Embryonic effects of radiation on ICR mice depending developmental stages

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Gu, Yeun Hwa; Kusama, Tomoko; Kai, Michiaki [University of Tokyo, Tokyo (Japan)

1995-06-15

The ICR pregnant mice were irradiated at 1.5Gy in every 6 hours in the period of organogenesis in order to classify the stage specificity of the embryonic effects of radiation and the stage of development differentiation of the primordium of each major organ. Intrauterine death, fetal body weight and external malformation in live fetuses were observed on day 18 of gestation. There was no statistically significant difference in the intrauterine mortality at any stage organogenesis. The fetal body weight of the mice irradiated in the intermediate stage of organogenesis showed significantly lower. There were specific highly sensitive stages in the incidences of each external malformation, that is exencephalia, open eyelid, cleft palate, anomalies of extremities and anomalies of the tail. At these stage, the primordial of the major organs are established in ICR mice.

Embryonic effects of radiation on ICR mice depending developmental stages

International Nuclear Information System (INIS)

Gu, Yeun Hwa; Kusama, Tomoko; Kai, Michiaki

1995-01-01

The ICR pregnant mice were irradiated at 1.5Gy in every 6 hours in the period of organogenesis in order to classify the stage specificity of the embryonic effects of radiation and the stage of development differentiation of the primordium of each major organ. Intrauterine death, fetal body weight and external malformation in live fetuses were observed on day 18 of gestation. There was no statistically significant difference in the intrauterine mortality at any stage organogenesis. The fetal body weight of the mice irradiated in the intermediate stage of organogenesis showed significantly lower. There were specific highly sensitive stages in the incidences of each external malformation, that is exencephalia, open eyelid, cleft palate, anomalies of extremities and anomalies of the tail. At these stage, the primordial of the major organs are established in ICR mice

Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.

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Katsiani, E; Garas, A; Skentou, C; Tsezou, A; Messini, C I; Dafopoulos, K; Daponte, A; Messinis, I E

2016-09-01

Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. MSCs derived from placental chorionic villi of the first trimester are likely to resemble, biologically, embryonic stem cells (ESC), due to the earlier development stage of placenta. In the present study long-term cultures of MSC-like cells were assessed in order to evaluate MSCs multipotent characteristics and molecular features during the period of culture. CV-cells obtained from 10 samples of chorionic villus displayed typical fibroblastoid morphology, undergone 20 passages during a period of 120 days, maintaining a stable karyotype throughout long term expansion. The cells were positive, for CD90, CD73, CD105, CD29, CD44, HLA ABC antigens and negative for CD14, CD34, AC133, and HLA DR antigens as resulted from the flow cytometry analysis. CV-cells were differentiated in adipocytes, osteoblasts, chondrocytes and neuronal cells under specific culture conditions. The expression of the ESC-gene markers POU5F1 (Oct-4) and NANOG was observed at earliest stages (4-12 passages) and not at the late stages (14-20 passages) by RT-PCR analysis. ZFP42 and SOX2 expression were not detected. Moreover, CV-cells were found to express GATA4 but not NES (Nestin). Chorionic villi-derived cells possess multipotent properties, display high proliferation rate and self-renew capacity, share common surface antigens with adult MSCs and express certain embryonics stem cells gene markers. These characteristics highlight chorionic villi as an attractive source of MSCs for the needs of regenerative medicine.

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

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Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

DEFF Research Database (Denmark)

Barraud, Perrine; Stott, Simon; Møllgård, Kjeld

2007-01-01

The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression....... Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain....... decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we...

Antigen-driven focal inflammatory death of malaria liver stages

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Ganchimeg eBayarsaikhan

2015-02-01

Full Text Available Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8+ T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8+ T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8+ T cells still remain obscure. Recently, it has been shown that specific effector CD8+ T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8+ T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.

Synthetic TLR4 agonists enhance functional antibodies and CD4+ T-cell responses against the Plasmodium falciparum GMZ2.6C multi-stage vaccine antigen

DEFF Research Database (Denmark)

Baldwin, Susan L; Roeffen, Will; Singh, Susheel K

2016-01-01

A subunit vaccine targeting both transmission and pathogenic asexual blood stages of Plasmodium falciparum, i.e., a multi-stage vaccine, could be a powerful tool to combat malaria. Here, we report production and characterization of the recombinant protein GMZ2.6C, which contains a fragment of the......-γ and TNF in response to GMZ2.6C. Both of these agonists have good safety records in humans....... of the sexual-stage protein Pfs48/45-6C genetically fused to GMZ2, an asexual vaccine antigen in advanced clinical development. To select the most suitable vaccine formulation for downstream clinical studies, GMZ2.6C was tested with various immune modulators in different adjuvant formulations (stable emulsions......, liposomes, and alum) in C57BL/6 mice. Some, but not all, formulations containing either the synthetic TLR4 agonist GLA or SLA elicited the highest parasite-specific antibody titers, the greatest IFN-γ responses in CD4+ TH1 cells, and the highest percentage of multifunctional CD4+ T cells expressing IFN...

[The characters and specific features of new human embryonic stem cells lines].

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Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

DEFF Research Database (Denmark)

Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

2017-01-01

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

Characterization of antigen-specific, Ia-restricted, L3T4+ cytolytic T lymphocytes and assessment of thymic influence on their self specificity

International Nuclear Information System (INIS)

Golding, H.; Munitz, T.I.; Singer, A.

1985-01-01

The goals of the present study were: (a) to generate antigen-specific L3T4+ cytolytic T lymphocytes (CTL), (b) to determine their major histocompatibility complex (MHC) restriction specificity, and (c) to assess the influence of thymic MHC determinants on their self specificity. The authors found that L3T4+ CTL specific for either trinitrophenyl (TNP)-modified self determinants or minor histocompatibility antigens could be generated from Lyt-2- responder T cells provided that the response cultures were supplemented with supernatants rich in helper factors. Such antigen-specific L3T4+ CTL were Ia-restricted by the criteria that they lysed only Ia+ target cells and that their lysis of Ia+ target cells was specifically inhibited by anti-Ia monoclonal antibodies. The relative frequency of L3T4+ pCTL was found to be only 5-10% of the total anti-TNP pCTL present in the spleens of normal mice. Finally, the authors utilized radiation bone marrow chimeras to assess the influence of the thymic haplotype on the self-Ia specificity of L3T4+ CTL. Both bulk culture and limiting dilution experiments revealed that the self-Ia specificity of L3T4+ anti-TNP CTL from F1----parent and A----B allogeneic chimeras was not markedly skewed toward the haplotype of the chimeric thymus. These results contrast with those obtained previously for L3T4+ anti-TNP Th cells and demonstrate that in the radiation bone marrow chimera model of T cell differentiation, the self specificity of Th cells but not pCTL is markedly influenced by the haplotype of the chimeric thymus

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

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Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Fetal- and uterine-specific antigens in human amniotic fluid.

Science.gov (United States)

Sutcliffe, R G; Brock, D J; Nicholson, L V; Dunn, E

1978-09-01

Removal of the major maternal serum proteins from second trimester amniotic fluid by antibody affinity chromatography revealed various soluble tissue antigens, of which two were fetal-specific skin proteins and another, of alpha2-mobility, was specific to the uterus, and was therefore designated alpha-uterine protein (AUP). These proteins could not be detected in maternal serum by antibody-antigen crossed electrophoresis. The concentration of AUP in amniotic fluid reached a maximum between 10 and 20 weeks of gestation, suggesting that there is an influx of uterine protein into the amniotic fluid at this stage of pregnancy.

Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

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Andrea Corsinotti

Full Text Available Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis.

RM2 antigen (beta1,4-GalNAc-disialyl-Lc4) as a new marker for prostate cancer.

Science.gov (United States)

Saito, Seiichi; Egawa, Shin; Endoh, Mareyuki; Ueno, Seiji; Ito, Akihiro; Numahata, Kenji; Satoh, Makoto; Kuwao, Sadahito; Baba, Shiro; Hakomori, Senitiroh; Arai, Yoichi

2005-05-20

Although prostate-specific antigen (PSA) has been widely used for early detection of prostate cancer, PSA has problems with specificity and prediction of pathological stage. Therefore, a new marker for prostate cancer is urgently required. We examined expression of a novel carbohydrate antigen, beta1,4-GalNAc-disialyl-Lc(4), defined by the monoclonal antibody RM2, in prostate cancer using 75 cases of radical prostatectomy specimens. RM2 immunoreactivity was negative to weak in all benign glands, and weak to moderate in high-grade prostatic intraepithelial neoplasia. In prostatic adenocarcinoma, RM2 immunoreactivity was negative to weak (lower expression) in 20 cases, and moderate to strong (higher expression) in 55 cases. A clear difference of RM2 expression level was observed between Gleason patterns 3 and >/=4. Higher expression of RM2 antigen was significantly associated with primary Gleason pattern >/=4, high Gleason score (>/=8), larger tumor volume and advanced tumor stage. Furthermore, 5-year PSA failure-free survival was significantly lower in the higher expression group. However, no significant relationship was observed between RM2 expression level and preoperative serum PSA. Western blot analysis in prostate cancer cell lines PC3 and LNCap revealed that major 49-kDa and minor 39-kDa glycoproteins were common to both cells, but there was an increase of 59- and 125-kDa glycoproteins unique to LNCap and an increase of 88- and 98-kDa glycoproteins unique to PC3. RM2 antigen is a new histological marker for prostate cancer that may reflect the Gleason grading system. Identification of the glycoproteins carrying the RM2 antigen will provide new insights into the properties of prostate cancer. (c) 2005 Wiley-Liss, Inc.

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses.

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Dowdall, S M J; Proudman, C J; Love, S; Klei, T R; Matthews, J B

2003-12-01

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.

NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

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Yoshie Kametani

Full Text Available Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD, which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg. After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2 cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP or keyhole limpet hemocyanin (KLH successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

International Nuclear Information System (INIS)

Abbas, A.K.; Haber, S.; Rock, K.L.

1985-01-01

Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

Possible presence of common tyvelose-containing glycans in Trichinella L1 larvae and embryonated eggs of several nematodes

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Dea-Ayuela M.A.

2001-06-01

Full Text Available A monoclonal antibody (mAb US4 recognising an epitope containing tyvelose within the T. spiralis L-1 muscle larvae (TSL-1 antigens was tested in western-blot against various antigenic preparations from different stages of the following nematodes: T. spiralis (L1,adult, T. muris (egg, L1, L3, adult, Ascaris suum (egg, adult, Toxocara canis (egg, adult, Anisakis simplex (L3 and Haemochus contortus (egg. Positive reaction was present in antigen preparations from L1 larvae of T. spiralis and T. muris and from embryonated eggs of T. muris, A. suum, T. canis and H. conlortus.

Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

DEFF Research Database (Denmark)

Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A

2016-01-01

. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells...... derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition....

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

Science.gov (United States)

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Activation of nickel-specific CD4+ T lymphocytes in the absence of professional antigen-presenting cells.

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Nasorri, Francesca; Sebastiani, Silvia; Mariani, Valentina; De Pità, Ornella; Puddu, Pietro; Girolomoni, Giampiero; Cavani, Andrea

2002-01-01

Allergic contact dermatitis ensues from exaggerated T cell responses to haptens. Dendritic cells are required for the initiation of hapten sensitization, but they may not be necessary for disease expression. Here we investigated the antigen-presenting cell requirement of nickel-specific CD4+ lymphocytes isolated from the blood of six allergic individuals. A significant proportion (42 out of 121; 35%) of the T cell clones proliferated in vitro to nickel also in the absence of professional antigen-presenting cells, suggesting a direct T-T hapten presentation. Antigen-presenting-cell-independent T cells showed a predominant T helper 1 phenotype. Nickel recognition by these T cells was major histocompatibility complex class II restricted, not influenced by CD28 triggering, independent from their state of activation, and did not require processing. The capacity of this T cell subset to be directly stimulated by nickel was not due to unique antigen-presenting properties, as both antigen-presenting-cell-dependent and antigen-presenting-cell-independent clones displayed comparable levels of HLA-DR, CD80, and CD86, and were equally capable of presenting nickel to antigen-presenting-cell-independent clones. In contrast, neither T cell types activated antigen-presenting-cell-dependent T lymphocytes. T-T presentation induced T cell receptor downregulation, CD25, CD80, CD86, and HLA-DR upregulation, and interferon-gamma release, although to a lesser extent compared to those induced by dendritic cell-T presentation. Following T-T presentation, the clones did not undergo unresponsiveness and maintained the capacity to respond to dendritic cells pulsed with antigen. In aggregate, our data suggest that antigen-presenting-cell-independent T cell activation can effectively amplify hapten- specific immune responses.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

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Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-03-30

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

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Wynne, E; Slocombe, J O; Wilkie, B N

1981-07-01

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

Science.gov (United States)

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Periconceptional maternal one-carbon biomarkers are associated with embryonic development according to the Carnegie stages.

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Parisi, F; Rousian, M; Koning, A H J; Willemsen, S P; Cetin, I; Steegers-Theunissen, R P M

2017-03-01

Is periconceptional maternal one-carbon (I-C) metabolism associated with embryonic morphological development in non-malformed ongoing pregnancies? Serum vitamin B12, red blood cell (RBC) folate and plasma total homocysteine (tHcy) are associated with embryonic development according to the Carnegie stages. Derangements in maternal I-C metabolism affect reproductive and pregnancy outcomes, as well as future health of the offspring. Between 2010 and 2014, women with singleton ongoing pregnancies were enrolled in a prospective periconceptional cohort study. A total of 234 pregnancies, including 138 spontaneous or IUI pregnancies with strict pregnancy dating and 96 pregnancies derived from IVF, ICSI or cryopreserved embryo transfer (IVF/ICSI pregnancies), underwent longitudinal transvaginal three-dimensional ultrasound (3D US) scans from 6+0 up to 10+2 weeks of gestation. Carnegie stages were defined using internal and external morphologic criteria in a virtual reality system. Maternal venous blood samples were collected at enrollment for serum vitamin B12, RBC folate and plasma tHcy assessment. Associations between biomarker concentrations and longitudinal Carnegie stages were investigated using linear mixed models. We performed a median of three 3D US scans per pregnancy (range 1-5) resulting in 600 good quality data sets for the Carnegie stage annotation (80.5%). Vitamin B12 was positively associated with embryonic development in the total study population (β = 0.001 (95% CI: 0.000; 0.002), P Carnegie stages only in IVF/ICSI pregnancies (β = 0.001 (95% CI: 0.0005; 0.0015), P < 0.05). In this group, low RBC folate concentrations (-2SD, 875.4 nmol/l) were associated with a 1.8-day delay (95% CI: 1.7-1.8) in development compared with high concentrations (+2SD, 2119.9 nmol/l). tHcy was negatively associated with embryonic development in the total study population (β = -0.08 (95% CI: -0.14; -0.02), P < 0.01), as well as in the IVF/ICSI subgroup (β = -0.08 (95% CI: -0

Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

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Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

2012-04-01

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

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Chhabra, Hemlata; Gupta, Priyanka; Verma, Paul J.; Jadhav, Sameer; Bellare, Jayesh R.

2014-01-01

We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold

Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

2014-04-01

We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

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Hinrich eAbken

2013-11-01

Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

Predictive value of different prostate-specific antigen-based markers in men with baseline total prostate-specific antigen <2.0 ng/mL.

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Fujizuka, Yuji; Ito, Kazuto; Oki, Ryo; Suzuki, Rie; Sekine, Yoshitaka; Koike, Hidekazu; Matsui, Hiroshi; Shibata, Yasuhiro; Suzuki, Kazuhiro

2017-08-01

To investigate the predictive value of various molecular forms of prostate-specific antigen in men with baseline prostate-specific antigen baseline prostate-specific antigen level baseline prostate-specific antigen- and age-adjusted men who did not develop prostate cancer. Serum prostate-specific antigen, free prostate-specific antigen, and [-2] proenzyme prostate-specific antigen were measured at baseline and last screening visit. The predictive impact of baseline prostate-specific antigen- and [-2] proenzyme prostate-specific antigen-related indices on developing prostate cancer was investigated. The predictive impact of those indices at last screening visit and velocities from baseline to final screening on tumor aggressiveness were also investigated. The baseline free to total prostate-specific antigen ratio was a significant predictor of prostate cancer development. The odds ratio was 6.08 in the lowest quintile baseline free to total prostate-specific antigen ratio subgroup. No serum indices at diagnosis were associated with tumor aggressiveness. The Prostate Health Index velocity and [-2] proenzyme prostate-specific antigen/free prostate-specific antigen velocity significantly increased in patients with higher risk D'Amico risk groups and higher Gleason scores. Free to total prostate-specific antigen ratio in men with low baseline prostate-specific antigen levels seems to predict the risk of developing prostate cancer, and it could be useful for a more effective individualized screening system. Longitudinal changes in [-2] proenzyme prostate-specific antigen-related indices seem to correlate with tumor aggressiveness, and they could be used as prognostic tool before treatment and during active surveillance. © 2017 The Japanese Urological Association.

Pathological Outcome following Radical Prostatectomy in Men with Prostate Specific Antigen Greater than 10 ng/ml and Histologically Favorable Risk Prostate Cancer.

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Yu, Jiwoong; Kwon, Young Suk; Kim, Sinae; Han, Christopher Sejong; Farber, Nicholas; Kim, Jongmyung; Byun, Seok Soo; Kim, Wun-Jae; Jeon, Seong Soo; Kim, Isaac Yi

2016-05-01

Active surveillance is now the treatment of choice in men with low risk prostate cancer. Although there is no consensus on which patients are eligible for active surveillance, prostate specific antigen above 10 ng/ml is generally excluded. In an attempt to determine the validity of using a prostate specific antigen cutoff of 10 ng/ml to counsel men considering active surveillance we analyzed a multi-institution database to determine the pathological outcome in men with prostate specific antigen greater than 10 ng/ml but histologically favorable risk prostate cancer. We queried a prospectively maintained database of men with histologically favorable risk prostate cancer who underwent radical prostatectomy between 2003 and 2015. The cohort was categorized into 3 groups based on prostate specific antigen level, including low-less than 10 ng/ml, intermediate-10 or greater to less than 20 and high-20 or greater. Associations of prostate specific antigen group with adverse pathological and oncologic outcomes were analyzed. Of 2,125 patients 1,327 were categorized with histologically favorable risk disease. However on multivariate analyses the rates of up staging and upgrading were similar between the intermediate and low prostate specific antigen groups. In contrast compared to the intermediate prostate specific antigen group the high group had higher incidences of up staging (p = 0.02) and upgrading to 4 + 3 or greater disease (p = 0.046). Biochemical recurrence-free survival rates revealed no pairwise intergroup differences except between the low and high groups. Patients with preoperatively elevated prostate specific antigen between 10 and less than 20 ng/ml who otherwise had histologically favorable risk prostate cancer were not at higher risk for adverse pathological outcomes than men with prostate specific antigen less than 10 ng/ml. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages.

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Gautam, A; Dubey, J P; Saville, W J; Howe, D K

2011-12-29

Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination. Copyright © 2011 Elsevier B.V. All rights reserved.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

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Laurence Rolland

1992-06-01

Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Vaccination Expands Antigen-Specific CD4+ Memory T Cells and Mobilizes Bystander Central Memory T Cells

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Li Causi, Eleonora; Parikh, Suraj C.; Chudley, Lindsey; Layfield, David M.; Ottensmeier, Christian H.; Stevenson, Freda K.; Di Genova, Gianfranco

2015-01-01

CD4+ T helper memory (Thmem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Thmem cells as expected, but was accompanied by parallel increase of Thmem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Thmem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Thmem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Thmem cells were activated (CD38High HLA-DR+), cycling or recently divided (Ki-67+), and apparently vulnerable to death (IL-7RαLow and Bcl-2 Low). In contrast, bystander Thmem cells were resting (CD38Low HLA-DR- Ki-67-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Thmem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines. PMID:26332995

File list: ALL.Emb.05.AllAg.Embryonic_heart [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Emb.05.AllAg.Embryonic_heart mm9 All antigens Embryo Embryonic heart SRX967652,...4,SRX1437348,SRX377683,SRX377685,SRX377687,SRX190172,SRX244285,SRX1100405,SRX337963 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_heart.bed ...

Function of the PHA-4/FOXA transcription factor during C. elegans post-embryonic development

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Chen Di

2008-02-01

Full Text Available Abstract Background pha-4 encodes a forkhead box (FOX A transcription factor serving as the C. elegans pharynx organ identity factor during embryogenesis. Using Serial Analysis of Gene Expression (SAGE, comparison of gene expression profiles between growing stages animals and long-lived, developmentally diapaused dauer larvae revealed that pha-4 transcription is increased in the dauer stage. Results Knocking down pha-4 expression by RNAi during post-embryonic development showed that PHA-4 is essential for dauer recovery, gonad and vulva development. daf-16, which encodes a FOXO transcription factor regulated by insulin/IGF-1 signaling, shows overlapping expression patterns and a loss-of-function post-embryonic phenotype similar to that of pha-4 during dauer recovery. pha-4 RNAi and daf-16 mutations have additive effects on dauer recovery, suggesting these two regulators may function in parallel pathways. Gene expression studies using RT-PCR and GFP reporters showed that pha-4 transcription is elevated under starvation, and a conserved forkhead transcription factor binding site in the second intron of pha-4 is important for the neuronal expression. The vulval transcription of lag-2, which encodes a ligand for the LIN-12/Notch lateral signaling pathway, is inhibited by pha-4 RNAi, indicating that LAG-2 functions downstream of PHA-4 in vulva development. Conclusion Analysis of PHA-4 during post-embryonic development revealed previously unsuspected functions for this important transcriptional regulator in dauer recovery, and may help explain the network of transcriptional control integrating organogenesis with the decision between growth and developmental arrest at the dauer entry and exit stages.

Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

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George Q Perrin

2016-01-01

Full Text Available The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8 vector expressing cytoplasmic ovalbumin (OVA into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

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MORALES Olga Lucía

2002-01-01

Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes.

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Shinomiya, A; Tanaka, M; Kobayashi, T; Nagahama, Y; Hamaguchi, S

2000-08-01

The medaka homolog of the Drosophila vasa gene, olvas (Oryzias latipes vas) was obtained using polymerase chain reaction of medaka cDNA from the testis and ovary. The spatio-temporal expression pattern of olvas transcripts was observed by in situ hybridization on gonads and embryos. The transcripts for olvas were exclusively detected in the cytoplasm of germ cells in the testis and ovary, not in gonadal somatic cells. In the early developmental stages, each blastomere possessed the maternal transcripts of olvas, which disappeared during gastrula stages. At the late gastrula stage, specific expression of olvas was observed only in germline cells located at the posterior shield. Embryos after the hybridization were examined histologically, and the distribution and migration path of primordial germ cells (PGC) during early stages of embryonic-body formation were revealed using the olvas gene as a germline cell marker. The PGC were translocated from the posterior shield to both sides of the embryonic body via the inner embryonic body in the medaka.

Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

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Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

2010-02-01

The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

4D atlas of the mouse embryo for precise morphological staging.

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Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark

2015-10-15

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development. © 2015. Published by The Company of Biologists Ltd.

Advanced prostatic carcinomas with low serum levels of prostate-specific antigen

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Cerović Snežana J.

2002-01-01

Full Text Available The serum levels of prostate-specific antigen (PSA represent a significant diagnostic and monitoring parameter of prostatic carcinoma (PC. The aim of the study was to establish correlation of serum PSA level in addition to grade, histological type, and clinical stage of PC in patients with normal or intermediary PSA serum level. In 37 untreated PC patients with preoperative serum PSA levels ranging between 0.1 and 9.6 ng/ml, paraffin-embedded tissue and serum samples were immunohistological studied and immunoassay for PSA was done. The most representative was poorly differentiated PC with D stage In serum samples from PC patients 27 (73.7% normal (≤ 4.0 ng/ml, and 10 (27.3% intermediate (4.1-10 ng/ml PSA levels were found Immunohistochemistry, in 36 PC (97.3% had demonstrated the expression of PSA. Our study results had shown low serum PSA levels in some patients with advanced poorly differentiated PC.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

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Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

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Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

2013-10-01

Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

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Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

2018-05-01

The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

Role of 4-1BB receptor in the control played by CD8(+ T cells on IFN-gamma production by Mycobacterium tuberculosis antigen-specific CD4(+ T Cells.

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Carla Palma

Full Text Available BACKGROUND: Antigen-specific IFN-gamma producing CD4(+ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+ T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+ T cells which suppressed IFN-gamma-secreting CD4(+ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+ T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+ T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+ T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+ T cells. The selective

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

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Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Prostate-specific antigen lowering effect of metabolic syndrome is influenced by prostate volume.

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Choi, Woo Suk; Heo, Nam Ju; Paick, Jae-Seung; Son, Hwancheol

2016-04-01

To investigate the influence of metabolic syndrome on prostate-specific antigen levels by considering prostate volume and plasma volume. We retrospectively analyzed 4111 men who underwent routine check-ups including prostate-specific antigen and transrectal ultrasonography. The definition of metabolic syndrome was based on the modified Adult Treatment Panel III criteria. Prostate-specific antigen mass density (prostate-specific antigen × plasma volume / prostate volume) was calculated for adjusting plasma volume and prostate volume. We compared prostate-specific antigen and prostate-specific antigen mass density levels of participants with metabolic syndrome (metabolic syndrome group, n = 1242) and without metabolic syndrome (non-prostate-specific antigen metabolic syndrome group, n = 2869). To evaluate the impact of metabolic syndrome on prostate-specific antigen, linear regression analysis for the natural logarithm of prostate-specific antigen was used. Patients in the metabolic syndrome group had significantly older age (P prostate volume (P prostate-specific antigen (non-metabolic syndrome group vs metabolic syndrome group; 1.22 ± 0.91 vs 1.15 ± 0.76 ng/mL, P = 0.006). Prostate-specific antigen mass density in the metabolic syndrome group was still significantly lower than that in the metabolic syndrome group (0.124 ± 0.084 vs 0.115 ± 0.071 μg/mL, P = 0.001). After adjusting for age, prostate volume and plasma volume using linear regression model, the presence of metabolic syndrome was a significant independent factor for lower prostate-specific antigen (prostate-specific antigen decrease by 4.1%, P = 0.046). Prostate-specific antigen levels in patients with metabolic syndrome seem to be lower, and this finding might be affected by the prostate volume. © 2016 The Japanese Urological Association.

Prostate specific antigen in the diagnosis and treatment of adenocarcinoma of the prostate. III. Radiation treated patients

International Nuclear Information System (INIS)

Stamey, T.A.; Kabalin, J.N.; Ferrari, M.

1989-01-01

Serum prostate specific antigen was determined (Yang polyclonal radioimmunoassay) in 183 men after radiation therapy for adenocarcinoma of the prostate. A total of 163 men had received 7,000 rad external beam radiotherapy and 20 had been implanted with iodine-125 seeds. Only 11 per cent of these 183 patients had undetectable prostate specific antigen levels at a mean interval of 5 years since completion of radiotherapy. Prostate specific antigen levels after radiotherapy were directly related to initial clinical stage and Gleason score before treatment. Multiple prostate specific antigen determinations were performed with time in 124 of 183 patients. During year 1 after radiotherapy prostate specific antigen levels were decreasing in 82 per cent of the patients but only 8 per cent continued to decrease beyond year 1. Of 80 patients observed greater than 1 year after completion of radiotherapy 51 per cent had increasing values and 41 per cent had stable values. Increasing prostate specific antigen values after radiotherapy were correlated with progression to metastastic disease and residual cancer on prostate biopsy. Total serum acid phosphatase levels were poorly related to prostate specific antigen levels, were less effective in discriminating patients with metastatic disease and provided no additional information beyond that provided by prostate specific antigen

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

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Ian A Cockburn

2010-05-01

Full Text Available Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Zinc oxide nanoparticle-enhanced ultrasensitive chemiluminescence immunoassay for the carcinoma embryonic antigen

International Nuclear Information System (INIS)

Pal, Souvik; Bhand, Sunil

2015-01-01

An ultrasensitive enzyme-linked immunosorbent assay (ELISA) is reported for the determination of carcinoma embryonic antigen (CEA) in human serum. It was realized using a microplate reader using a 384-well plate. Monoclonal antibody (Ab) against CEA (1° Ab) acting as the capture probe was immobilized on zinc oxide nanoparticles (ZnO-NPs) in the form of self-assembled monolayers (SAMs). CEA captured by 1° Ab was quantified using a sandwich ELISA wherein a polyclonal second antibody against CEA (2° Ab) was used for detection and quantified using an HRP-labeled secondary antibody (3° Ab). The ZnO-NPs-CEA capture probe was deposited on the bottom of the wells in order to enhance capture of CEA. A 3-fold enhancement in the chemiluminescence (CL) signal of luminol is found (compared to a conventional ELISA). CEA can be quantified by this method in concentrations as low as 1 pg · mL −1 . The upper limit of detection is 20 ng · mL −1 . The use of ZnO-NPs also imparts improved thermal stability. When stored at 4 °C in phosphate-buffered saline of pH 7.4, the probe displays stability of up to 30 days. (author)

Posttreatment prostatic-specific antigen doubling time as a surrogate endpoint for prostate cancer-specific survival: An analysis of Radiation Therapy Oncology Group Protocol 92-02

International Nuclear Information System (INIS)

Valicenti, Richard K.; DeSilvio, Michelle; Hanks, Gerald E.; Porter, Arthur; Brereton, Harmar; Rosenthal, Seth A.; Shipley, William U.; Sandler, Howard M.

2006-01-01

Purpose: We evaluated whether posttreatment prostatic-specific antigen doubling time (PSADT) was predictive of prostate cancer mortality by testing the Prentice requirements for a surrogate endpoint. Methods and Materials: We analyzed posttreatment PSA measurements in a cohort of 1,514 men with localized prostate cancer (T2c-4 and PSA level Cox = 0.002), PSADT Cox Cox Cox Cox = 0.4). The significant posttreatment PSADTs were also significant predictors of CSS (p Cox < 0.001). After adjusting for T stage, Gleason score and PSA, all of Prentice's requirements were not met, indicating that the effect of PSADT on CSS was not independent of the randomized treatment. Conclusions: Prostatic specific antigen doubling time is significantly associated with CSS, but did not meet all of Prentice's requirements for a surrogate endpoint of CSS. Thus, the risk of dying of prostate cancer is not fully explained by PSADT

The value of prostate specific antigen and prostate specific antigen density in the diagnosis ad treatment of prostate cancer

International Nuclear Information System (INIS)

Hu Guoying; Yu Mingqi; Feng Xinli

2001-01-01

To study the clinical value of prostate specific antigen (PSA) and prostate specific antigen density (PSAD), the PSA levels of pre-and post-treatment were measured in 28 cases with prostate cancer (Pca) and 80 patients with being Prostate hyperplasia (BPH). PASD was measured in 18 cases Pca and 50 cases BPH of them. The results suggest that the sensitivity, specificity and accuracy of diagnosis for Pca were 85.7%, 80.0% and 81.4%, respectively. The false positive rate was 20%. PSAD is superior to PSA in distinguishing prostate cancer from benign prostate hyperplasia. The false positive rate was only 6%. But in the clinical application, the authors should combine PASD with other materials. The regular observation of post therapeutic PSA is of great value to the earlier discovery of local recurrence and metastasis as well as the judgement of curative effect and prognosis

Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii

International Nuclear Information System (INIS)

Grujic, Ognjen; Grigg, Michael E.; Boulanger, Martin J.

2008-01-01

Preliminary X-ray diffraction studies of the bradyzoite-specific surface antigen BSR4 from T. gondii are described. Toxoplasma gondii is an important global pathogen that infects nearly one third of the world’s adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 Å resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4 1 2 1 2 or P4 3 2 1 2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 Å 3 Da −1

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

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Ahmed Kamel El-Sayed

2014-11-01

Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model

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Prabitha Natarajan

2017-08-01

Full Text Available Approximately 3–10% of human red blood cell (RBC transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that “non-responders” may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken

Specialized mouse embryonic stem cells for studying vascular development.

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Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

2014-01-01

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

Prostate Specific Membrane Antigen (PSMA) Targeted Bio-orthogonal Therapy for Metastatic Prostate Cancer

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0595 TITLE: Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate Cancer...Sep 2016 - 14 Sep 2017 4. TITLE AND SUBTITLE Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate

Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

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Kuo-Ching Sheng

2013-01-01

Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

Functional, Antigen-Specific Stem Cell Memory (TSCM CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

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Cheleka A. M. Mpande

2018-03-01

Full Text Available BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM, which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM or effector (TEFF T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb, the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+ were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also

Generation of hematopoietic lineage cells from embryonic like cells

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Gholam Reza Khamisipour

2014-10-01

Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

Anvendelse af prostataspecifikt antigen. En oversigt

DEFF Research Database (Denmark)

Brasso, K; Skaarup, P; Roosen, Jens Ulrik

1998-01-01

Since it was first introduced, measurement of prostate specific antigen has gained increasing interest, and prostate specific antigen is regarded as being the best tumour marker available. The antigen lacks cancer specificity, limiting the usefulness in early diagnosis, The use of prostate specific...... antigen in early diagnosis, staging, and in monitoring patients with prostate cancer is reviewed....

The Hamster Model for Identification of Specific Antigens of Taenia solium Tapeworms

Science.gov (United States)

Ochoa-Sánchez, Alicia; Jiménez, Lucía; Landa, Abraham

2011-01-01

Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm) and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs) and excretion-secretion antigens (E/S Ag) obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis. PMID:22253530

The Hamster Model for Identification of Specific Antigens of Taenia solium Tapeworms

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Alicia Ochoa-Sánchez

2011-01-01

Full Text Available Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs and excretion-secretion antigens (E/S Ag obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis.

Screening Immunomodulators To Skew the Antigen-Specific Autoimmune Response.

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Northrup, Laura; Sullivan, Bradley P; Hartwell, Brittany L; Garza, Aaron; Berkland, Cory

2017-01-03

Current therapies to treat autoimmune diseases often result in side effects such as nonspecific immunosuppression. Therapies that can induce antigen-specific immune tolerance provide an opportunity to reverse autoimmunity and mitigate the risks associated with global immunosuppression. In an effort to induce antigen-specific immune tolerance, co-administration of immunomodulators with autoantigens has been investigated in an effort to reprogram autoimmunity. To date, identifying immunomodulators that may skew the antigen-specific immune response has been ad hoc at best. To address this need, we utilized splenocytes obtained from mice with experimental autoimmune encephalomyelitis (EAE) in order to determine if certain immunomodulators may induce markers of immune tolerance following antigen rechallenge. Of the immunomodulatory compounds investigated, only dexamethasone modified the antigen-specific immune response by skewing the cytokine response and decreasing T-cell populations at a concentration corresponding to a relevant in vivo dose. Thus, antigen-educated EAE splenocytes provide an ex vivo screen for investigating compounds capable of skewing the antigen-specific immune response, and this approach could be extrapolated to antigen-educated cells from other diseases or human tissues.

Advances in prostate-specific membrane antigen PET of prostate cancer.

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Bouchelouche, Kirsten; Choyke, Peter L

2018-05-01

In recent years, a large number of reports have been published on prostate-specific membrane antigen (PSMA)/PET in prostate cancer (PCa). This review highlights advances in PSMA PET in PCa during the past year. PSMA PET/computed tomography (CT) is useful in detection of biochemical recurrence, especially at low prostate-specific antigen (PSA) values. The detection rate of PSMA PET is influenced by PSA level. For primary PCa, PSMA PET/CT shows promise for tumour localization in the prostate, especially in combination with multiparametric MRI (mpMRI). For primary staging, PSMA PET/CT can be used in intermediate and high-risk PCa. Intraoperative PSMA radioligand guidance seems promising for detection of malignant lymph nodes. While the use of PSMA PET/MRI in primary localized disease is limited to high and intermediate-risk patients and localized staging, in the recurrence setting, PET/MRI can be particularly helpful when the lesions are subtle. PSMA PET/CT is superior to choline PET/CT and other conventional imaging modalities. Molecular imaging with PSMA PET continues to pave the way for personalized medicine in PCa.However, large prospective clinical studies are still needed to fully evaluate the role of PSMA PET/CT and PET/MRI in the clinical workflow of PCa.

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

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Gunter Rappl

Full Text Available Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+ CD57(+ CD7(- phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

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Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

2008-09-01

A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

Antigenic determinants of prostate-specific antigen (PSA) and development of assays specific for different forms of PSA.

OpenAIRE

Nilsson, O.; Peter, A.; Andersson, I.; Nilsson, K.; Grundstr?m, B.; Karlsson, B.

1997-01-01

Monoclonal antibodies were raised against prostate-specific antigen (PSA) by immunization with purified free PSA, i.e. not in complex with any protease inhibitor (F-PSA) and PSA in complex with alpha1-anti-chymotrypsin (PSA-ACT). Epitope mapping of PSA using the established monoclonal antibody revealed a complex pattern of independent and partly overlapping antigenic domains in the PSA molecule. Four independent antigenic domains and at least three partly overlapping domains were exposed both...

Impact of obesity on the predictive accuracy of prostate-specific antigen density and prostate-specific antigen in native Korean men undergoing prostate biopsy.

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Kim, Jae Heon; Doo, Seung Whan; Yang, Won Jae; Lee, Kwang Woo; Lee, Chang Ho; Song, Yun Seob; Jeon, Yoon Su; Kim, Min Eui; Kwon, Soon-Sun

2014-10-01

To evaluate the impact of obesity on the biopsy detection of prostate cancer. We retrospectively reviewed data of 1182 consecutive Korean patients (≥50 years) with serum prostate-specific antigen levels of 3-10 ng/mL who underwent initial extended 12-cores biopsy from September 2009 to March 2013. Patients who took medications that were likely to influence the prostate-specific antigen level were excluded. Receiver operating characteristic curves were plotted for prostate-specific antigen and prostate-specific antigen density predicting cancer status among non-obese and obese men. A total of 1062 patients (mean age 67.1 years) were enrolled in the analysis. A total of 230 men (21.7%) had a positive biopsy. In the overall study sample, the area under the receiver operator characteristic curve of serum prostate-specific antigen for predicting prostate cancer on biopsy were 0.584 and 0.633 for non-obese and obese men, respectively (P = 0.234). However, the area under the curve for prostate-specific antigen density in predicting cancer status showed a significant difference (non-obese 0.696, obese 0.784; P = 0.017). There seems to be a significant difference in the ability of prostate-specific antigen density to predict biopsy results between non-obese and obese men. Obesity positively influenced the overall ability of prostate-specific antigen density to predict prostate cancer. © 2014 The Japanese Urological Association.

Cell surface carbohydrate changes during embryonic and fetal skin development

DEFF Research Database (Denmark)

Dabelsteen, Erik; Holbrook, K; Clausen, H

1986-01-01

Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N-acetyllac......Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N...

Analysis of GAGE, NY-ESO-1 and SP17 cancer/testis antigen expression in early stage non-small cell lung carcinoma

International Nuclear Information System (INIS)

Gjerstorff, Morten F; Pøhl, Mette; Olsen, Karen E; Ditzel, Henrik J

2013-01-01

The unique expression pattern and immunogenic properties of cancer/testis antigens make them ideal targets for immunotherapy of cancer. The MAGE-A3 cancer/testis antigen is frequently expressed in non-small cell lung cancer (NSCLC) and vaccination with MAGE-A3 in patients with MAGE-A3-positive NSCLC has shown promising results. However, little is known about the expression of other cancer/testis antigens in NSCLC. In the present study the expression of cancer/testis antigens GAGE, NY-ESO-1 and SP17 was investigated in patients with completely resected, early stage, primary NSCLC. Tumor biopsies from normal lung tissue and from a large cohort (n = 169) of NSCLC patients were examined for GAGE, NY-ESO-1 and SP17 protein expression by immunohistochemical analysis. The expression of these antigens was further matched to clinical and pathological features using univariate cox regression analysis. GAGE and NY-ESO-1 cancer/testis antigens were not expressed in normal lung tissue, while SP17 was expressed in ciliated lung epithelia. The frequency of GAGE, NY-ESO-1 and SP17 expression in NSCLC tumors were 26.0% (44/169), 11.8% (20/169) and 4.7% (8/169), respectively, and 33.1% (56/169) of the tumors expressed at least one of these antigens. In general, the expression of GAGE, NY-ESO-1 and SP17 was not significantly associated with a specific histotype (adenocarcinoma vs. squamous cell carcinoma), but high-level GAGE expression (>50%) was more frequent in squamous cell carcinoma (p = 0.02). Furthermore, the frequency of GAGE expression was demonstrated to be significantly higher in stage II-IIIa than stage I NSCLC (17.0% vs. 35.8%; p = 0.02). Analysis of the relation between tumor expression of GAGE and NY-ESO-1 and survival endpoints revealed no significant associations. Our study demonstrates that GAGE, NY-ESO-1 and SP17 cancer/testis antigens are candidate targets for immunotherapy of NSCLC and further suggest that multi-antigen vaccines may be beneficial

Prostate-Specific Antigen (PSA) Test: MedlinePlus Lab Test Information

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... medlineplus.gov/labtests/prostatespecificantigenpsatest.html Prostate-Specific Antigen (PSA) Test To use the sharing features on this ... enable JavaScript. What is a prostate-specific antigen (PSA) test? A prostate-specific antigen (PSA) test measures ...

Isolation of chicken embryonic stem cell and preparation of chicken chimeric model.

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Zhang, Yani; Yang, Haiyan; Zhang, Zhentao; Shi, Qingqing; Wang, Dan; Zheng, Mengmeng; Li, Bichun; Song, Jiuzhou

2013-03-01

Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.

Function of JARID2 in bovines during early embryonic development

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Yao Fu

2017-12-01

Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

Anterior Visceral Endoderm SMAD4 Signaling Specifies Anterior Embryonic Patterning and Head Induction in Mice

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Li, Cuiling; Li, Yi-Ping; Fu, Xin-Yuan; Deng, Chu-Xia

2010-01-01

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4Co/Co;TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4Co/Co;TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction. PMID:20941375

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

DEFF Research Database (Denmark)

Vejlsted, Morten; Offenberg, Hanne Kjær; Thorup, Flemming

2006-01-01

was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers......In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around...... gastrulation, days 8-17 postinsemination, introducing a steromicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency...

Comparison of serum prostate specific antigen levels and bone scintigraphy in patients with prostate carcinoma

International Nuclear Information System (INIS)

Bielickaite, J.; Zadeikaite, R.; Jurkiene, N. and others

2003-01-01

The aim of this study was to analyze the levels of serum prostate specific antigen in patients with and without bone metastases detected by means of bone scintigraphy and to determine the highest prostate specific antigen level in patients without bone metastases. The 50 patients consecutively diagnosed of prostate cancer between 1999 and 2001 in our institution made up the study population. Prostate specific antigen plasmatic levels were determined and bone scintigraphy was performed (whole body study after 99mTc-methyl-diphosphonate administration) in all the patients. In patients with positive bone scans (n=23), the mean prostate specific antigen level was 71.4±35.2 ng/ml and was significantly (p<0.00005) higher than in 14 patients with negative bone scans (mean prostate specific antigen level was 10.1±10.5 ng/ml). Suspicious lesions were found in 13 patients and their mean prostate specific antigen level was 8.5±7.7 ng/ml. Regarding prostate specific antigen levels, no statistically significant differences were found between patients with suspicious lessons and normal bone scans. The highest determined prostate specific antigen level in patients without bone metastases was 18 ng/ml. The bone scintigraphy should be performed in all patients with prostate specific antigen level above 18 ng/ml, but it is of limited value in patients with prostate specific antigen level below 18 ng/ml. (author)

Developing antigen-specific therapies in multiple sclerosis: a tale of Tantalus or Ulysses?

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van Noort JM

1999-10-01

Autoreactive T-cell responses directed to myelin proteins in the central nervous system are widely believed to be crucial in the pathology of multiple sclerosis (MS). However, effective ways of selectively targeting these T-cells in order to alter the clinical course of MS in a predictable manner has yet to be demonstrated. This review discusses two recent developments of crucial importance to the rational development of antigen-specific therapy in MS. The very idea of antigen-specific therapy in MS has long faced the challenge of determinant spreading, i.e., the development of novel autoimmune responses as the consequence of tissue damage. This phenomenon has led many to expect that in ongoing MS, many different pathogenic specificities would accumulate. Obviously, this would render antigen-specific therapy very difficult. Recent data now suggest that determinant spreading is most likely to be a transient phenomenon limited only to the first stages of tissue damage. A second development has changed our perspective on the specificity of individual T-cells and, thus, on the suitability of various ways to implement antigen-specific therapy. Evidence is rapidly accumulating that T-cell receptors are much more cross-reactive than previously assumed. This notion poses unexpected challenges to therapeutic approaches in MS that are based on selective targeting of autoreactive TCR. Vaccination with TCR peptides, administration of anti-TCR antibodies and development of therapeutically altered peptide ligands all depend on a significant level of predictability of pathogenic TCR. With such predictability now turning out to be much lower than was previously hoped, selective TCR-directed strategies for intervention may therefore turn out to be much less effective than anticipated. In the development of antigen-specific therapies, the use of whole protein tolerogens now seems to be the most promising route. Oral, intranasal or iv. administration of antigen remain viable options

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

Science.gov (United States)

Moguche, Albanus O; Musvosvi, Munyaradzi; Penn-Nicholson, Adam; Plumlee, Courtney R; Mearns, Helen; Geldenhuys, Hennie; Smit, Erica; Abrahams, Deborah; Rozot, Virginie; Dintwe, One; Hoff, Søren T; Kromann, Ingrid; Ruhwald, Morten; Bang, Peter; Larson, Ryan P; Shafiani, Shahin; Ma, Shuyi; Sherman, David R; Sette, Alessandro; Lindestam Arlehamn, Cecilia S; McKinney, Denise M; Maecker, Holden; Hanekom, Willem A; Hatherill, Mark; Andersen, Peter; Scriba, Thomas J; Urdahl, Kevin B

2017-06-14

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Antigen-specific T8+ human clone of cells with a nonspecific augmenting function on the T4 cell-B cell helper interaction

International Nuclear Information System (INIS)

Brines, R.D.; Sia, D.Y.; Lehner, T.

1987-01-01

The authors isolated a T8 + T3 + Ia + clone of cells from the peripheral blood mononuclear cells of a healthy subject. The clone was expanded and maintained with autologous feed cells, interleukin 2, and a streptococcal antigen. The T8 + clone of cells responded specifically to the streptococcal antigen, in the absence of accessory cells,and released a soluble factor. Both the cloned cells and the corresponding soluble factor expressed augmenting helper but not suppressor activity. The augmenting helper activity for B cell antibody synthesis was demonstrable only in the presence of autologous T 4 cells. Radioimmunoassay was used to measure antibodies. Although stimulation of the T8 + cloned cells was antigen-specific, the resulting soluble factor elicited nonspecific antibody synthesis in the presence of T4 and B cells. The T8 + cloned cell-derived factor was adsorbed by B cells but not by T4 cells. Preliminary studies suggest that the factor has the properties of a B cell growth factor. They suggest that the T8 + population consists of functionally heterogeneous cell subsets, some that have suppressor function and others that augment the T4 + helper-inducer activity in B cell antibody synthesis

Do embryonic polar bodies commit suicide?

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Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

2014-02-01

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

Immunohistochemical expression of embryonal marker TRA-1-60 in carcinoma in situ and germ cell tumors of the testis

DEFF Research Database (Denmark)

Giwercman, Alexander; Andrews, P W; Jørgensen, N

1993-01-01

Testicular cancer is preceded by the noninvasive stage of carcinoma in situ (CIS). According to a recent hypothesis, testicular CIA cells are germ cells transformed in fetal life. The idea of an embryonal origin of testicular germ cell neoplasia would be strengthened by the finding of antigenic...

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Science.gov (United States)

Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

2013-01-01

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

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Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

2011-04-01

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Immunogenetic mechanisms driving norovirus GII.4 antigenic variation.

Directory of Open Access Journals (Sweden)

Lisa C Lindesmith

Full Text Available Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs. Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987-1997, contemporary (2004-2009, and broad (1987-2009. NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294-298 and 368-372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393-395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing

Research on the comparative value of radio-immunoanalyses of three embryonic antigens: alpha 1 foeto-protein ''α1FP'' alpha 2H-isoferritine ''α2HF'' and carcino-embryonnic antigen ''CEA'' for diagnonis and post-therapy observation

International Nuclear Information System (INIS)

Rimbaut, C.; Rudant, C.; Buffe, D.

Alpha 1 foeto-protein, specific for 2 kinds of tumour (hepatoblastemo, teratoma), is useful in radio-immunoanalysis for the very early diagnosis of a tumour revival. Alpha 2 HF isoferritine and carcino-embryonic antigen, proteins non-specific to cancer, nevertheless possess in common the property of increasing in cases of tumours with liver metastases, though one is more specific to widespread secreting tumours while the other seems to be connected rather with an early reactivity of a tumour whatever its nature or origin. Finally a wide variation in these protein fractions is observed from one individual to another and it is therefore extremely important to make comparative determinations within a given series, the tendency of the evolution curve to rise or not being the important element in post-treatment supervision [fr

A novel classifier based on three preoperative tumor markers predicting the cancer-specific survival of gastric cancer (CEA, CA19-9 and CA72-4).

Science.gov (United States)

Guo, Jing; Chen, Shangxiang; Li, Shun; Sun, Xiaowei; Li, Wei; Zhou, Zhiwei; Chen, Yingbo; Xu, Dazhi

2018-01-12

Several studies have highlighted the prognostic value of the individual and the various combinations of the tumor markers for gastric cancer (GC). Our study was designed to assess establish a new novel model incorporating carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 72-4 (CA72-4). A total of 1,566 GC patients (Primary cohort) between Jan 2000 and July 2013 were analyzed. The Primary cohort was randomly divided into Training set (n=783) and Validation set (n=783). A three-tumor marker classifier was developed in the Training set and validated in the Validation set by multivariate regression and risk-score analysis. We have identified a three-tumor marker classifier (including CEA, CA19-9 and CA72-4) for the cancer specific survival (CSS) of GC (ptumor marker classifier is closely associated with the CSS of GC and may serve as a novel model for future decisions concerning treatments.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

Science.gov (United States)

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

Directory of Open Access Journals (Sweden)

Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Prognostic value of determination of carcinoembryonic antigen and α-fetoprotein level in blood plasma in patients with cancer stomach

International Nuclear Information System (INIS)

Smyslova, V.N.; Vygonnyj, I.I.

1986-01-01

60 donors and 129 patients with cancer stomach were examined. Tumor antigens were determined in blood plasma by the method of radioimmunoassay. The upper boundary of the norm of alpha-fetoprotein (AFP) and carcino-embryonic antigen (CEA) is 12 ng/ml. Increased concentration of antigens studied is detected in most patients. It is established that the level of antigens increases depending on generalization of the process, cancer stage, tumor propagation in the stomach wall, patient's age. High volumes of AFP and CEA after operation give evidence about non-radicality of operation and bad prognosis

Diagnostic Accuracy of 64Copper Prostate-specific Membrane Antigen Positron Emission Tomography/Computed Tomography for Primary Lymph Node Staging of Intermediate- to High-risk Prostate Cancer: Our Preliminary Experience.

Science.gov (United States)

Cantiello, Francesco; Gangemi, Vincenzo; Cascini, Giuseppe Lucio; Calabria, Ferdinando; Moschini, Marco; Ferro, Matteo; Musi, Gennaro; Butticè, Salvatore; Salonia, Andrea; Briganti, Alberto; Damiano, Rocco

2017-08-01

To assess the diagnostic accuracy of 64 Copper prostate-specific membrane antigen ( 64 Cu-PSMA) positron emission tomography/computed tomography (PET/CT) in the primary lymph node (LN) staging of a selected cohort of intermediate- to high-risk prostate cancer (PCa) patients. An observational prospective study was performed in 23 patients with intermediate- to high-risk PCa, who underwent 64 Cu-PSMA PET/CT for local and lymph nodal staging before laparoscopic radical prostatectomy with an extended pelvic LN dissection. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for LN status of 64 Cu-PSMA PET/CT were calculated using the final pathological findings as reference. Furthermore, we evaluated the correlation of intraprostatic tumor extent and grading with 64 Cu-PSMA intraprostatic distribution. Pathological analysis of LN involvement in 413 LNs harvested from our study cohort identified a total of 22 LN metastases in 8 (5%) of the 23 (35%) PCa patients. Imaging-based LN staging in a per-patient analysis showed that 64 Cu-PSMA PET/CT was positive in 7 of 8 LN-positive patients (22%) with a sensitivity of 87.5%, specificity of 100%, PPV of 100%, and NPV of 93.7%, considering the maximum standardized uptake value (SUV max ) at 4 hours as our reference. Receiver operating characteristic curve was characterized by an area under the curve of 0.938. A significant positive association was observed between SUV max at 4 hours with Gleason score, index, and cumulative tumor volume. In our intermediate- to high-risk PCa patients study cohort, we showed the high diagnostic accuracy of 64 Cu-PSMA PET/CT for primary LN staging before radical prostatectomy. Copyright © 2017 Elsevier Inc. All rights reserved.

Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation.

Science.gov (United States)

Liang, Yuh-Jin; Kuo, Huan-Hsien; Lin, Chi-Hung; Chen, Yen-Ying; Yang, Bei-Chia; Cheng, Yuan-Yuan; Yu, Alice L; Khoo, Kay-Hooi; Yu, John

2010-12-28

A systematic survey of expression profiles of glycosphingolipids (GSLs) in two hESC lines and their differentiated embryoid body (EB) outgrowth with three germ layers was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In addition to the well-known hESC-specific markers stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb(4)Cer, Lc(4)Cer, fucosyl Lc(4)Cer, Globo H, and disialyl Gb(5)Cer. During hESC differentiation into EBs, MS analysis revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series, which was not as evident by immunostaining with antibodies against SSEA-3 and SSEA-4, owing to their cross-reactivities with various glycosphingolipids. Such a switch was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways by the up-regulation of ganglio-series-related GTs with simultaneous down-regulation of globo- and lacto-series-related GTs. Thus, these results provide insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation. In addition, unique glycan structures uncovered by MS analyses may serve as surface markers for further delineation of hESCs and help identify of their functional roles not only in hESCs but also in cancers.

Prostatic specific antigen. From its early days until becoming a prostate cancer biomarker.

Science.gov (United States)

Dellavedova, T

2016-01-01

Prostate-specific antigen (PSA) has been since the mid 80's the most commonly used biomarker for measuring current and future risk of prostate cancer, for its early detection and to measure response to treatments and detecting recurrence in all stages of the disease. PSA's early development came along with progress in the field of immunology, which allowed detection and study of antigens from different tissues and fluids when injecting them into rabbits to promote immune response. Rubin Flocks in 1960 was the first to investigate and discover prostate-specific antigens in benign and malignant tissue. Some years later, Hara, a Japanese forensic investigator, found 'gamma seminoprotein', that he used to detect human semen in rape cases. However, his work published in Japanese did not reach the Englishspeaking scientific community. In 1970 Ablin discovered both in prostatic fluid and tissue what he called "prostate-specific antigen", but he didn't characterize or describe it. Investigators Li and Beling, and Sensabaugh, approached the current PSA, but they were limited by available technology at that time. Dr T Ming Chu led a research team on prostate cancer in New York, USA and published their results in 1979. He finally received the patent for the discovery of "human purified prostate antigen" in 1984. Due to this work, the Food and Drug Administration (FDA), in USA, approved the use of PSA for monitoring recurrence after treatment. It was later known that PSA was not prostate-specific since it was produced in other tissues and fluids, but it was recognized that it was human species-specific. Works by Papsidero and Stamey showed new indications and utilities for PSA, but it was Catalona who first used it as a marker for prostate cancer in 1991. Thanks to these advances FDA authorized in 1994 the clinical use of PSA for early detection of prostate cancer.

Intensified follow-up in colorectal cancer patients using frequent Carcino-Embryonic Antigen (CEA) measurements and CEA-triggered imaging : Results of the randomized "CEAwatch" trial

NARCIS (Netherlands)

Verberne, C. J.; Zhan, Z.; van den Heuvel, E.; Grossmann, I.; Doornbos, P. M.; Havenga, K.; Manusama, E.; Klaase, J.; van der Mijle, H. C. J.; Lamme, B.; Bosscha, K.; Baas, P.; van Ooijen, B.; Nieuwenhuijzen, G.; Marinelli, A.; van der Zaag, E.; Wasowicz, D.; de Bock, G. H.; Wiggers, T.

Aim: The value of frequent Carcino-Embryonic Antigen (CEA) measurements and CEA-triggered imaging for detecting recurrent disease in colorectal cancer (CRC) patients was investigated in search for an evidence-based follow-up protocol. Methods: This is a randomized-controlled multicenter prospective

Prostate-Specific Antigen (PSA) Test

Science.gov (United States)

... Cancer Prostate Cancer Screening Research Prostate-Specific Antigen (PSA) Test On This Page What is the PSA ... parts of the body before being detected. The PSA test may give false-positive or false-negative ...

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Science.gov (United States)

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

OpenAIRE

Wynne, E; Slocombe, J O; Wilkie, B N

1981-01-01

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected als...

V-set and Ig domain-containing 4 (VSIG4)-expressing hepatic F4/80+ cells regulate oral antigen-specific responses in mouse.

Science.gov (United States)

Shin, Wonhwa; Jeon, Youkyoung; Choi, Inhak; Kim, Yeon-Jeong

2018-04-01

Oral tolerance can prevent unnecessary immune responses against dietary antigens. Members of the B7 protein family play critical roles in the positive and/or negative regulation of T cell responses to interactions between APCs and T cells. V-set and Ig domain-containing 4 (VSIG4), a B7-related co-signaling molecule, has been known to act as a co-inhibitory ligand and may be critical in establishing immune tolerance. Therefore, we investigated the regulation of VSIG4 signaling in a food allergy and experimental oral tolerance murine models. We analyzed the contributions of the two main sites involved in oral tolerance, the mesenteric lymph node (MLN) and the liver, in VSIG4-mediated oral tolerance induction. Through the comparative analysis of major APCs, dendritic cells (DCs) and macrophages, we found that Kupffer cells play a critical role in inducing regulatory T cells (Tregs) and establishing immune tolerance against oral antigens via VSIG4 signaling. Taken together, these results suggest the possibility of VSIG4 signaling-based regulation of orally administered antigens. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensitivity and specificity of three Onchocerca volvulus cloned antigens in diagnosis of onchocerciasis

International Nuclear Information System (INIS)

Atti, Miska Elyemen; TR, Unnasch; Mukhtar, M.M.

1999-01-01

Onchcerciasis is endemic in three geographical regions in Sudan, and presented with variable clinical reactions. The current diagnosis of onchocerciasis is based on detection of live microflaraie in skin snips in addition to clinical signs and history of living in endemic regions. Different serological diagnostic trials using crude soluble antigens of O. volvulus have shown variable degrees of cross reaction with other nematodes co-endemic in the same area. We have studied the sensitivity and specificity of RAL-2, calreticulin and PDI O. volvulus cloned antigens using the ELISA techniques. Eighty serum samples of Onchocerciasis patients, 20 non endemic normal controls and 42 samples of patients of other endemic deseas including Leishmanoasis, malaria, tuberculosis and shestosomiasis were tested RAL-2 gave the best Ig G response, with 83.75% sensitivity and 91. 66% specificity. PDI sensitivity was 20% and specificity 91.66%, while calreticulum showed sensitivity of 37.5% and specificity of 73.30%. IgG3 subclasses was not significantly different from controls while IgG4 was significantly higher in patients. The sensitivity of RAL-2 for IgG4 was 90% with a specificity of 100%. For PDI sensitivity was 25% and 100% specificity while calreticulin resulted in sensitivity of 75% and specificity of 100%. Low levels of circulating IgE to RAL-2 antigen were detected. For all antigens there was significant correlation with gender, age, microflarial load or presence of nodules. (Author)

The performance characteristics of prostate-specific antigen and prostate-specific antigen density in Chinese men.

Science.gov (United States)

Teoh, Jeremy Yc; Yuen, Steffi Kk; Tsu, James Hl; Wong, Charles Kw; Ho, Brian Sh; Ng, Ada Tl; Ma, Wai-Kit; Ho, Kwan-Lun; Yiu, Ming-Kwong

2017-01-01

We investigated the performance characteristics of prostate-specific antigen (PSA) and PSA density (PSAD) in Chinese men. All Chinese men who underwent transrectal ultrasound-guided prostate biopsy (TRUS-PB) from year 2000 to 2013 were included. The receiver operating characteristic (ROC) curves for both PSA and PSAD were analyzed. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) at different cut-off levels were calculated. A total of 2606 Chinese men were included. For the ROC, the area under curve was 0.770 for PSA (P specificity of 14.1%, PPV of 29.5%, and NPV of 86.9%; PSAD of 0.12 ng ml-1 cc-1 had sensitivity of 94.5%, specificity of 26.6%, PPV of 32.8%, and NPV of 92.7%. On multivariate logistic regression analyses, PSA cut-off at 4.5 ng ml-1 (OR 1.61, 95% CI 1.05-2.45, P= 0.029) and PSAD cut-off at 0.12 ng ml-1 cc-1 (OR 6.22, 95% CI 4.20-9.22, Pprostate cancer detection on TRUS-PB. In conclusion, the performances of PSA and PSAD at different cut-off levels in Chinese men were very different from those in Caucasians. PSA of 4.5 ng ml-1 and PSAD of 0.12 ng ml-1 cc-1 had near 95% sensitivity and were significant predictors of prostate cancer detection in Chinese men.

Predictive value of prostate-specific antigen for prostate cancer

DEFF Research Database (Denmark)

Shepherd, Leah; Borges, Alvaro Humberto; Ravn, Lene

2014-01-01

INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics and predict......INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics...... and predictive value of PSA in HIV+ men. METHODS: Men with PCa (n=21) and up to two matched controls (n=40) with prospectively stored plasma samples before PCa (or matched date in controls) were selected. Cases and controls were matched on date of first and last sample, age, region of residence and CD4 count...... at first sample date. Total PSA (tPSA), free PSA (fPSA), testosterone and sex hormone binding globulin (SHBG) were measured. Conditional logistic regression models investigated associations between markers and PCa. Sensitivity and specificity of using tPSA >4 µg/L to predict PCa was calculated. Mixed...

Development of a new in vivo kit for detection of prostate specific antigen in human serum using immunoradiometric assay method

International Nuclear Information System (INIS)

Babaei, M. H.; Behradkia, P.; Shafii, M.; Movla, M.; Forutan, H.; Najafi, R.

2006-01-01

Prostate is a leading site for the cancer incidence, accounted for 31.0% of new cancer cases in men. Prostate-specific antigen is widely used in the detection and monitoring of the prostate cancer. Currently, immunoassay is used to detect Prostate-specific antigen in human serum. This technique is based on the interaction between antibody and antigen. The varied immunoassay formats and equipment to run the assays allow the users to measure the analytes rapidly, with the flexibility to run a small or a large number of samples. Among different immunoassay methods, immunoradiometric assay is a more sensitive and valuable detection approach. This study has been made in 4 parts: (1) purification of Prostate-specific antigen from seminal fluid; (2) preparation of hybridoma cells which secrete monoclonal antibody (mAb) against Prostate-specific antigen , (3) selection of pair monoclonal antibody among those antibodies, and finally (4) design of an immunoradiometric assay kit and it's quality control . The results of this study were: (1) obtaining a huge amount of Prostate-specific antigen as semi-purified and purified, that is a valuable material for preparation of standard kits; (2) preparation of 8 kinds of monoclonal antibodies; (3) finding 4 pairs of monoclonal antibodies which react with different epitopes on Prostate-specific antigen molecule; and (4) preparation of immunoradiometric assay kit for measuring Prostate-specific antigen concentration in human serum

Peculiarities of Embryonic and Post-Embryonic Development of Оesophagostomum dentatum (Nematoda, Strongylidae Larvae Cultured in Vitro

Directory of Open Access Journals (Sweden)

Yevstafieva V. А.

2017-02-01

Full Text Available Morphometric peculiarities of the development of Оesophagostomum dentatum Rudolphi, 1803 from egg to infective larva were studied under laboratory conditions at various temperatures. The determined optimum temperature for embryonic and post-embryonic development of О. dentatum larvae from domestic pig (Sus scrofa domesticus Linnaeus, 1758 is 22 °С. At this temperature, 81 % of larvae develop to the third stage (L3 on the 10th day. Temperatures of 24 °С and 20 °С are less favorable for the development of the nematode, at those temperatures only 67 and 63 % of larvae, respectively, reached infective stage by the 10th day of cultivation. Embryonic development of О. dentatum eggs is characterized by their lengthening (by 8.87-9.50 %, р < 0.01 and widening (by 6.77-9.35 %, р < 0.05-0.01, and post-embryonic larval development is associated with lengthening (by 4.59-17.33 %, р < 0.01-0.001.

Complexity of type-specific 56 kDa antigen CD4 T-cell epitopes of Orientia tsutsugamushi strains causing scrub typhus in India.

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Arunachalam Ramaiah

Full Text Available Orientia tsutsugamushi (Ots is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III and three spacer conserved domains (SVDI, SVDII/III, SVDIII. The total number (152 of antigenic types (amino acid variants varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435 of the predicted CD4 T-cell epitopes (TCEs from all the six domains had high binding affinities (HBA to at least one of the prevalent Indian human leukocyte antigen (HLA alleles. A surprisingly high proportion (61% of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.

Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

International Nuclear Information System (INIS)

Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

1987-01-01

To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

Directory of Open Access Journals (Sweden)

Pilar T. V. Florentino

2018-04-01

Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels, it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

Radioimmunoassay for a human prostate specific antigen

International Nuclear Information System (INIS)

Machida, T.; Miki, M.; Ohishi, Y.; Kido, A.; Morikawa, J.; Ogawa, Y.

1983-01-01

As a marker for prostatic cancer, a prostate-specific antigen was purified from human prostatic tissues. Double antibody radioimmunoassay utilizing immune reaction was developed on the basis of the purified prostatic antigen (PA). Measurement results have revealed that PA radioimmunoassay is much better than prostatic acid phosphatase (PAP) radioimmunoassay in the diagnosis of prostatic cancer

File list: ALL.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: ALL.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Emb.20.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_palates.bed ...

File list: ALL.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Emb.50.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_palates.bed ...

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

Science.gov (United States)

Reichman, David E; Park, Laura; Man, Limor; Redmond, David; Chao, Kenny; Harvey, Richard P; Taketo, Makoto M; Rosenwaks, Zev; James, Daylon

2018-01-08

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs. © 2018. Published by The Company of Biologists Ltd.

Prostate-specific antigen superior serum marker for prostatic carcinoma

Energy Technology Data Exchange (ETDEWEB)

Heaney, J A; Allen, M A; Keane, T; Duffy, J J

1987-05-01

A new immunoradiometric assay based on dual monoclonal antibody reaction system (Hybritech-TANDEM/sup R/) was used to measure serum levels of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) in 39 patients with prostatic carcinoma (CaP), in 57 with benign prostatic hyperplasia (BPH) and in 14 without prostatic disease. Serum PSA was elevated in 82% of patients with CaP while PAP was elevated in only 54%. In this and other studies, PSA is superior to conventional serum markers in sensitivity, prediction of CaP stage and in longitudinal monitoring of disease. A 16% false positive rate precludes PSA as a screening test. The assay used was found to be simple and reliable.

Prostate-specific antigen superior serum marker for prostatic carcinoma

International Nuclear Information System (INIS)

Heaney, J.A.; Allen, M.A.; Keane, T.; Duffy, J.J.

1987-01-01

A new immunoradiometric assay based on dual monoclonal antibody reaction system (Hybritech-TANDEM R ) was used to measure serum levels of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) in 39 patients with prostatic carcinoma (CaP), in 57 with benign prostatic hyperplasia (BPH) and in 14 without prostatic disease. Serum PSA was elevated in 82% of patients with CaP while PAP was elevated in only 54%. In this and other studies, PSA is superior to conventional serum markers in sensitivity, prediction of CaP stage and in longitudinal monitoring of disease. A 16% false positive rate precludes PSA as a screening test. The assay used was found to be simple and reliable. (author)

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Science.gov (United States)

Reyes, L; Hartikka, J; Bozoukova, V; Sukhu, L; Nishioka, W; Singh, G; Ferrari, M; Enas, J; Wheeler, C J; Manthorpe, M; Wloch, M K

2001-06-14

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos.

Directory of Open Access Journals (Sweden)

Rong-Ping Zhang

Full Text Available Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM and the pectoral muscle (PM of two genetically different duck breeds, Heiwu duck (H and Peking duck (P, at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P at the same developmental stage (embryonic 15 days. Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG were used to functionally annotate DEGs (differentially expression genes. The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05. In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05. In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only

Chemiluminescence immunoassay for prostate-specific antigen

International Nuclear Information System (INIS)

Zhang Xuefeng; Liu Yibing; Jia Juanjuan; Xu Wenge; Li Ziying; Chen Yongli; Han Shiquan

2008-01-01

The chemiluminescence immunoassay (CLIA) for serum total prostate-specific antigen (T-PSA) was developed. The reaction of luminol with hydrogen peroxide was introduced into this chemiluminescence system. The detection limit is established as 0.12 μg/L (n=10, mean of zero standard + 2SD) and the analytical recovery of PSA is 83.8%-118.7%. The intra-assay and inter-assay CVs vary from 4.4%-5.0% and 6.2%-11.7%, respectively. The experimental correlation coefficient of dilution is found to be 0.999. Compared with immunoradiometric assay (IRMA) kits, the correlative equation is y=1.07x+0.68, and correlation coefficient r=0.97. The standard range for the method is 1.5-80 μg/L, and it presents good linearity. (authors)

Carcino-embryonic antigen in monitoring the growth of human colon adenocarcinoma tumour cells SK-CO-1 and HT-29 in vitro and in nude mice

DEFF Research Database (Denmark)

Sölétormos, G; Fogh, J M; Sehested-Hansen, B

1997-01-01

A set of experimental model systems were designed to investigate (a) the inter-relationship between growth of two human cancer cell lines (SK-CO-1, HT-29) and carcino-embryonic antigen (CEA) kinetics; and (b) whether neoplastic growth or CEA concentration is modulated by human growth hormone (hGH...

Effect of Heamolysis on Prostate-Specific Antigen

OpenAIRE

Sağlam, Hasan S.; Köse, Osman; Özdemir, Fatma; Adsan, Öztuğ

2012-01-01

Purpose. We have investigated the effect of haemolysis on free and total prostate-specific antigen (PSA) in daily clinical practice. Materials and Methods. Thirty-nine consecutive men were enrolled in this study. With an 18 gauge (G) needle 4 cc of blood samples were drawn from the right arm and 2 cc of it was expelled gently in a Vacutainer for regular PSA assay and the remaining was emptied into a second tube for complete haemolysis. Simultaneously 2 cc of more blood were taken with a 26 G ...

Salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy for prostate cancer. A single-center experience

International Nuclear Information System (INIS)

Yoshida, Takahiro; Nakayama, Masashi; Suzuki, Osamu

2011-01-01

The aim of this study was to investigate the efficacy and prognostic factors of salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy for prostate cancer at a single center in Japan. A retrospective review of the medical records of 51 patients who underwent salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy was carried out. Salvage radiotherapy was undergone for the single indication of at least two consecutive prostate-specific antigen elevations >0.1 ng/ml. Salvage radiotherapy was delivered to the prostatic bed at a total dose of 60 or 64 Gy. Late toxicity was scored according to the Common Terminology Criteria for Adverse Events 3.0. A total dose of 60 and 64 Gy were administered to 26 and 25 patients, respectively. The median prostate-specific antigen level at the initiation of radiotherapy was 0.29 ng/ml (range, 0.11-1.10 ng/ml). With a median follow-up of 57.3 months (range, 9.9-134.0 months), the prostate-specific antigen relapse-free rate at 5 years was 50.7%. Multivariate analysis using Cox's proportional hazards regression model revealed that the Gleason score at radical prostatectomy ≥8 significantly predicted prostate-specific antigen relapse after salvage radiotherapy (hazard ratio 4.531; 95% confidence interval 1.413-14.535; P=0.011). The prostate-specific antigen relapse-free rate at 5 years in the Gleason score at radical prostatectomy ≤7 and at radical prostatectomy ≥8 was 62.7 and 15.4%, respectively. Salvage radiotherapy was effective for prostate-specific antigen relapse after radical prostatectomy with tolerable toxicities in Japanese patients. A high Gleason score seemed to be a poor prognostic factor. (author)

A radioimmunoassay for human antibody specific for microbial antigens

International Nuclear Information System (INIS)

Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

1977-01-01

A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

Effectiveness of the combined evaluation of KLK3 genetics and free-to-total prostate specific antigen ratio for prostate cancer diagnosis.

Science.gov (United States)

Zambon, Carlo-Federico; Prayer-Galetti, Tommaso; Basso, Daniela; Padoan, Andrea; Rossi, Elisa; Secco, Silvia; Pelloso, Michela; Fogar, Paola; Navaglia, Filippo; Moz, Stefania; Zattoni, Filiberto; Plebani, Mario

2012-10-01

Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate

In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

Directory of Open Access Journals (Sweden)

Kaori Yamauchi

Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

Immunization against Capillaria hepatica: The effects of primary infections, X-irradiated stages, non-embryonated eggs and soluble egg extracts

Energy Technology Data Exchange (ETDEWEB)

Zahner, H.; Schmidt, H.; Laemmler, G.; Geyer, E.

1980-01-01

The worm reproductivity was significantly suppressed in a sublethal challenge infection given 11 days after a primary infection of Mastomys natalensis with 50, 150, 400, and 800 eggs per animal. The administration of 600 X-irradiated (2.2 Krd) embryonated eggs 36 days before challenge as well as an intraperitoneal injection of non-embryonated eggs 12, 10, 8, 6, 4, and 2 days before challenge also reduced significantly the egg production of a weak (50 eggs/animal) infection. No effect was observed on a moderate challenge (300 eggs/animal). The effect was not markedly enhanced by the repeated administration of X-irradiated eggs or by the combination of X-irradiated infective eggs and non-embryonated eggs. Immunization of mice with soluble egg extracts resulted in significant reduction of egg production determined 60 days after challenge. Two hundred and thirty eggs of C. hepatica/g bodyweight proved to be a lethal infection dose for M. natalensis. The animals died between 20 and 35 days after infection. After single infections with 50, 150, 400, or 800 eggs per animal the mortality of Mastomys challenged 36 or 52 days later was reduced to 0-30%. Using X-irradiated embryonated eggs for immunization only repeated administration led to protection in 70 to 80%, of the animals. About 40% of the animals could be protected by the intraperitoneal injection of non-embryonated eggs. If death occurred it was delayed. The combination of X-irradiated stages and eggs did not enhance the protection.

Radiation dose response in patients with favorable localized prostate cancer (Stage T1-T2, biopsy Gleason ≤6, and pretreatment prostate-specific antigen ≤10)

International Nuclear Information System (INIS)

Kupelian, Patrick A.; Buchsbaum, Jeffrey C.; Reddy, Chandana A.; Klein, Eric A.

2001-01-01

Purpose: To study the radiation dose response as determined by biochemical relapse-free survival in patients with favorable localized prostate cancers, i.e., Stage T1-T2, biopsy Gleason score (bGS) ≤6, and pretreatment prostate-specific antigen (iPSA) ≤10 ng/mL. Methods and Materials: A total of 292 patients with favorable localized prostate cancer were treated with radiotherapy alone between 1986 and 1999. The median age was 69 years. Sixteen percent of cases (n=46) were African-American. The distribution by clinical T stage was as follows: T1/T2A, 243 (83%); and T2B/T2C, 49 (17%). The distribution by iPSA was as follows: ≤4 ng/mL, 49 (17%); and >4 ng/mL, 243 (83%). The mean iPSA level was 6.2 (median, 6.4). The distribution by bGS was as follows: ≤5 in 89 cases (30%) and 6 in 203 cases (70%). The median radiation dose was 70.0 Gy (range, 63.0-78.0 Gy). Doses of ≤70.0 Gy were delivered in 175 cases, 70.2-72.0 Gy in 24 cases, 74 Gy in 30 cases, and 78 Gy in 63 cases. For patients receiving 2 =5.7), and radiation dose (p=0.021, χ 2 =5.3) were independent predictors of outcome. Age (p=0.94), race (p=0.89), stage (p=0.45), biopsy GS (p=0.40), and radiation technique (p=0.45) were not. Conclusion: There is a clear radiation dose response in patients with favorable localized prostate cancers (i.e., Stage T1-T2, biopsy Gleason score ≤6, and iPSA ≤10 ng/mL). At least 74 Gy should be delivered to the prostate and periprostatic tissues. With our cohort of patients, longer follow-up will be needed to assess the importance of doses exceeding 74 Gy

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation

International Nuclear Information System (INIS)

Ouyang, Liliang; Yao, Rui; Mao, Shuangshuang; Sun, Wei; Chen, Xi; Na, Jie

2015-01-01

With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies. (paper)

Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation.

Science.gov (United States)

Ouyang, Liliang; Yao, Rui; Mao, Shuangshuang; Chen, Xi; Na, Jie; Sun, Wei

2015-11-04

With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies.

Prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in active surveillance patients.

Science.gov (United States)

Iremashvili, Viacheslav; Barney, Shane L; Manoharan, Murugesan; Kava, Bruce R; Parekh, Dipen J; Punnen, Sanoj

2016-04-01

To analyze the association between prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in prostate cancer patients on active surveillance, and to study the effect of prediagnostic prostate-specific antigen values on the predictive performance of prostate-specific antigen velocity and prostate-specific antigen doubling time. The study included 137 active surveillance patients with two or more prediagnostic prostate-specific antigen levels measured over a period of at least 3 months. Two sets of analyses were carried out. First, the association between prostate-specific antigen kinetics calculated using only the prediagnostic prostate-specific antigen values and the risk of biopsy progression was studied. Second, using the same cohort of patients, the predictive value of prostate-specific antigen kinetics calculated using only post-diagnostic prostate-specific antigens and compared with that of prostate-specific antigen kinetics based on both pre- and post-diagnostic prostate-specific antigen levels was analyzed. Of 137 patients included in the analysis, 37 (27%) had biopsy progression over a median follow-up period of 3.2 years. Prediagnostic prostate-specific antigen velocity of more than 2 ng/mL/year and 3 ng/mL/year was statistically significantly associated with the risk of future biopsy progression. However, after adjustment for baseline prostate-specific antigen density, these associations were no longer significant. None of the tested prostate-specific antigen kinetics based on combined pre- and post-diagnostic prostate-specific antigen values were statistically significantly associated with the risk of biopsy progression. Historical prediagnostic prostate-specific antigens seems to be not clinically useful in patients diagnosed with low-risk prostate cancer on active surveillance. © 2016 The Japanese Urological Association.

File list: ALL.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: ALL.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: ALL.Emb.10.AllAg.Embryonic_pancreas [Chip-atlas[Archive

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Lifescience Database Archive (English)

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IgG isotypic antibodies to crude Plasmodium falciparum blood-stage ...

African Journals Online (AJOL)

Methods: Levels of IgG (IgG1-IgG4) and IgM to crude P. falciparum blood stage antigen ... dosage influenced P. falciparum-specific isotypic antibody responses to blood stage .... exposed Swedish donors. ..... with adverse pregnancy outcomes.

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Lifescience Database Archive (English)

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File list: ALL.Emb.10.AllAg.Embryonic_testis [Chip-atlas[Archive

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File list: ALL.Emb.20.AllAg.embryonic_skin [Chip-atlas[Archive

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File list: ALL.Emb.10.AllAg.embryonic_skin [Chip-atlas[Archive

Lifescience Database Archive (English)

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Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration

Directory of Open Access Journals (Sweden)

Kamil Kruczek

2017-06-01

Full Text Available The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs. Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

Time--temperature relation of embryonic development in the northwestern salamander, Ambystoma gracile

Energy Technology Data Exchange (ETDEWEB)

Brown, H A

1976-04-01

A field and laboratory study on temperature-related embryonic development of Ambystoma gracile was made on a population from northwestern Washington. Natural spawning began in the beaver pond during early March, and the duration of embryonic development (stages 1 to 46) was about 62 days. Average water temperature in the pond during embryonic development was 8.5/sup 0/C (range, 4.4 to 14.3/sup 0/C). The laboratory data of embryonic development at constant temperatures show that the limits of temperature tolerance are about 5 to 22.5/sup 0/C. Rate of development was measured by determining time required to develop from first cleavage (stage 2) to gill circulation (stage 37); representative rates are 12.7 days at 20/sup 0/C, 27 days at 12/sup 0/C, and 89 days at 7/sup 0/C. Embryos of A. gracile have the slowest rate of development when compared with embryos of four other species of Ambystoma (maculatum, mexicanum, tigrinum, and jeffersonianum) and with embryos of three Pacific Northwest frogs (Ascaphus truei, Rana aurora, and Hyla regilla).

Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

DEFF Research Database (Denmark)

Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

2016-01-01

Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...

File list: ALL.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: ALL.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: ALL.Emb.10.AllAg.Embryonic_heart [Chip-atlas[Archive

Lifescience Database Archive (English)

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Usefulness of transrectal ultrasound in diagnosing prostate cancer: comparison with digital rectal examination, prostate-specific antigen and prostate-specific antigen density

International Nuclear Information System (INIS)

Yoon, Jung Hwan; Kim, Bo Hyun; Choi, Sang Hee; Kim, Seung Hoon; Choi, Han Yong; Chai, Soo Eung; Yoon, Hye Kyung; Lee, Soon Jin; Choo, In Wook; Kim, Bo Kyung

1998-01-01

To determine the usefulness of transrectal ultrasonography (TRUS) in diagnosing prostate cancer by comparing the sensitivity, specificity, accuracy, and positive and negative predictive values of TRUS with those of serum prostate-specific antigen (PSA), prostate-specific antigen density (PSAD) and digital rectal examination (DRE). Two hundred and ten consecutive patients underwent TRUS-guided prostate biopsy due to elevated PSA and/or abnormal findings on TRUS or DRE. The TRUS findings were analyzed and correlated with pathological diagnosis. PSAD was calculated by dividing the serum PSA level by the prostate volume calculated on TRUS. The sensitivity, specificity, accuracy, and positive and negative predictive values of TRUS were compared with those of PSA, PSAD and DRE. Using ROC curve analysis, the combinations of these diagnostic methods were also evaluated for the determination of efficacy in diagnosing prostate cancer. The sensitivity and specificity of serum PSA (cut-off level, 4ng/ml), PSAD (cut-off level, 0.15ng/ml/cm 3 ), DRE, and TRUS were 96%/17%, 96%/37%, 72%/62%, and 89%/68%, respectively. On TRUS, the sensitivity and specificity of low echoic lesions and those of irregular outer margin were 89%/69%, and 60%/90%, respectively. TRUS was statistically more accurate than other diagnostic methods. Of the combinations of diagnostic methods, TRUS and PSAD were most accurate. TRUS demonstrated lower sensitivity but higher specificity than PSA or PSAD. Although it is an accurate modality for the diagnosis of prostate cancer, it cannot be used as a confirmative test due to its relatively low positive predictive value. A combination of diagnostic methods and random biopsy is needed in patients in whom prostate cancer is suspected.=20

Oncogenic cancer/testis antigens

DEFF Research Database (Denmark)

Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

2015-01-01

Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer....../testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...... immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic...

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations

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Murphy, Sean C.; Kas, Arnold; Stone, Brad C.; Bevan, Michael J.

2013-01-01

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins. PMID:23530242

Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2012-01-01

of the study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The antigens used were 4 ESAT-6 family members, 4 latency proteins, 4 secreted proteins including Ag85B, 3 other antigens and PPDj......Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection can be detected by measuring antigen specific cell mediated immune responses by the interferon gamma (IFN-γ) assay. Available IFN-γ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives...... of the infected and non-infected herds were significantly (Passay using PPDj did not correlate with the results using the novel antigens since 5 of the 17 animals that were positive to PPDj were...

Stem cell-specific expression of Dax1 is conferred by STAT3 and Oct3/4 in embryonic stem cells

International Nuclear Information System (INIS)

Sun Chuanhai; Nakatake, Yuhki; Ura, Hiroki; Akagi, Tadayuki; Niwa, Hitoshi; Koide, Hiroshi; Yokota, Takashi

2008-01-01

Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocysts. An orphan nuclear receptor, Dax1, is specifically expressed in undifferentiated ES cells and plays an important role in their self-renewal. The regulatory mechanism of Dax1 expression in ES cells, however, remains unknown. In this study, we found that STAT3 and Oct3/4, essential transcription factors for ES cell self-renewal, are involved in the regulation of Dax1 expression. Suppression of either STAT3 or Oct3/4 resulted in down-regulation of Dax1. Reporter assay identified putative binding sites for these factors in the promoter/enhancer region of the Dax1 gene. Chromatin immunoprecipitation analysis suggested the in vivo association of STAT3 and Oct3/4 with the putative sites. Furthermore, gel shift assay indicated that these transcription factors directly bind to their putative binding sites. These results suggest that STAT3 and Oct3/4 control the expression of Dax1 to maintain the self-renewal of ES cells

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

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Beauvais, Anne; Beau, Remi; Candoni, Anna; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Zanetti, Eleonora; Quadrelli, Chiara; Codeluppi, Mauro; Guaraldi, Giovanni; Pagano, Livio; Caira, Morena; Giovane, Cinzia Del; Maccaferri, Monica; Stefani, Alessandro; Morandi, Uliano; Tazzioli, Giovanni; Girardis, Massimo; Delia, Mario; Specchia, Giorgina; Longo, Giuseppe; Marasca, Roberto; Narni, Franco; Merli, Francesco; Imovilli, Annalisa; Apolone, Giovanni; Carvalho, Agostinho; Comoli, Patrizia; Romani, Luigina; Latgè, Jean Paul; Luppi, Mario

2013-01-01

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned

All-trans retinoic acid directs urothelial specification of murine embryonic stem cells via GATA4/6 signaling mechanisms.

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Joshua R Mauney

2010-07-01

Full Text Available The urinary bladder and associated tract are lined by the urothelium, a transitional epithelium that acts as a specialized permeability barrier that protects the underlying tissue from urine via expression of a highly specific group of proteins known as the uroplakins (UP. To date, our understanding of the developmental processes responsible for urothelial differentiation has been hampered due to the lack of suitable models. In this study, we describe a novel in vitro cell culture system for derivation of urothelial cells from murine embryonic stem cells (ESCs following cultivation on collagen matrices in the presence all trans retinoic acid (RA. Upon stimulation with micromolar concentrations of RA, ESCs significantly downregulated the pluripotency factor OCT-4 but markedly upregulated UP1A, UP1B, UP2, UP3A, and UP3B mRNA levels in comparison to naïve ESCs and spontaneously differentiating controls. Pan-UP protein expression was associated with both p63- and cytokeratin 20-positive cells in discrete aggregating populations of ESCs following 9 and 14 days of RA stimulation. Analysis of endodermal transcription factors such as GATA4 and GATA6 revealed significant upregulation and nuclear enrichment in RA-treated UP2-GFP+ populations. GATA4-/- and GATA6-/- transgenic ESC lines revealed substantial attenuation of RA-mediated UP expression in comparison to wild type controls. In addition, EMSA analysis revealed that RA treatment induced formation of transcriptional complexes containing GATA4/6 on both UP1B and UP2 promoter fragments containing putative GATA factor binding sites. Collectively, these data suggest that RA mediates ESC specification toward a urothelial lineage via GATA4/6-dependent processes.

Analysis of antigen-specific B-cell memory directly ex vivo.

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McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

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Patrick R. Adair

2017-09-01

Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model.

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Zineb Belcaid

Full Text Available Glioblastoma (GBM is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4 blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137 is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response.Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse

A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

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Miller, A

1977-01-01

The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

International Nuclear Information System (INIS)

Yoshikawa, Tomoaki; Imazu, Susumu; Gao Jianqing; Hayashi, Kazuyuki; Tsuda, Yasuhiro; Shimokawa, Mariko; Sugita, Toshiki; Niwa, Takako; Oda, Atushi; Akashi, Mitsuru; Tsutsumi, Yasuo; Mayumi, Tadanori; Nakagawa, Shinsaku

2004-01-01

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Science.gov (United States)

Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Discoveries and application of prostate-specific antigen, and some proposals to optimize prostate cancer screening

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Tokudome S

2016-05-01

Full Text Available Shinkan Tokudome,1 Ryosuke Ando,2 Yoshiro Koda,3 1Department of Nutritional Epidemiology, National Institute of Health and Nutrition, Shinjuku-ku, Tokyo, 2Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya, 3Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan Abstract: The discoveries and application of prostate-specific antigen (PSA have been much appreciated because PSA-based screening has saved millions of lives of prostate cancer (PCa patients. Historically speaking, Flocks et al first identified antigenic properties in prostate tissue in 1960. Then, Barnes et al detected immunologic characteristics in prostatic fluid in 1963. Hara et al characterized γ-semino-protein in semen in 1966, and it has been proven to be identical to PSA. Subsequently, Ablin et al independently reported the presence of precipitation antigens in the prostate in 1970. Wang et al purified the PSA in 1979, and Kuriyama et al first applied an enzyme-linked immunosorbent assay for PSA in 1980. However, the positive predictive value with a cutoff figure of 4.0 ng/mL appeared substantially low (~30%. There are overdiagnoses and overtreatments for latent/low-risk PCa. Controversies exist in the PCa mortality-reducing effects of PSA screening between the European Randomized Study of Screening for Prostate Cancer (ERSPC and the US Prostate, Lung, Colorectal, and Ovarian (PLCO Cancer Screening Trial. For optimizing PCa screening, PSA-related items may require the following: 1 adjustment of the cutoff values according to age, as well as setting limits to age and screening intervals; 2 improving test performance using doubling time, density, and ratio of free: total PSA; and 3 fostering active surveillance for low-risk PCa with monitoring by PSA value. Other items needing consideration may include the following: 1 examinations of cell proliferation and cell cycle markers

Normal embryonic stages of the Longnose Gar, Lepisosteus osseus

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Ballard William W

2001-04-01

Full Text Available Abstract Background Gaps exist in the modern literature that describes patterns of development in living groups of actinopterygian fishes. Relatively recent descriptions of development exist for the teleost fishes, bowfin, sturgeon, paddlefish and bichirs. Such literature dealing with the gars is to be found in older work, done approximately a century ago. The present study concerns the gars, of which the garpike, Lepisosteus osseus, is a representative example. Results The embryonic period of life of this fish is divided, as required for experimentation, into 34 stages, from fertilization to exhaustion of the yolk supply. Diagnostic structural characteristics are cited for each stage, and the rate of development is indicated. Conclusions Three features of development are especially noted that compare or contrast with other members of the Neopterygii, and with the Chondrostei. These are meroblastic cleavage, a well-defined yolk syncytial layer (ysl, and a pit at the posterodorsal edge of the blastoderm, which defines an overhanging dorsal lip. Meroblastic cleavage and the ysl in the garpike show an affinity to those character states in the teleosts, though not with Amia, the other neopterygian fish. The posterodorsal pit and dorsal lip are reminiscent of similar features in the Chondrostei. Lepisosteus is unique among the Neopterygii with respect to this character state. Such comparisons set the stage for a broader understanding of the mechanisms for development in these organisms, and of the evolutionary relationships between them.

An Analytical Study of Prostate-Specific Antigen Dynamics.

Science.gov (United States)

Esteban, Ernesto P; Deliz, Giovanni; Rivera-Rodriguez, Jaileen; Laureano, Stephanie M

2016-01-01

The purpose of this research is to carry out a quantitative study of prostate-specific antigen dynamics for patients with prostatic diseases, such as benign prostatic hyperplasia (BPH) and localized prostate cancer (LPC). The proposed PSA mathematical model was implemented using clinical data of 218 Japanese patients with histological proven BPH and 147 Japanese patients with LPC (stages T2a and T2b). For prostatic diseases (BPH and LPC) a nonlinear equation was obtained and solved in a close form to predict PSA progression with patients' age. The general solution describes PSA dynamics for patients with both diseases LPC and BPH. Particular solutions allow studying PSA dynamics for patients with BPH or LPC. Analytical solutions have been obtained and solved in a close form to develop nomograms for a better understanding of PSA dynamics in patients with BPH and LPC. This study may be useful to improve the diagnostic and prognosis of prostatic diseases.

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

Science.gov (United States)

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

Identification of antigenic proteins of setaria cervi by immunoblotting technique

International Nuclear Information System (INIS)

Kaushal, N.A.; Kaushal, D.C.; Ghatak, S.

1987-01-01

Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125 I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites

Engineering antigen-specific immunological tolerance.

Energy Technology Data Exchange (ETDEWEB)

Kontos, Stephan; Grimm, Alizee J.; Hubbell, Jeffrey A.

2015-05-01

Unwanted immunity develops in response to many protein drugs, in autoimmunity, in allergy, and in transplantation. Approaches to induce immunological tolerance aim to either prevent these responses or reverse them after they have already taken place. We present here recent developments in approaches, based on engineered peptides, proteins and biomaterials, that harness mechanisms of peripheral tolerance both prophylactically and therapeutically to induce antigenspecific immunological tolerance. These mechanisms are based on responses of B and T lymphocytes to other cells in their immune environment that result in cellular deletion or ignorance to particular antigens, or in development of active immune regulatory responses. Several of these approaches are moving toward clinical development, and some are already in early stages of clinical testing.

Clinical use and primary evaluation of tumor marker-free prostate specific antigen

International Nuclear Information System (INIS)

Wu Junyuan; Gao Xiuying; Kong Linghua; Su Ping; Guo Xinrong

2002-01-01

Free-prostate specific antigen (fPSA)/total prostate specific antigen (tPSA) ratio was evaluated in clinical utility. Serum tPSA and fPSA level were measured by electro-chemo-luminescence (ECL) immunoassay and fPSA/tPSA ratio was calculated. Samples were drawn from 38 patients with Pca, 68 patients with BPH and 43 health men. Results showed serum tPSA > 4.0 μg/L as only cut off for diagnosis Pca, sensitivity and specificity of fPSA/tPSA ratio were 84.2%, 75.0% respectively. But fPSA/tPSA ratio 20.0 μg/L; they were 93.6%, 89.9% when serum tPSA was 2-20 μg/L. fPSA/tPSA ratio may greatly raised accurate rate for diagnosis prostate cancer when tPSA level between 2-20 μg/L and no value to other patients

Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

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Chiou-Yueh Yeh

Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

Long-term Maintenance of CD4 T Cell Memory Responses to Malaria Antigens in Malian Children Coinfected with Schistosoma haematobium

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Kirsten E. Lyke

2018-02-01

Full Text Available Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP Malian children, aged 4–8 years, are protected from malaria compared to matched schistosomiasis-negative (SN children. The effect of concomitant schistosomiasis upon acquisition of T cell memory is unknown. We examined antigen-specific T cell frequencies in 48 Malian children aged 4–14 to a pool of malaria blood stage antigens, and a pool of schistosomal antigens, at a time point during a malaria episode and at a convalescent time point ~6 months later, following cessation of malaria transmission. CD4+ T cell-derived memory responses, defined as one or more significant cytokine (IFN-γ, TNF-α, IL-2, and/or IL-17A responses, was measured to schistoma antigens in 18/23 SP children at one or both time points, compared to 4/23 SN children (P < 0.0001. At the time of malaria infection, 12/24 SN children and 15/23 SP children (P = 0.29 stimulated with malaria antigens demonstrated memory recall as defined by CD4-derived cytokine production. This compares to 7/23 SN children and 16/23 SP children (P = 0.009 at the convalescent timepoint. 46.2% of cytokine-producing CD4+ T cells expressed a single cytokine after stimulation with malaria antigen during the malaria episode. This fell to 40.9% at follow-up with a compensatory rise of multifunctional cytokine secretion over time, a phenomenon consistent with memory maturation. The majority (53.2–59.5% of responses derived from CD45RA−CD62L− effector memory T cells with little variation in the phenotype depending upon the time point or the study cohort. We conclude that detectable T cell memory responses can be measured against both malaria and schistosoma antigens and that the presence of Schistosoma haematobium may be associated with long-term maintenance of T memory to malaria.

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

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Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP10025–33 peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma

International Nuclear Information System (INIS)

Chang, Xiaoli; Xia, Chang-Qing

2015-01-01

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP100 25–33 peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP100 25–33 peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleen cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP100 25–33 peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP100 25–33 were significantly increased compared to control groups. Tumor antigen, GP100 25–23 specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy. - Highlights: • Infusion of GP100 25–33 -coupled spleen cells leads to potent anti-melanoma immunity. • GP100 25–33 -coupled spleen cell treatment induces antigen-specific IFN-γ-producing CD8 T cells. • This approach takes advantage of homing nature of immune cells.

Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors

Energy Technology Data Exchange (ETDEWEB)

Ertürk, Gizem [Department of Biotechnology, Lund University, Lund (Sweden); Department of Biology, Hacettepe University, Ankara (Turkey); Hedström, Martin [Department of Biotechnology, Lund University, Lund (Sweden); CapSenze HB, Medicon Village, SE-223 63 Lund (Sweden); Tümer, M. Aşkın [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Ankara (Turkey); Mattiasson, Bo, E-mail: Bo.Mattiasson@biotek.lu.se [Department of Biotechnology, Lund University, Lund (Sweden); CapSenze HB, Medicon Village, SE-223 63 Lund (Sweden)

2015-09-03

Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL{sup −1}–100 ng mL{sup −1}. The detection limits were found as 8.0 × 10{sup −5} ng mL{sup −1} (16 × 10{sup −17} M) and 6.0 × 10{sup −4} ng mL{sup −1} (12 × 10{sup −16} M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations. - Highlights: • Microcontact imprinting method was used for preparing the sensor chip for capacitive biosensing. • High sensitivity was obtained. • Good selectivity was

Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors

International Nuclear Information System (INIS)

Ertürk, Gizem; Hedström, Martin; Tümer, M. Aşkın; Denizli, Adil; Mattiasson, Bo

2015-01-01

Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL"−"1–100 ng mL"−"1. The detection limits were found as 8.0 × 10"−"5 ng mL"−"1 (16 × 10"−"1"7 M) and 6.0 × 10"−"4 ng mL"−"1 (12 × 10"−"1"6 M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations. - Highlights: • Microcontact imprinting method was used for preparing the sensor chip for capacitive biosensing. • High sensitivity was obtained. • Good selectivity was demonstrated. • Stability of

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

Science.gov (United States)

Soucie, Erinn L; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J-C; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H

2016-02-12

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. Copyright © 2016, American Association for the Advancement of Science.

Age-Specific Cutoff Value for the Application of Percent Free Prostate-Specific Antigen (PSA) in Chinese Men with Serum PSA Levels of 4.0–10.0 ng/ml

Science.gov (United States)

Xie, Liping; He, Dalin; Zhou, Liqun; Xu, Chuanliang; Gao, Xu; Ren, Shancheng; Wang, Fubo; Ma, Lulin; Wei, Qiang; Yin, Changjun; Tian, Ye; Sun, Zhongquan; Fu, Qiang; Ding, Qiang; Zheng, Junhua; Ye, Zhangqun; Ye, Dingwei; Xu, Danfeng; Hou, Jianquan; Xu, Kexin; Yuan, Jianlin; Gao, Xin; Liu, Chunxiao; Pan, Tiejun; Sun, Yinghao

2015-01-01

Objective The influence of age on the performance of percent free prostate-specific antigen (%fPSA) in diagnosing prostate cancer (PCa) in East Asians is controversial. We tested the diagnostic performance of %fPSA in a multi-center biopsy cohort in China and identified the proper age-specific cutoff values to avoid unnecessary biopsies. Methods Consecutive patients with a prostate-specific antigen (PSA) level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml who underwent transrectal ultrasound-guided or transperineal prostate biopsy were enrolled from 22 Chinese medical centers from Jan 1, 2010 to Dec 31, 2013. The diagnostic accuracy of PSA and %fPSA was determined using the area under the receiver operating characteristic (ROC) curve (AUC). Age-specific cutoff values were calculated using ROC curve analysis. Results The median %fPSA was much lower in younger patients compared with older patients with a PSA level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml. The AUC of %fPSA was higher than PSA only in older patients. In patients aged 50 to 59 years, %fPSA failed to improve the diagnosis compared with PSA in these two PSA ranges. Age-specific cutoff values were 24%, 27% and 32% for patients aged 60–69, 70–79 and ≥80 years, respectively, to reduce unnecessary biopsies in men with PSA levels of 4.0–10.0 ng/ml to detect 90% of all PCa. Conclusions The effectiveness of %fPSA is correlated with age in the Chinese population. Age-specific cutoff values would help avoid unnecessary biopsies in the Chinese population. PMID:26091007

vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster

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Andrew D. Renault

2012-08-01

Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster.

Science.gov (United States)

Renault, Andrew D

2012-10-15

Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

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Kurosawa Nobuyuki

2012-09-01

Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

Prostate-specific antigen testing accuracy in community practice

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Adams-Cameron Meg

2002-10-01

Full Text Available Abstract Background Most data on prostate-specific antigen (PSA testing come from urologic cohorts comprised of volunteers for screening programs. We evaluated the diagnostic accuracy of PSA testing for detecting prostate cancer in community practice. Methods PSA testing results were compared with a reference standard of prostate biopsy. Subjects were 2,620 men 40 years and older undergoing (PSA testing and biopsy from 1/1/95 through 12/31/98 in the Albuquerque, New Mexico metropolitan area. Diagnostic measures included the area under the receiver-operating characteristic curve, sensitivity, specificity, and likelihood ratios. Results Cancer was detected in 930 subjects (35%. The area under the ROC curve was 0.67 and the PSA cutpoint of 4 ng/ml had a sensitivity of 86% and a specificity of 33%. The likelihood ratio for a positive test (LR+ was 1.28 and 0.42 for a negative test (LR-. PSA testing was most sensitive (90% but least specific (27% in older men. Age-specific reference ranges improved specificity in older men (49% but decreased sensitivity (70%, with an LR+ of 1.38. Lowering the PSA cutpoint to 2 ng/ml resulted in a sensitivity of 95%, a specificity of 20%, and an LR+ of 1.19. Conclusions PSA testing had fair discriminating power for detecting prostate cancer in community practice. The PSA cutpoint of 4 ng/ml was sensitive but relatively non-specific and associated likelihood ratios only moderately revised probabilities for cancer. Using age-specific reference ranges and a PSA cutpoint below 4 ng/ml improved test specificity and sensitivity, respectively, but did not improve the overall accuracy of PSA testing.

SENSITIVITY AND SPECIFICITY OF URINARY BLADDER CANCER ANTIGEN FOR DIAGNOSIS OF BLADDER TUMOR;A COMPARATIVE STUDY WITH URINARY CYTOLOGY

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K. Radkhah

2005-06-01

Full Text Available Cystoscopy and urinary cytology are currently the basis for diagnosis and ‎follow-up of bladder tumors. Research to find a sensitive and specific tumor ‎marker for diagnosis of bladder tumor is actively underway, however, due to low sensitivity ‎and high cost of cytology. This cross-sectional study was performed in 65 patients to evaluate whether urinary bladder ‎cancer (UBC antigen level can predict the presence of active bladder tumor. In patients with ‎inactive tumor, UBC antigen level was determined in addition to standard cystoscopy ‎and cytology for follow-up. Patients with active tumor were ‎subjected to standard treatment and UBC antigen level determination. UBC antigen ‎ levels were measured by ELISA, using monoclonal antibodies ‎specific for UBC antigen. As a control group, UBC antigen level ‎was also determined in 65 persons who had been referred for urinalysis for other reasons. ‎UBC antigen level more than 1 μg/L which was regarded as ‎positive was found in 49.4% of the patients. In control group, 96.9% had UBC antigen < 1μg/L‎. Mean UBC antigen level in patients was ‎3.77 μg/L while it was 0.508 μg/L in controls (P < 0.0001. Sensitivity of ‎UBC antigen was 53.3% and its specificity was 40%. Sensitivity and specificity of urinary cytology was 17.3% and 88.2%, respectively. This difference was statistically ‎significant (P < 0.001. UBC antigen is more sensitive than urinary cytology, although cytology still ‎retains its priority in specificity. It is not yet recommended to replace UBC antigen for ‎cytology due to its low specificity and not favorable sensitivity.

Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

International Nuclear Information System (INIS)

Kamali Sarvestani, E.; Ghaderi, A.

2002-01-01

Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

I-125 input into antibodies molecules specific to australian antigen

International Nuclear Information System (INIS)

Abdukayumov, A. M.; Chistyakov, P.G.; Garajshina, G. R.

1999-01-01

There are experimental data on I-125 input into antibodies molecules specific to superficial antigen of hepatitis B virus (australian antigen). Three ways of input are submitted: with the help of T chloramine usage, Bolton-Hunter Reagent and with the help of iodogen. There are also comparative characteristics of iodized products obtained: molar radioactivity, radiochemical frequency, immuno - reactivity. The report also discusses advantages and disadvantages of the used methods for inputting I-125 into antibodies to australian antigen in order to study the possibility of creating radio immunological test system for detecting superficial antigen of B hepatitis

Insights into the biological features of the antigenic determinants recognized by four monoclonal antibodies in redia and adult stages of the liver fluke Fasciola hepatica.

Science.gov (United States)

Alba, Annia; Sánchez, Jorge; Hernández, Hilda; Mosqueda, Maryani; Rodríguez, Suanel Y; Capó, Virginia; Otero, Oscar; Alfonso, Carlos; Marcet, Ricardo; Sarracent, Jorge

2016-09-01

Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites

Matrix Metalloproteinase-2, Squamous Cell Carcinoma Antigen, and Tissue Polypeptide-Specific Antigen Expression in Egyptian Patients with Cervical Carcinoma: Relationship with Prognosis

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Maha Imam Ahmed

2004-01-01

Full Text Available Matrix metalloproteinases (MMPs, a family of proteolytic enzymes produced by both stromal and tumor cells, appear to have a key role in the events leading to local invasion and metastasis by malignant neoplasms. In the present study, we evaluated the role of MMP-2, squamous cell carcinoma antigen (SCCA, and tissue polypeptide – specific antigen (TPS in cervical neoplasia. Using Western blotting and enzyme immunoassay (EIA, we analyzed 50 patients with cervical carcinoma (CC and 25 normal controls for expression of MMP-2 in tissue cell lysates. We also quantified SCCA and TPS with microparticle immunoassay and EIA, respectively. The results were correlated with human papilloma virus (HPV infection, clinicopathological findings, and disease outcome. The cutoff point for each marker was estimated from receiver operating characteristic curves. Logistic regression analysis was performed to estimate the odds ratio (OR and 95% confidence interval (CI for each marker. MMP-2, SCCA, and TPS protein expression were significantly higher in patients with CC than in normal controls. While TPS was the best marker for discriminating between patients and controls, MMP-2 was associated with an advanced tumor stage (OR, 13.9 [95% CI, 1.4-133.9] and poor histological grade (OR, 10.2 [95% CI, 1.7-60.5]. Moreover, independent of the effect of an advanced CC stage and grade, the patients' age, and the presence of HPV infection, MMP-2 was considered a strong predictor for CC recurrence (OR, 8.1 [95% CI, 1.3- 49.1]. Tissue markers may be used to select high-risk patients for early detection of and adjuvant therapy for recurrence. Our MMP-2 findings are particularly relevant to the development of protease inhibitors as a new cancer therapy approach.

Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

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Daniel P Depledge

2010-09-01

Full Text Available A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes.We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are

Serum CEA (carcino-embryonic antigen) monitoring after surgery for cancer of the rectum and colon

International Nuclear Information System (INIS)

Reginster, J.Y.; Desaive, C.; Collette, J.; Zangerle, P.F.; Denis, D.; Franchimont, P.

1984-01-01

Fifty four patients, operated for colorectal cancer have been followed up for 2 to 100 months after surgery by carcino-embryonic antigen (CEA) determinations and classical, clinical, biological, radiological, echographical, isotopical and tomoscanninvestigations. Each new serum sample has been assayed for CEA with previously collected samples within the same patients. This repetition of CEA on the same samples allows to check the good reproducibility of CEA radioimmunoassay (variation coefficient between assay is less than 10%) and to get a complete profile of CEA level evaluation within the same assay. There is a good correlation between clinical evolution and CEA levels. In 42 patients, CEA levels remained or became normal ( 20 ng ml) at the same time or before clinical and/or paraclinical evidences for metastases or local recurrence. These results showed CEA assay in a quantitative parameter to assess the follow-up of colorectal cancer complementary to clinical, biological, radiological, echographical and isotopical criterias [fr

SSX2-4 expression in early-stage non-small cell lung cancer

DEFF Research Database (Denmark)

Greve, K B V; Pøhl, M; Olsen, K E

2014-01-01

The expression of cancer/testis antigens SSX2, SSX3, and SSX4 in non-small cell lung cancers (NSCLC) was examined, since they are considered promising targets for cancer immunotherapy due to their immunogenicity and testis-restricted normal tissue expression. We characterized three SSX antibodies...... was only detected in 5 of 143 early-stage NSCLCs, which is rare compared to other cancer/testis antigens (e.g. MAGE-A and GAGE). However, further studies are needed to determine whether SSX can be used as a prognostic or predictive biomarker in NSCLC....

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

Science.gov (United States)

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

Energy Technology Data Exchange (ETDEWEB)

Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan); Chiba, Joe, E-mail: chibaj@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan)

2009-09-11

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

[Research of Embryonic Mortality Stages of Drosophila melanogaster Depending on Age and Starvation of an Imago].

Science.gov (United States)

Kostenko, V V; Kolot, N V; Vorobyova, L I

2015-01-01

Influence of age of parents and duration of starvation on egg production and demonstration of embryonic mortality at different stages of egg development has been studied. It is shown that, with increasing age of organisms, the overall egg production reduces and the percentage of embryonic mortality increases at 0-5.5 and 5.5-17 h of development. An increase in the duration of starvation also promotes a reduction in egg production in 3- and 10-day-old adult D. melanogaster compared with short-term starvation. A statistically significant effect of factors, such as the allelic state of the white locus, the genetic background, the age of the parents, and the duration of starvation, on all studied parameters was established.

Radiation and chemical effects on viral transformation and tumor antigen expression. Annual progress report, August 1, 1978--May 1, 1979

International Nuclear Information System (INIS)

Coggin, J.H. Jr.

1979-01-01

Studies aimed at the biological, biochemical, and immunologic characterization of fetal antigens (EA) in hamsters and mice and locating and determining the distribution of fetal antigens in tumor tissues and in developing fetuses have been underway for several months. Progress has been made in isolating embryonic or fetal antigens from fetuses and from tumor cells. We have developed and reported a reliable lymphocyte transformation assay (LTA) which meets our needs in routinely assaying cell free tumor associated antigen (TAA) preparations from fetal and tumor cells. The assay correlated with transplantation resistance assays and has appropriate specificity. We have also developed the staph-A protein binding assay utilizing anti-serum derived against embryonic antigens present on SV40 tumor cells. In other studies, we have reported increases and perturbations in thymocytes during viral and chemical oncogenesis in hamsters, have developed a simple technique for preserving functional lymphocytes sensitized against TAA by freezing for use in our model system work, have reported the cross-reactivity of tranplantation resistance antigen on a spectrum of chemically induced tumors previously believed to only contain individually specific TSTAs and have recently reported the cross-reactivity of papovavirus induced transplantation resistance antigen in sarcoma cells induced by different viruses. We have concluded our studies of glycosyltransferases in the membranes of developing fetuses and noted no differences in their levels with advancing days of gestation using whold embryo cell populations

Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

Science.gov (United States)

Tian, Xinghui; Kaufman, Dan S

2008-07-01

Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

OpenAIRE

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Celine; Dolle, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-01-01

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and pro...

Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection

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Janice K. P. Lam

2018-03-01

Full Text Available Long term carriers were shown to generate robust polyfunctional T cell (PFC responses against lytic and latent antigens of Epstein-Barr virus (EBV. However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM] and asymptomatic (AS EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6–12 months. In conclusion, EBV antigen-specific CD4+ and CD

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

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Sean A Gray

Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

Prostate-Specific Antigen and Prostate-Specific Antigen Velocity as Threshold Indicators in 11C-Acetate PET/CTAC Scanning for Prostate Cancer Recurrence

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Dusing, Reginald W.; Peng, Warner; Lai, Sue-Min; Grado, Gordon L.; Holzbeierlein, Jeffrey M.; Thrasher, J. Brantley; Hill, Jacqueline; Van Veldhuizen, Peter J.

2014-01-01

Purpose The aim of this study was to identify which patient characteristics are associated with the highest likelihood of positive findings on 11C-acetate PET/computed tomography attenuation correction (CTAC) (PET/CTAC) scan when imaging for recurrent prostate cancer. Methods From 2007 to 2011, 250 11C-acetate PET/CTAC scans were performed at a single institution on patients with prostate cancer recurrence after surgery, brachytherapy, or external beam radiation. Of these patients, 120 met our inclusion criteria. Logistic regression analysis was used to examine the relationship between predictability of positive findings and patients’ characteristics, such as prostate-specific antigen (PSA) level at the time of scan, PSA kinetics, Gleason score, staging, and type of treatment before scan. Results In total, 68.3% of the 120 11C-acetate PET/CTAC scans were positive. The percentage of positive scans and PSA at the time of scanning and PSA velocity (PSAV) had positive correlations. The putative sensitivity and specificity were 86.6% and 65.8%, respectively, when a PSA level greater than 1.24 ng/mL was used as the threshold for scanning. The putative sensitivity and specificity were 74% and 75%, respectively, when a PSAV level greater than 1.32 ng/mL/y was used as the threshold. No significant associations were found between scan positivity and age, PSA doubling time, Gleason score, staging, or type of treatment before scanning. Conclusions This retrospective study suggests that threshold models of PSA greater than 1.24 ng/mL or PSAV greater than 1.32 ng/mL per year are independent predictors of positive findings in 11C-acetate PET/CTAC imaging of recurrent prostate cancer. PMID:25036021

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

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Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

2014-01-01

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

Directory of Open Access Journals (Sweden)

Maria Domina

Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria

2014-12-04

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Antigen entrapped in the escheriosomes leads to the generation of CD4(+) helper and CD8(+) cytotoxic T cell response.

Science.gov (United States)

Syed, Faisal M; Khan, Masood A; Nasti, Tahseen H; Ahmad, Nadeem; Mohammad, Owais

2003-06-02

In previous study, we demonstrated the potential of Escherichia coli (E. coli) lipid liposomes (escheriosomes) to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells including professional antigen presenting cells. Our present study demonstrates that antigen encapsulated in escheriosomes could be successfully delivered simultaneously to the cytosolic as well as endosomal processing pathways of antigen presenting cells, leading to the generation of both CD4(+) T-helper and CD8(+) cytotoxic T cell response. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (egg PC) liposomes, just like antigen-incomplete Freund's adjuvant (IFA) complex, has inefficient access to the cytosolic pathway of MHC I-dependent antigen presentation and failed to generate antigen-specific CD8(+) cytotoxic T cell response. However, both egg PC liposomes as well as escheriosomes-encapsulated antigen elicited strong humoral immune response in immunized animals but antibody titre was significantly higher in the group of animals immunized with escheriosomes-encapsulated antigen. These results imply usage of liposome-based adjuvant as potential candidate vaccine capable of eliciting both cell-mediated as well as humoral immune responses. Furthermore, antigen entrapped in escheriosomes stimulates antigen-specific CD4(+) T cell proliferation and also enhances the level of IL-2, IFN-gamma and IL-4 in the immunized animals.

Alternative pathway for the development of Vα14+ NKT cells directly from CD4-CD8- thymocytes that bypasses the CD4+CD8+ stage.

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Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Aihara, Minako; Ozawa, Ritsuko; Kojo, Satoshi; Harada, Michishige; Endo, Takaho A; Watanabe, Takashi; Ohara, Osamu; Taniguchi, Masaru

2017-03-01

Although invariant V α 14 + natural killer T cells (NKT cells) are thought to be generated from CD4 + CD8 + double-positive (DP) thymocytes, the developmental origin of CD4 - CD8 - double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (T H 1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.

Prostatic specific antigen for prostate cancer detection

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Lucas Nogueira

2009-10-01

Full Text Available Prostate-specific antigen (PSA has been used for prostate cancer detection since 1994. PSA testing has revolutionized our ability to diagnose, treat, and follow-up patients. In the last two decades, PSA screening has led to a substantial increase in the incidence of prostate cancer (PC. This increased detection caused the incidence of advanced-stage disease to decrease at a dramatic rate, and most newly diagnosed PC today are localized tumors with a high probability of cure. PSA screening is associated with a 75% reduction in the proportion of men who now present with metastatic disease and a 32.5% reduction in the age-adjusted prostate cancer mortality rate through 2003. Although PSA is not a perfect marker, PSA testing has limited specificity for prostate cancer detection, and its appropriate clinical application remains a topic of debate. Due to its widespread use and increased over-detection, the result has been the occurrence of over-treatment of indolent cancers. Accordingly, several variations as regards PSA measurement have emerged as useful adjuncts for prostate cancer screening. These procedures take into consideration additional factors, such as the proportion of different PSA isoforms (free PSA, complexed PSA, pro-PSA and B PSA, the prostate volume (PSA density, and the rate of change in PSA levels over time (PSA velocity or PSA doubling time. The history and evidence underlying each of these parameters are reviewed in the following article.

Prostatic specific antigen for prostate cancer detection.

Science.gov (United States)

Nogueira, Lucas; Corradi, Renato; Eastham, James A

2009-01-01

Prostate-specific antigen (PSA) has been used for prostate cancer detection since 1994. PSA testing has revolutionized our ability to diagnose, treat, and follow-up patients. In the last two decades, PSA screening has led to a substantial increase in the incidence of prostate cancer (PC). This increased detection caused the incidence of advanced-stage disease to decrease at a dramatic rate, and most newly diagnosed PC today are localized tumors with a high probability of cure. PSA screening is associated with a 75% reduction in the proportion of men who now present with metastatic disease and a 32.5% reduction in the age-adjusted prostate cancer mortality rate through 2003. Although PSA is not a perfect marker, PSA testing has limited specificity for prostate cancer detection, and its appropriate clinical application remains a topic of debate. Due to its widespread use and increased over-detection, the result has been the occurrence of over-treatment of indolent cancers. Accordingly, several variations as regards PSA measurement have emerged as useful adjuncts for prostate cancer screening. These procedures take into consideration additional factors, such as the proportion of different PSA isoforms (free PSA, complexed PSA, pro-PSA and B PSA), the prostate volume (PSA density), and the rate of change in PSA levels over time (PSA velocity or PSA doubling time). The history and evidence underlying each of these parameters are reviewed in the following article.

[New Radiopharmaceuticals Based on Prostate-Specific Inhibitors of Membrane Antigen for Diagnostics and Therapy of Metastatic Prostate Cancer].

Science.gov (United States)

Vlasova, O P; German, K E; Krilov, V V; Petriev, V M; Epstein, N B

2015-01-01

About 10.7% cases of prostate cancer were registered in Russia in 2011 (40,000 patients). More than half of cancer cases were revealed in advanced (III-IV) stages when metastases inevitably developed quickly. Clinical problem of early diagnostics and treatment of metastatic prostate cancer is still not solved. Anatomical imaging techniques have low sensitivity and specificity for the detection of this disease. Metabolic visualization methods which use prostate specific antigen (PSA) as a marker are also ineffective. This article describes prostate-specific membrane antigens (PSMA) that are proposed as a marker for diagnostics and therapy of prostate cancer. The most promising PSMA-based radiopharmaceutical agent for diagnostics has been developed and clinically tested in the European countries. These pharmaceuticals are based on small peptide molecules modified with urea, and have the highest affinity to PSMA. Favorable phannacokinetics, rapid accumulation in the tumor and rapid excretion from the body are beneficial features of these pharmaceuticals.

Prostate specific antigen velocity does not aid prostate cancer detection in men with prior negative biopsy.

Science.gov (United States)

Vickers, Andrew J; Wolters, Tineke; Savage, Caroline J; Cronin, Angel M; O'Brien, M Frank; Roobol, Monique J; Aus, Gunnar; Scardino, Peter T; Hugosson, Jonas; Schröder, Fritz H; Lilja, Hans

2010-09-01

Prostate specific antigen velocity has been proposed as a marker to aid in prostate cancer detection. We determined whether prostate specific antigen velocity could predict repeat biopsy results in men with persistently increased prostate specific antigen after initial negative biopsy. We identified 1,837 men who participated in the Göteborg or Rotterdam section of the European Randomized Screening study of Prostate Cancer and who underwent 1 or more subsequent prostate biopsies after an initial negative finding. We evaluated whether prostate specific antigen velocity improved predictive accuracy beyond that of prostate specific antigen alone. Of the 2,579 repeat biopsies 363 (14%) were positive for prostate cancer, of which 44 (1.7%) were high grade (Gleason score 7 or greater). Prostate specific antigen velocity was statistically associated with cancer risk but had low predictive accuracy (AUC 0.55, p <0.001). There was some evidence that prostate specific antigen velocity improved AUC compared to prostate specific antigen for high grade cancer. However, the small increase in risk associated with high prostate specific antigen velocity (from 1.7% to 2.8% as velocity increased from 0 to 1 ng/ml per year) had questionable clinical relevance. Men with prior negative biopsy are at lower risk for prostate cancer at subsequent biopsies with high grade disease particularly rare. We found little evidence to support prostate specific antigen velocity to aid in decisions about repeat biopsy for prostate cancer. 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100{sub 25–33} peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma

Energy Technology Data Exchange (ETDEWEB)

Chang, Xiaoli [Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing (China); Xia, Chang-Qing, E-mail: cqx65@yahoo.com [Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing (China); Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL32610 (United States)

2015-12-04

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP100{sub 25–33} peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP100{sub 25–33} peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleen cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP100{sub 25–33} peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP100{sub 25–33} were significantly increased compared to control groups. Tumor antigen, GP100{sub 25–23} specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy. - Highlights: • Infusion of GP100{sub 25–33}-coupled spleen cells leads to potent anti-melanoma immunity. • GP100{sub 25–33}-coupled spleen cell treatment induces antigen-specific IFN-γ-producing CD8 T cells. • This approach takes advantage of homing nature of immune cells.

Feasibility of minimally invasive radical prostatectomy in prostate cancer patients with high prostate-specific antigen. Feasibility and 1-year outcomes

International Nuclear Information System (INIS)

Do, M.; Ragavan, N.; Dietel, A.; Liatsikos, E.; Stolzenburg, J.U.; Anderson, C.; McNeill, A.

2012-01-01

Urologists are cautious to offer minimally invasive radical prostatectomy in prostate cancer patients with high prostate-specific antigen (and therefore anticipated to have locally advanced or metastatic disease) because of concerns regarding lack of complete cure after minimally invasive radical prostatectomy and of worsening of continence if adjuvant radiotherapy is used. A retrospective review of our institutional database was carried out to identify patients with prostate specific antigen (PSA) ≥20 ng/mL who underwent minimally invasive radical prostatectomy between January 2002 and October 2010. Intraoperative, pathological, functional and short-term oncological outcomes were assessed. Overall, 233 patients met study criteria and were included in the analysis. The median prostate-specific antigen and prostate size were 28.5 ng/mL and 47 mL, respectively. Intraoperative complications were the following: rectal injury (0.86%) and blood transfusion (1.7%). Early postoperative complications included prolonged (>6 days) catheterization (9.4%), hematoma (4.7%), deep venous thrombosis (0.86%) and lymphocele (5.1%). Late postoperative complications included cerebrovascular accident (0.4%) and anastomotic stricture (0.8%). Pathology revealed poorly differentiated cancer in 48.9%, pT3/pT4 disease in 55.8%, positive margins in 28.3% and lymph node disease in 20.2% of the cases. Adverse pathological findings were more frequent in patients with prostate-specific antigen >40 ng/mL and (or) in those with locally advanced disease (pT3/pT4). In 62.2% of the cases, adjuvant radiotherapy was used. At 1-year follow up, 80% of patients did not show evidence of biochemical recurrence and 98.8% of them had good recovery of continence. Minimally invasive radical prostatectomy might represent a reasonable option in prostate cancer patients with high prostate-specific antigen as a part of a multimodality treatment approach. (author)

Characterization of a human antigen specific helper factor

International Nuclear Information System (INIS)

Richardson, B.

1986-01-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with 35 S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants

CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens

OpenAIRE

Sharma, Sharad K.; Roumanes, David; Almudevar, Anthony; Mosmann, Tim R.; Pichichero, Michael E.

2013-01-01

We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing...

Equivalent biochemical failure-free survival after external beam radiation therapy or radical prostatectomy in patients with a pretreatment prostate specific antigen of > 4-20 ng/ml

International Nuclear Information System (INIS)

D'Amico, Anthony V.; Whittington, Richard; Kaplan, Irving; Beard, Clair; Jiroutek, Michael; Malkowicz, S. Bruce; Wein, Alan; Coleman, C. Norman

1997-01-01

Purpose: Biochemical failure-free survival stratified by the pretreatment prostate-specific antigen (PSA) and biopsy Gleason score (bGl) is determined for prostate cancer patients managed definitively with external beam radiation therapy or radical retropubic prostatectomy. Methods and Materials: A Cox regression multivariable analysis evaluating the variables of PSA, bGl, and clinical stage was used to evaluate the end point of time to PSA failure in 867 and 757 consecutive prostate cancer patients managed definitively with external beam radiation therapy or radical retropubic prostatectomy, respectively. PSA failure-free survival was determined using Kaplan-Meier analysis. Comparisons were made using the log rank test. Results: The pretreatment PSA, bGl, and clinical stage (T3,4 vs. T1,T2) were found to be independent predictors of time to post-treatment PSA failure for both surgically and radiation managed patients using Cox regression multivariable analysis. Patients with a pretreatment PSA of > 4 ng/ml and ≤ 20 ng/ml could be classified into risk groups for time to post-therapy PSA failure: low = PSA > 4-10 ng/ml and bGl ≤ 4; intermediate = PSA > 4-10 and bGl 5-7; or PSA > 10-20 ng/ml and bGl ≤ 7; high = PSA > 4-20 ng/ml and bGl ≥ 8. Two-year PSA failure-free survival for surgically managed and radiation-managed patients, respectively, were 98% vs. 92% (p = 0.45), 77% vs. 81% (p = 0.86), and 51% vs. 53% (p = 0.48) for patients at low, intermediate, and high risk for post-therapy PSA failure. Conclusions: There was no statistical difference in the 2-year PSA failure-free survival for potentially curable patients managed definitively with surgery or radiation therapy when a retrospective comparison stratifying for the pretreatment PSA and bGl was performed

Stepwise development of hematopoietic stem cells from embryonic stem cells.

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Kenji Matsumoto

Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Science.gov (United States)

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Establishment of immunoradiometric assay for free prostate-specific antigen

International Nuclear Information System (INIS)

Ma Lianxue

2009-01-01

An immunoradiometric assay (IRMA) of free prostate specific antigen (F-PSA) in serum was established. One monoclonal antibody against total PSA (T-PSA) was coated on the plastic tubes, the other against F-PSA was labeled with 125 I. The sensitivity of assay was 0.04 μg/L (n=20, +2s), the CVs were 2.9%-4.0% for the intra-assay and 3.5%-10.5% for the inter-assay and the average recovery was 102.7%. The correlative equation comparing with the FPSA-RIA (CIS BIO) is y=0.965 1 χ -0.001 1, and r=0.996 4. This F-PSA IRMA is a sensitive and precise method in detecting F-PSA and fit for the vitro assay. (authors)

Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

DEFF Research Database (Denmark)

Wright, A; Andrews, N; Bardsley, K

2011-01-01

The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

Characterization of glycolipid galactosyltransferases from embryonic chicken brain

International Nuclear Information System (INIS)

Kyle, J.W.

1985-01-01

Glycolipid galactosyltransferases (GalT-3 and GalT-4) were solubilized from a membrane fraction isolated from embryonic chicken brain. The profiles of specific activity and total units per brain of GalT-3 and GalT-4 varied with embryonic age. GalT-4 had the highest specific activity at 9 days of embryonic development and showed a steady decrease until hatching. GalT-3 showed a gradual increase in specific activity. Both GalT3 and GalT-4 showed a steady increase in total units per brain throughout embryonic development. The solubilized enzymes could be separated using gel filtration, ion exchange chromatography or affinity chromatography on α-lactalbumin-agarose. Data obtained in the study imply that GalT-4 is involved in both glycoprotein and glycolipid biosynthesis. Glycosphingolipid products from GalT-3 and GalT-4 catalyzed reactions labeled with [ 14 C]galactose comigrated with authentic GMI and nLcOse 4 Cer, when examined by thin layer chromatography and autoradiography. Studies with galactosidases revealed that all of the enzyme products formed by GalT-3 and GalT-4 contained a [ 14 C]-galactose in a β anomeric linkage. Periodate oxidation studies of Gal-[ 14 C]GlcNAc, formed by purified GalT-4 using [ 14 C]GlcNAc as the acceptor, demonstrated that approximately 70% of the linkage formed was Galβ1-4GlcNAc and 30% was Galβ1-3GlcNAc. Studies on the susceptibility of [ 14 C]Gal-GlcNAc to base catalyzed β-elimination also suggested the presence of approximately 30% Galβ1-3GlcNAc

Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells.

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Beatriz Suárez-Alvarez

2010-04-01

Full Text Available Human embryonic stem cells (hESCs are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM components and NKG2D ligands (NKG2D-L in hESCs, induced pluripotent stem cells (iPSCs and NTera2 (NT2 teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1 and tapasin (TPN components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs. Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Science.gov (United States)

Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

2010-01-01

Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

Prostate-specific antigen-based prostate cancer screening: Past and future.

Science.gov (United States)

Alberts, Arnout R; Schoots, Ivo G; Roobol, Monique J

2015-06-01

Prostate-specific antigen-based prostate cancer screening remains a controversial topic. Up to now, there is worldwide consensus on the statement that the harms of population-based screening, mainly as a result of overdiagnosis (the detection of clinically insignificant tumors that would have never caused any symptoms), outweigh the benefits. However, worldwide opportunistic screening takes place on a wide scale. The European Randomized Study of Screening for Prostate Cancer showed a reduction in prostate cancer mortality through prostate-specific antigen based-screening. These population-based data need to be individualized in order to avoid screening in those who cannot benefit and start screening in those who will. For now, lacking a more optimal screening approach, screening should only be started after the process of shared decision-making. The focus of future research is the reduction of unnecessary testing and overdiagnosis by further research to better biomarkers and the value of the multiparametric magnetic resonance imaging, potentially combined in already existing prostate-specific antigen-based multivariate risk prediction models. © 2015 The Japanese Urological Association.

CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

Science.gov (United States)

Chen, Peter; Mesci, Aruz; