosphate-starvation inducible fructose-1,6-bisphosphatase gene in common bean nodules correlates with phosphorus use efficiency

While increased P-hydrolysing acid phosphatases (APase) activity in bean nodules is well documented under phosphorus (P) limitation, gene expression and subcellular localization patterns within the N2-fixing nodule tissues are poorly understood. The aim of this research was to track the enzyme activity along with the intra-nodular localization of fructose-1,6-bisphosphatase (FBPase), and its contribution to P use efficiency (PUE) under symbiotic nitrogen fixation (SNF) in Phaseolus vulgaris. The FBPase transcript were localized in situ using RT-PCR and the protein activity was measured in nodules of two contrasting recombinant inbred lines (RILs) of P. vulgaris, namely RILs 115 (P-efficient) and 147 (P-inefficient), that were grown under sufficient versus deficient P supply. Under P-deficiency, higher FBPase transcript fluorescence was found in the inner cortex as compared to the infected zone of RIL115. In addition, both the specific FBPase and total APase enzyme activities significantly increased in both RILs, but to a more significant extent in RIL115 as compared to RIL147. Furthermore, the increased FBPase activity in nodules of RIL115 positively correlated with higher use efficiency of both the rhizobial symbiosis (23%) and P for SNF (14% calculated as the ratio of N2 fixed per nodule total P content). It is concluded that the abundant tissue-specific localized FBPase transcript along with induced enzymatic activity provides evidence of a specific tolerance mechanism where N2-fixing nodules overexpress under P-deficiency conditions. Such a mechanism would maximise the intra-nodular inorganic P fraction necessary to compensate for large amount of P needed during the SNF process.

Semicoherent Multipopulation Mortality Modeling: The Impact on Longevity Risk Securitization

AbstractMultipopulation mortality models play an important role in longevity risk transfers involving more than one population. Most of the existing multi‐population mortality models are built on the hypothesis of coherence, which assumes that there always exists a force that brings the mortality differential between any two populations back to a constant long‐term equilibrium level. This hypothesis prevents diverging long‐term forecasts, which do not seem to be biologically reasonable. However, the coherence assumption may be perceived by market participants as too strong and is in fact not always supported by empirical observations. In this article, we introduce a new concept called “semicoherence,” which is less stringent in the sense that it permits the mortality trajectories of two related populations to diverge, as long as the divergence does not exceed a specific tolerance corridor, beyond which mean reversion will come into effect. We further propose to produce semicoherent mortality forecasts by using a vector threshold autoregression. The proposed modeling approach is illustrated with mortality data from U.S. and English and Welsh male populations, and is applied to several pricing and hedging scenarios.

Reprogramming the Local Lymph Node Microenvironment Promotes Tolerance that Is Systemic and Antigen Specific

Many experimental therapies for autoimmune diseases, such as multiple sclerosis (MS), aim to bias Tcells toward tolerogenic phenotypes without broad suppression. However, the link between local signal integration in lymph nodes (LNs) and the specificity of systemic tolerance is not well understood. We used intra-LN injection of polymer particles to study tolerance as a function of signals in the LN microenvironment. In a mouse MS model, intra-LN introduction of encapsulated myelin self-antigen and a regulatory signal (rapamycin) permanently reversed paralysis after one treatment during peak disease. Therapeutic effects were myelin specific, required antigen encapsulation, and were less potent without rapamycin. This efficacy was accompanied by local LN reorganization, reduced inflammation, systemic expansion of regulatory Tcells, and reduced Tcell infiltration to the CNS. Our findings suggest that local control over signaling in distinct LNs can promote cell types and functions that drive tolerance that is systemic but antigen specific.

