Determination Of Expression Research Articles

K-theoretic Chern class formulas for vexillary degeneracy loci

Using raising operators and geometric arguments, we establish formulas for the K-theory classes of degeneracy loci in classical types. We also find new determinantal and Pfaffian expressions for classical cases considered by Giambelli: the loci where a generic matrix drops rank, and where a generic symmetric or skew-symmetric matrix drops rank.In an appendix, we construct a K-theoretic Euler class for even-rank vector bundles with quadratic form, refining the Chow-theoretic class introduced by Edidin and Graham. We also establish a relation between top Chern classes of maximal isotropic subbundles, which is used in proving the type D degeneracy locus formulas.

Role of Multivalency and Antigenic Threshold in Generating Protective Antibody Responses.

Vaccines play a vital role in protecting our communities against infectious disease. Unfortunately, some vaccines provide only partial protection or in some cases vaccine-mediated immunity may wane rapidly, resulting in either increased susceptibility to that disease or a requirement for more booster vaccinations in order to maintain immunity above a protective level. The durability of antibody responses after infection or vaccination appears to be intrinsically determined by the structural biology of the antigen, with multivalent protein antigens often providing more long-lived immunity than monovalent antigens. This forms the basis for the Imprinted Lifespan model describing the differential survival of long-lived plasma cell populations. There are, however, exceptions to this rule with examples of highly attenuated live virus vaccines that are rapidly cleared and elicit only short-lived immunity despite the expression of multivalent surface epitopes. These exceptions have led to the concept that multivalency alone may not reliably determine the duration of protective humoral immune responses unless a minimum number of long-lived plasma cells are generated by reaching an appropriate antigenic threshold of B cell stimulation. Examples of long-term and in some cases, potentially lifelong antibody responses following immunization against human papilloma virus (HPV), Japanese encephalitis virus (JEV), Hepatitis B virus (HBV), and Hepatitis A virus (HAV) provide several lessons in understanding durable serological memory in human subjects. Moreover, studies involving influenza vaccination provide the unique opportunity to compare the durability of hemagglutinin (HA)-specific antibody titers mounted in response to antigenically repetitive whole virus (i.e., multivalent HA), or detergent-disrupted “split” virus, in comparison to the long-term immune responses induced by natural influenza infection. Here, we discuss the underlying mechanisms that may be associated with the induction of protective immunity by long-lived plasma cells and their importance in future vaccine design.

Genetically engineered probiotic Saccharomyces cerevisiae strains mature human dendritic cells and stimulate Gag-specific memory CD8+ T cells ex vivo.

Recombinant Saccharomyces cerevisiae strains expressing HIV antigens have shown promising pre-clinical results. Probiotic S. cerevisiae strains naturally induce gut immunity; thus, genetically engineered probiotic strains could be used to stimulate immune responses against HIV in the mucosa. Probiotic strains have a higher rate of heterologous protein production, meaning higher antigen's epitope expression levels per yeast cell. We expressed HIV-1 Gag protein in the probiotic yeasts' surface, which was eagerly phagocytosed by and induced type 1 polarization of human monocyte-derived dendritic cells (DCs) from healthy donors in vitro. We further matured DCs derived from HIV-1+ donors with transformed yeasts and incubated them with autologous T cells. Only DCs matured with Gag-expressing probiotic strains were able to efficiently present antigen to CD8+ T cells and induced their clonal expansion. Our results show that genetically engineered probiotic S. cerevisiae strains are a promising vaccination strategy against HIV.

Determinantal expressions and recurrence relations for Fubini and Eulerian polynomials

In the paper, by virtue of an explicit formula for higher order derivatives of the ratio between two differentiable functions in terms of the Hessenberg determinant, the author presents determinantal expressions and recurrence relations for generalized Fubini polynomials. Hereafter, by virtue of relations between Eulerian polynomials and the (generalized) Fubini numbers and polynomials, the author derives a determinantal expression for Eulerian polynomials and several recurrence relations for the Fubini and Eulerian polynomials.