Current Concepts in Tissue Engineering: Skin and Wound

Pure regenerative healing with little to no donor morbidity remains an elusive goal for both surgeon and patient. The ability to engineer and promote the development of like tissue holds so much promise, and efforts in this direction are slowly but steadily advancing. Products selected and reviewed reflect historical precedence and importance and focus on current clinically available products in use. Emerging technologies we anticipate will further expand our therapeutic options are introduced. The topic of tissue engineering is incredibly broad in scope, and as such the authors have focused their review on that of constructs specifically designed for skin and wound healing. A review of pertinent and current clinically related literature is included. Products such as biosynthetics, biologics, cellular promoting factors, and commercially available matrices can be routinely found in most modern health care centers. Although to date no complete regenerative or direct identical soft-tissue replacement exists, currently available commercial components have proven beneficial in augmenting and improving some types of wound healing scenarios. Cost, directed specificity, biocompatibility, and bioburden tolerance are just some of the impending challenges to adoption. Quality of life and in fact the ability to sustain life is dependent on our most complex and remarkable organ, skin. Although pure regenerative healing and engineered soft-tissue constructs elude us, surgeons and health care providers are slowly gaining comfort and experience with concepts and strategies to improve the healing of wounds.

P068 Transplant tolerance in heart transplant recipients: It is all in the genes

Aim The “holy grail” of transplantation is donor specific tolerance in the absence of immunosuppression. There are no cases reported of survival without immunosuppression in heart transplant recipients. This prospective study identifies a group of heart transplant recipients who have developed tolerance to the transplanted organ. We hypothesize that these recipients were pre-disposed to survival without immunosuppression. Identification of a gene signature in these recipients could help understand the mechanism of development of transplant tolerance in heart transplant recipients. Methods We identified 8 cardiac transplant patients with a history of at least 7 days without immunosuppression (TOL group). Comparator groups included (i) 120 days post-transplant patients without rejection (11 pts), (ii) 5 years post-transplant patients with normal graft function (15 pts) (iii) patients with acute cellular rejection ISHLT 2R or greater (5 pts). Blood samples were obtained and total RNA extracted from PBMCs was reverse transcribed to cDNA. The cDNA was overlaid onto 96-well PCR plates containing customized gene-specific PCR primer pairs for Treg regulation genes and analyzed using quantitative Real Time PCR. Results We observed 15 genes which were differentially expressed between the TOL and rejector (REJX) groups. As shown in Table 1, genes that play an important role in the regulation of Tregs that is central to the development of a potentially tolerant state showed a significant higher expression in the TOL group as compared to REJX group. Conclusions This is the first report of a group of heart transplant patients surviving more than transiently without immunosuppression. The existence of these patients and the documented gene expression differences supports further research into mechanisms and potential ways to identify the development of tolerance in heart transplant patients. Download : Download high-res image (89KB) Download : Download full-size image

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid and quantitative detection of aflatoxin B1 in crops

Contamination of grains and other crops by aflatoxin B1 (AFB1), a highly toxic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus, poses a serious threat to human health and is an important food safety issue. In this study, a competitive up-converting phosphor technology-based lateral flow (AFB1-UPT-LF) assay was developed for rapid detection of AFB1. Detection sensitivity of the proposed assay can reach 0.03ngmL−1 for standard AFB1 solutions, with the coefficients of variation (CV) less than 10% (from 1.0 to 9.4%). A good linearity (r=0.9889) was observed for quantification of AFB1 from 0.03 to 1000ngmL−1. Except for aflatoxin M1, no cross-reactivity was found with the abrin, ricin, ochratoxin A, botulinum toxin, shiga toxin 1, shiga toxin 2, and staphylococcal enterotoxin B, even at high concentrations of 100 or 1000ngmL−1. After optimizing the extraction of AFB1, the assay showed good tolerance to various crop samples, with the detection limit (from 0.1 to 5ngg−1) lower than the corresponding maximum residue level (MRL) set in China. The AFB1-UPT-LF assay provides a promising tool for rapid on-site detection of AFB1 because of its high sensitivity, specificity, and sample tolerance.

Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model.

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-γ) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrient- and oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h(-1)) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 μm. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance.

Life-history strategies constrain invertebrate community tolerance to multiple stressors: A case study in the Ebro basin

ContextMultiple stressors constitute a serious threat to aquatic ecosystems, particularly in the Mediterranean region where water scarcity is likely to interact with other anthropogenic stressors. Biological traits potentially allow the unravelling of the effects of multiple stressors. However, thus far, trait-based approaches have failed to fully deliver on their promise and still lack strong predictive power when multiple stressors are present. GoalWe aimed to quantify specific community tolerances against six anthropogenic stressors and investigate the responses of the underlying macroinvertebrate biological traits and their combinations. MethodsWe built and calibrated boosted regression tree models to predict community tolerances using multiple biological traits with a priori hypotheses regarding their individual responses to specific stressors. We analysed the combinations of traits underlying community tolerance and the effect of trait association on this tolerance. ResultsOur results validated the following three hypotheses: (i) the community tolerance models efficiently and robustly related trait combinations to stressor intensities and, to a lesser extent, to stressors related to the presence of dams and insecticides; (ii) the effects of traits on community tolerance not only depended on trait identity but also on the trait associations emerging at the community level from the co-occurrence of different traits in species; and (iii) the community tolerances and the underlying trait combinations were specific to the different stressors. ConclusionThis study takes a further step towards predictive tools in community ecology that consider combinations and associations of traits as the basis of stressor tolerance. Additionally, the community tolerance concept has potential application to help stream managers in the decision process regarding management options.

Numerical prediction of distortions during machining of large aluminium aeronautical parts

Aluminium alloy structural aerospace parts are usually large monolithic parts machined from rolled plates or preformed parts. Previous to machining, several manufacturing steps (forming, heat treatments and mechanical stress relief operations) are performed, resulting in the creation of residual stresses. During machining, a redistribution of the residual stresses occurs due to the material removal. This residual stress redistribution is the main reason for machining errors such as dimensional variations and post‐machining distortions. In order to predict the mechanical behaviour of the workpiece during machining and the final part quality in taking into consideration both the machining parameters (fixture layout and machining sequence) and the residual stress redistribution, a specific numerical tool has been developed [1]. Using this numerical tool, an analysis of the influence of the initial residual stresses as well as of the fixture layout and the machining sequences (tool path) on the machining quality of parts machined from an AIRWARE®2050‐T84 alloy rolled plate is performed. The numerical results are compared with experimental ones in terms of both post‐machining distortions and dimensional errors. Similar results are obtained for both, showing that the developed numerical tool allows to predict dimensional and geometrical errors due to the redistribution of the residual stresses during machining. The machining of these cases and results obtained are then analysed, demonstrating the feasibility to adapt and to optimize the machining process plan to ensure conformity of the part with the tolerance specifications.

Methodology for aluminium part machining quality improvement considering mechanical properties and process conditions

The manufacturing of structural aluminium alloy parts requires several steps of both forming processes and heat treatments. Before machining, which is usually the last step of the manufacturing, the workpiece has thus undergone multiple manufacturing steps involving unequal plastic deformations which are source of residual stresses. During machining, where up to 90% of the initial workpiece volume can be removed, the mechanical equilibrium of the part evolves constantly with the redistribution of the initial residual stresses. For thick, large and complex parts in highly alloyed aluminium, this redistribution of the residual stresses can leads to an unexpected behaviour of the workpiece and is the main reason for both workpiece deflections (during machining) and post-machining distortions (after unclamping). These two phenomena can lead to the nonconformity of the part with the geometrical and dimensional tolerance specifications and therefore to the rejection of the part or to additional conforming steps. As a consequence, the mechanical behaviour of the workpiece has to be considered during the definition of the machining process plan to improve the machining accuracy and the robustness of the process and thus to ensure the conformity of the machined part with the dimensional and geometrical specifications, i.e. to ensure the desired machining quality. In this paper, the numerical tool developed in [1] is used to conduct an analysis on the influence of the initial workpiece residual stress state, of the fixture layout as well as of the machining sequence on the machining quality. This analysis is performed on a part which has been specially designed and which can be considered as being representative of real aerospace parts. Several comparisons with experimental results are performed, one of them using digital image correlation (DIC) measurements. Results obtained show a good agreement, validating both the prediction of the behaviour of the workpiece during machining and the prediction of the machined part geometry. Based on the results of this analysis, a classification of the parameters has been performed depending on their influence on the machining quality. A first methodology allowing to define machining process plans adapted to the initial workpiece stress state has then been created based on the previous classification. This methodology is composed of a procedure and basic guidelines which are both presented in detail. An example of an application of this methodology is then introduced, demonstrating the benefits of the approach developed in this work.

Inducing Specific Immune Tolerance to Prevent Type 1 Diabetes in NOD Mice.

Proinsulin is the first autoantigen in type 1 diabetes (T1D). We reasoned that coupling hematopoietic stem cells (HSCs) transplantation with ex vivo transduction of syngeneic HSCs with lentiviral vectors to express proinsulin II could prevent T1D in nonobese diabetic (NOD) mice. Hematopoietic stem cells were isolated from 6- to 8-week-old NOD female mice and transduced in vitro with lentiviral vectors encoding proinsulin II. Preconditioned 3- to 4-week-old female NOD mice were transplanted with transduced or nontransduced HSCs and compared with age-matched unmanipulated control. The insulitis, T1D development, and immune reconstitution were assessed. The mean (SD) insulitis score was significantly reduced (1.156 [0.575] vs 2.156 [0.892] or 3.043 [0.728], P = 0.009 or <0.001), and diabetes was nearly completely prevented (1/13 vs 5/12 or 4/9, P = 0.031 or 0.013) in recipients of transduced HSCs expressing proinsulin II as compared with recipients of nontransduced HSCs or unmanipulated control. Sialitis, reconstitution of peripheral blood leukocytes, and in vitro recall responses to ovalbumin were not different between 3 groups of mice. Syngeneic transplantation of HSCs transduced ex vivo with lentiviral vectors to encode proinsulin II is a novel strategy to prevent T1D.

Development of the 19 X-STR loci multiplex system and genetic analysis of a Zhejiang Han population in China.

The 19 X-STRs multiplex system is a PCR-based amplification kit that facilitates simultaneous amplification of 19 X-chromosomal STR loci (i.e. DXS7423, DXS10148, DXS10159, DXS6809, DXS7424, DXS8378, DXS10164, DXS10162, DXS7132, DXS10079, DXS6789, DXS101, DXS10103,DXS10101, HPTRB, DXS10075, DXS10074, DXS10135, and DXS10134). Eleven loci were extensively used in an Investigator Qiagen Argus X-12 (DXS7423, DXS10148, DXS8378, DXS10162, DXS7132, DXS10079, DXS10103, DXS10101, HPTRB, DXS10074, and DXS10135). In this research, the multiplex system was tested for detection sensitivity, DNA mixtures, inhibitor tolerance and species specificity; SWGDAM Validation Guidelines - Approved December 2012 were followed for the human fluorescent STR multiplex PCR reagent. Samples from 181 unrelated Zhejiang Han individuals (121 males and 60 females) were typed using this multiplex system. The results show that this 19X-STRs multiplex system is a robust and reliable amplification means to facilitate forensic and human identification testing.

TH-CD-207A-02: Implementation of Live EPID-Based Inspiration Level Assessment (LEILA) for Deepinspiration Breath-Hold (DIBH) Monitoring Using MV Fluoroscopy

Purpose: As prior work has shown that current DIBH monitoring approaches using surrogate measures (marker block on chest) do not always correspond with the clinical quantity of interest (lung depth, LD), a software tool and workflow are introduced to use MV fluoroscopy during treatment for real-time / Live EPID-based Inspiration Level Assessment (LEILA). Methods: A prototype software tool calculates and displays the LD during the treatment of left sided breast cancer. Calculations are based on MV cine images which are acquired with the treatment beam thereby not incurring any additional imaging dose. Image capture and processing are implemented using a dedicated frame grabber computer. The calculation engine automatically detects image orientation and includes provisions for large treatment fields that exceed the size of the EPID panel. LD is measured along a line profile in the middle of the field. LEILA's interface displays the current MV image, a reference image (DRR), the current LD, as well as a trace of LD over treatment time. The display includes patient specific LD tolerances. Tolerances are specified for each field and loaded before the treatment. A visual warning is generated when the tolerance is exceeded. LEILA is initially run in parallel with current DIBH techniques. When later run by itself DIBH setup will be done using skin marks and room laser. Results: Offline tests of LEILA confirmed accurate automatic LD measurement for a variety of patient geometries. Deployment of the EPID during all left sided breast treatments was well tolerated by patients and staff during a multi-month pilot. The frame grabber provides 11 frames-per-second; the MATLAB based LEILA prototype software can analyze five frames-per-second standalone on standard desktop hardware. Conclusion: LEILA provides an automated approach to quantitatively monitor LD on MV images during DIBH treatment. Future improvements include a database and further speed optimization.

Novel Research on a Fibration Knowledge-Based Solution for Engineered Surface Tolerances within the Modern GPS Context

Geometrical product specification and verification (GPS) is the only worldwide tolerancing specification language available for unambiguously communicating functional requirements and precisely expressing geometrical tolerances of workpieces on technical drawings. Although the understanding and application of GPS are of vital importance for modern manufacturing industry, the latest GPS standards contain huge amounts of data, information and knowledge that are obscurely linked together, so they are very difficult for designers, engineers and metrologists to understand, remember and comprehensively use them. This is especially true for surface texture that is one of the most complicated geometrical characteristics in GPS domain. To overcome the implementation barrier of GPS surface texture, a novel knowledge-based solution supported formally by a unified knowledge modeling of the complete GPS surface texture standard chain has been researched. This knowledge model can serve as a formal basis for developing more intelligent and reliable tolerance design and measurement systems. This paper starts with an introduction on how to build up a formal knowledge model based on category theory, called categorical fibration model, to unified acquisition and representation of all surface texture knowledge. It then moves on to define a recursive knowledge refinement process for the devised model to enable the recursive multi-level GPS knowledge transformations and refinements. This knowledge model is validated naturally by checking rules identified from category theoretic notions. Finally, this paper presents a general framework for applying the modeled GPS knowledge in practice, from the aspects of the integrated and complete modeling of all surface texture knowledge, detailed knowledge acquisition and modeling examples for four sub-knowledge bases of the proposed software solution, core functional features of this software, and a comprehensive case study for testing its validity and usability. This knowledge-based solution facilitates life cycle collaboration and integration of the complete GPS surface texture chain to reduce product development cost and lead time, and to ensure functional requirements of geometrical workpieces.

Manufacturing signature in variational and vector-loop models for tolerance analysis of rigid parts

To control and to manage the geometric deviations along the product life cycle, the first step is to consider, during the design stage, the tolerance specification, the tolerance allocation and the tolerance analysis. Many approaches of the literature for tolerance analysis of rigid assemblies exist, and different commercial computer-aided tolerancing (CAT) software packages were developed with those models. However, there is a growing interest in considering working conditions and operating windows in CAT. As a response to these needs, skin model concept was proposed. The aim of this paper is to connect a point cloud-based discrete geometry framework (i.e. a skin model representation) to the manufacturing processes, in order to bring the CAT simulation tools closer to reality. In this work, the effect of a manufacturing process on solving a stack-up function is investigated throughout circular profiles obtained by a turning process. A case study has been defined and solved using two literature models, the variational model and the vector-loop one, by considering the manufacturing signature. The results have been compared to those obtained by the same models without considering the manufacturing signature. Monte Carlo simulations have been carried out by solving the stack-up functions into Matlab® software, and statistical analyses have been carried out by Minitab® software.