Гетеротопическая ксенотрансплантация фрагментов аорты новорожденных поросят после гипотермической инкубации в консервирующих растворах

Nowadays the porcine vessels and heart valves have been widely applied in transplantology. Due to a high level of Gal-α-1,3-Gal (α-Gal epitope) expression, the porcine bioprostheses are specially treated prior to transplantation either to remove this epitope or to deactivate it. Our previous findings showed that certain media, used for hypothermic storage (HS), reduced the α-Gal epitope expression in aortic tissue in vitro. Here, we studied the HS impact in the histidine-tryptophan-ketoglutarate (HTK), Euro-Collins and Dulbecco’s Modified Eagle Medium (DMEM) solutions on aortic graft integrity in vivo. The analysis of aortic wall histological structure and the evaluation of the immunoproteasome subunits LMP2 and LMP7 expression level demonstrated the aortic fragments, subjected to HS in Euro-Collins solution to day 7 after xenotransplantation to cause a less pronounced immune response as compared to the HTK- and DMEM-incubated xenografts. This may be partially due to the Euro-Collins medium composition. Our findings may be of interest for ela-borating the novel methods for donor material pre-transplantation preparation. Probl Cryobiol Cryomed 2019; 29(1): 073–087

Mutational and Antigenic Landscape in Tumor Progression and Cancer Immunotherapy.

Evolving neoplasms accumulate non-synonymous mutations at a high rate, potentially enabling the expression of antigenic epitopes that can be recognized by the immune system. Since they are not covered by central tolerance, such tumor neoantigens (TNAs) should be under robust immune control as they surge. However, genetic defects that impair cancer cell eradication by the immune system coupled with the establishment of local immunosuppression can enable TNA accumulation, which is generally associated with improved clinical sensitivity to various immunotherapies. Here, we explore how tumor-intrinsic factors and immunological processes shape the mutational and antigenic landscape of evolving neoplasms to influence clinical responses to immunotherapy, and propose strategies to achieve robust immunological control of the disease despite disabled immunosurveillance.

Dual-isotope imaging allows in vivo immunohistochemistry using radiolabelled antibodies in tumours

While radiolabelled antibodies have found great utility as PET and SPECT imaging agents in oncological investigations, a notable shortcoming of these agents is their propensity to accumulate non-specifically within tumour tissue. The degree of this non-specific contribution to overall tumour uptake is highly variable and can ultimately lead to false conclusions. Therefore, in an effort to obtain a reliable measure of inter-individual differences in non-specific tumour uptake of radiolabelled antibodies, we demonstrate that the use of dual-isotope imaging overcomes this issue, enables true quantification of epitope expression levels, and allows non-invasive in vivo immunohistochemistry. The approach involves co-administration of (i) an antigen-targeting antibody labelled with zirconium-89 (89Zr), and (ii) an isotype-matched non-specific control IgG antibody labelled with indium-111 (111In). As an example, the anti-HER2 antibody trastuzumab was radiolabelled with 89Zr, and co-administered intravenously together with its 111In-labelled non-specific counterpart to mice bearing human breast cancer xenografts with differing HER2 expression levels (MDA-MB-468 [HER2-negative], MDA-MB-231 [low-HER2], MDA-MB-231/H2N [medium-HER2], and SKBR3 [high-HER2]). Simultaneous PET/SPECT imaging using a MILabs Vector4 small animal scanner revealed stark differences in the intratumoural distribution of [89Zr]Zr-trastuzumab and [111In]In-IgG, highlighting regions of HER2-mediated uptake and non-specific uptake, respectively. Normalisation of the tumour uptake values and tumour-to-blood ratios obtained with [89Zr]Zr-trastuzumab against those obtained with [111In]In-IgG yielded values which were most strongly correlated (R = 0.94; P = 0.02) with HER2 expression levels for each breast cancer type determined by Western blot and in vitro saturation binding assays, but not non-normalised uptake values. Normalised intratumoural distribution of [89Zr]Zr-trastuzumab correlated well with intratumoural heterogeneity HER2 expression.

Abstract IA17: Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors

Abstract We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. Our largest clinical experience is in treating human Acute Myelogenous Leukemia (AML). After identifying that WT1, a gene associated with promoting leukemic transformation, is over-expressed in human leukemic stem cells, and demonstrating in a clinical trial that in vitro expanded WT1-specific CD8 T cell clones can be safely transferred, exhibit anti-leukemic activity, and provide therapeutic benefit to AML patients, we extensively screened normal human repertoires and isolated a high affinity TCR specific for WT1 for genetically engineering CD8 T cells to reproducibly create cells with high avidity for leukemic cells. We have initially pursued this strategy in a 2 Arm trial for leukemia patients either at high risk of relapse (Arm 1) after hematopoietic cell transplant (HCT) or who have already relapsed after HCT (Arm 2). The prophylactic arm is now completed, with very encouraging results- all patients treated with engineered T cells remain alive and relapse free at a median of 38 months, compared to a relapse rate of ~50% in a concurrent matched cohort. Results in relapsed patients (Arm 2) have been less effective, and we have used high-dimensional analyses including single cell RNAseq both to elucidate the reasons for failure to eradicate the leukemia and to design strategies to overcome these obstacles. Our results have identified several mechanisms by which the leukemia escapes, and we have been testing approaches that employ further genetic modification of the T cells to enhance efficacy. The predominant reason for leukemia progression despite targeted therapy with T cells is the inability of the T cells to persist and maintain function in the context of encountering a rapidly proliferating myeloid leukemia. This reflects both engagement of pathways inhibitory to T cells by the leukemic cells, and apoptosis of the T cells from repetitive stimulation. We are addressing this issue by creating immuno-modulatory fusion proteins (IFPs) that have the ectodomain of an inhibitory or death receptor fused to a survival costimulatory domain. Results with two such IFPs will be discussed, a CD200R/CD28 fusion that binds the inhibitory ligand CD200 commonly expressed on leukemic cells but provides a CD28 costimulatory signal and a Fas/4-1BB fusion that binds FasL but rather than induce death promotes proliferation and survival. A more uncommon reason explaining progression is loss of expression of the WT1 epitope being targeted. This has occurred in 2 patients, but for distinct reasons. In one patient this reflected loss of a component of the immunoproteasome, and we have now isolated a TCR that recognizes an epitope not dependent on the immunoproteasome. In a second patient the level of WT1 expression declined- interestingly, this patient was treated with Vidaza, which can increase expression of WT1, and post-Vidaza the transferred T cells persisting in the patient’s bone marrow recognized and responded in vivo to the relapsing leukemia. The approaches and technologies we are developing and testing in leukemia are also applicable to solid tumors, and preclinical studies in pancreatic and ovarian cancers will be discussed. Citation Format: Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Kelly Paulson, Rachel Perret, Leah Schmidt, Thomas Schmitt, Ingunn Stromnes, Aude Chapuis, Philip D. Greenberg. Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA17.

Shifted Cauchy numbers

We introduce and study shifted Cauchy numbers as different natural extension of the classical Cauchy numbers, in particular, in terms of determinantal expressions. We give several arithmetical or combinatorial properties. We also show a determinant expression of Cauchy numbers of the second kind extensively.

Restricted r-Stirling numbers and their combinatorial applications

We study set partitions with r distinguished elements and block sizes found in an arbitrary index set S. The enumeration of these (S, r)-partitions leads to the introduction of (S, r)-Stirling numbers, an extremely wide-ranging generalization of the classical Stirling numbers and the r-Stirling numbers. We also introduce the associated (S, r)-Bell and (S, r)-factorial numbers. We study fundamental aspects of these numbers, including recurrence relations and determinantal expressions. For S with some extra structure, we show that the inverse of the (S, r)-Stirling matrix encodes the Möbius functions of two families of posets. Through several examples, we demonstrate that for some S the matrices and their inverses involve the enumeration sequences of several combinatorial objects. Further, we highlight how the (S, r)-Stirling numbers naturally arise in the enumeration of cliques and acyclic orientations of special graphs, underlining their ubiquity and importance. Finally, we introduce related (S, r) generalizations of the poly-Bernoulli and poly-Cauchy numbers, uniting many past works on generalized combinatorial sequences.

Development of a Neoantigen Prediction Tool for Patient Stratification in Immuno-Oncology Trials

Immunotherapeutic agents are quickly becoming a routine aspect of treatment paradigms. However, despite clinical successes, cancer immunotherapy faces major challenges. One such challenge is that efficacy in most patients is unpredictable. Many immunotherapy treatments have demonstrated efficacy in only select cancer types. Variability in patient response indicates that immunotherapy needs to be patient-specific in order to be most effective.Identifying biomarkers that have value in predicting benefit from treatment with immunotherapy has been difficult. Few predictive biomarkers for immunotherapy treatments are robustly validated for use in clinical trials. Pivotal trials reveal that treatment benefits with checkpoint blockers is not solely restricted to PD-L1-positive patients, indicating the existence of other unknown biomarkers that could be predictive of response. Similarly, tumor mutational burden (TMB) has limitations, as it is not able to effectively segregate patients that are likely to respond to immunotherapeutic agents in tumor types that are relatively mutationally dormant.Increasingly, evidence suggests that tumor immunogenicity may be a strong biomarker for immunotherapy patient selection. In short, the abundance of predicted immunogenic mutations may be useful in predicting patients likely to benefit from checkpoint blockade and related immunotherapies.To address this need for a more specific biomarker, we have designed an assay and bioinformatic work-flow utilizing a multimodal neo-antigen prediction approach that combines data on somatic variants, RNA expression, and compatibility of resultant epitope with host HLA type.In summary, whole exome and whole transcriptome sequencing are performed on a patient tumor sample, and HLA typing is performed on a matched germline patient sample. The somatic variants (tumor-specific mutations) identified by exome sequencing are compared to the RNA sequencing data to identify the most prevalent variants in the transcriptome occurring in the most highly expressed regions. These highly expressed mutations are most likely to be translated into mutant peptides that can interact with MHC molecules and be subsequently presented on the tumor cell surface as neoantigens. The subject's HLA type is then determined using the seq2hla computational tool. Next, a molecular modeling tool, NetMHC4.0, compares the structures of the candidate mutant peptides to the HLA molecule structures and generates a goodness of fit prediction. A higher binding affinity between mutant peptide and HLA molecule corresponds to a greater likelihood of this complex existing on the cell surface as a neoantigen. This data - the DNA sequencing, RNA expression and binding affinity calculation - is combined via a series of filters to generate an immunogenicity score associated with each tumor mutation / predicted mutant peptide. These candidate neoantigens are then returned as a rank order list for each case. This information then can be used to guide targeted therapies and to stratify patients with higher immunogenicity scores for immunotherapy.To test our bioinformatic pipeline, we utilized a subset of multiple myeloma samples. Such analysis yielded a rank list of predicted neoantigens for each tumor sample, with associated immunogenicity scores for each prediction. Additionally, TMB was calculated for these samples. We compared the number of predicted neoantigens from our workflow to the TMB of the tumors as a proxy for this assay's performance against a current clinically utilized biomarker (TMB). The numbers of predicted neoantigens for the samples ranged from 19 to 61 (Average number of 41 neoantigens per sample), and the TMB scores for these samples respectively were between 7 and 13 mutations per megabase. Comparing these results using Pearson Correlation method yields a strong R squared value of 0.91. Among top ranking neoantigens were peptides associated with TP53, SIK3, ATM and NOTCH2 genes among others, and representing known frequently mutated genes in multiple myeloma. Therefore, our neoantigen predictor demonstrates promise as a reliable tool to identify markers of tumor immunogenicity. These preliminary results suggest that further validation of our process is warranted and may yield a new method for use in patient stratification and response prediction in immuno-oncology trials. DisclosuresNo relevant conflicts of interest to declare.

Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation.Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these.Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv.In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks.To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML.Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. DisclosuresNeri:Philochem AG: Equity Ownership.

Targeting αGal epitopes for multi-species embryo immunosurgery

Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo immunosurgery.

Genetic and environmental factors affecting the expression of \u03b1-gliadin canonical epitopes involved in celiac disease in a wide collection of spelt (Triticum aestivum ssp. spelta) cultivars and landraces

BackgroundCeliac disease (CD) is an autoimmune disorder affecting genetically predisposed individuals whose dietary gluten proteins trigger an inflammatory reaction in the small intestine. Gluten is found in the seeds of cereals like bread wheat (Triticum aestivum ssp. aestivum) and spelt (Triticum aestivum ssp. spelta). The development of new varieties lacking immunogenic peptides is one of the strategies currently investigated to address the CD problem. Among gluten proteins, α-gliadins display the strongest immunogenicity with four main T-cell stimulatory epitopes. The objective of this work was to study the expression of α-gliadin epitopes related to CD in a wide collection of 121 spelt accessions (landraces and varieties, spring and winter accessions) from different provenances, and to analyze the correlation between the presence of epitope sequences in gDNA and their expression (cDNA). The effect of environmental factors (harvest year and N fertilization) on the epitope expression was also investigated.ResultsTaqMan probes targeting the canonical form of the epitopes were used to evaluate the epitope expression levels. Significant variations in the amount of epitope transcripts were identified between accessions and according to the provenances. Spring accessions showed a significantly higher immunogenicity than winter ones and no influence of spelt breeding on the epitope expression levels could be assessed when comparing landraces and varieties from Northwestern Europe. No correlation was observed between quantitative PCR results obtained from cDNA and gDNA for 45 accessions tested, stressing the need to use markers focusing on epitope transcripts rather than on genomic sequences. A relative stability of the amount of epitopes expressed by a same accession across four harvest years was detected. The fertilization strategy, evaluated through seven N fertilization modalities applied to two commercial spelt varieties, did not influence the epitope expression of the first variety, whereas it had a slight effect for the second one.ConclusionsThe results obtained in this work showed that the CD-related epitope expression greatly fluctuated among the spelt accessions studied. This expression was not correlated to the epitope genomic occurrence and environmental factors had almost no influence on the amount of epitope transcripts.

Gal epitope expression and immunological properties in iGb3S deficient mice

The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.

Toxoplasma Does Not Secrete the GRA16 and GRA24 Effectors Beyond the Parasitophorous Vacuole Membrane of Tissue Cysts.

After invasion, Toxoplasma resides in a parasitophorous vacuole (PV) that is surrounded by the PV membrane (PVM). Once inside the PV, tachyzoites secrete dense granule proteins (GRAs) of which some, such as GRA16 and GRA24, are transported beyond the PVM likely via a putative translocon. However, once tachyzoites convert into bradyzoites within cysts, it is not known if secreted GRAs can traffic beyond the cyst wall membrane. We used the tetracycline inducible system to drive expression of HA epitope tagged GRA16 and GRA24 after inducing stage conversion and show that these proteins are not secreted beyond the cyst wall membrane. This suggests that secretion of GRA beyond the PVM is not important for the tissue cyst stage of Toxoplasma.

Strong hydrophobicity enables efficient purification of HBc VLPs displaying various antigen epitopes through hydrophobic interaction chromatography

Hepatitis B core virus-like particle (HBc-VLP) has become a carrier for expression and presentation of foreign epitopes as vaccine candidates. Efficient purification is necessary for preparation of HBc-VLP and its derivatives with various foreign epitopes. In previous reports, HBc-VLP was mainly purified with ion exchange chromatography. In this study, we developed a platform purification technique based on hydrophobic interaction chromatography (HIC). The underlying principle is the strong hydrophobicity on the surface of HBc-VLP, which was found by mathematical calculation and experimental measurement. Based on HIC, a complete downstream process, from feedstock supernatant to the final pure product, was developed involving a heat pretreatment, an HIC, an ultrafiltration concentration and a size exclusion chromatography. The total recovery of HBc-VLP was 41.92% with nearly 100% purity. HIC was also applicable to three HBc-based vaccine candidates displaying epitope from nuclear protein (NP) and matrix protein 2 (M2e) of the influenza A virus, as well as from ovalbumin (OVA). Optimal HIC condition for these three recombinant VLPs was rationally designed by analysis on their surface hydrophobicity, which was influenced upon insertion of the foreign epitope. By applying the same process developed for HBc-VLP, satisfactory results were achieved for product recovery, purity and host cell DNA removal, thus it is possible to become a platform technique for various HBc-VLP based vaccine candidates.

Identifying Stable Fragments of Arabidopsis thaliana Cellulose Synthase Subunit 3 by Yeast Display.

Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown. Here, yeast surface display (YSD) is used to determine the full-length expression of A. thaliana cellulose synthase 3 (AtCesA3) fragments. The level of stably-folded AtCesA3 fragments displayed on the yeast surface are determined using flow cytometric analysis of differential surface expression of epitopes flanking the AtCesA3 fragment. This technique provides a fast and simple method for examining folding and expression of protein domains and fragments of complex proteins.

P146 Variations in the expression of antibody epitopes associated with dqb1*03:01 alleles in B cells

Aim To evaluate variations of the expression of antibody epitopes associated with DQB1*03:01 alleles in B cells. Methods Antibody tests were performed using Luminex Single Antigen Bead (SAB) assay on serum samples treated with DTT. XMs were performed by flow cytometry, using lymphocytes from peripheral blood of healthy volunteers, which were treated with pronase. Results Four sera reacted strongly with beads carrying the following alleles: DQB1*03:01-DQA1*06:01, DQB1*03:01-DQA1*05:05, and DQB1*03:01-DQA1*05:03 (Figure 1). Sera JM and RH reacted strongly with B cells from all 6 donors who were positive for DQB1*03:01 indicating that these cells express adequate amount of DQ molecules (Figure 2A). However, XMs of sera CG and SR with the 4 heterozygous DQB1*03:01 B cells showed weak or no reactivity respectively (Figure 2B). In comparison to XM reactivity from sera JM and RH the XMs with sera SR and CG exhibited a 25-50-fold lower Δ MESF values (Figure 2A versus 2B). This unexpected low reactivity of sera SR and CG with B cells is striking since their reactivity with SAB is similar to that of serum JM except with the DQB1*03:01-DQA1*03:01 allele (Figure 1). Notably, SR and CG reacted with B cells that were homozygous for DQB1*03:01 (Figure 2B, Donor 1 and 2) indicating that the reactivity of these sera with the SAB was not due to denatured epitopes. Conclusions These results suggest that the observed variable expression of DQB1*03:01 epitope on B cells could be due to variation in the conformation of this epitope on the same DQ molecules with a potential impact on antibody affinity to this epitope. The discrepancy between SAB MFI values and XM reactivity is important to consider in assessing HLA compatibility and lymphocyte crossmatching.

Canine mesenchymal stem cells from synovium have a higher chondrogenic potential than those from infrapatellar fat pad, adipose tissue, and bone marrow.

Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA.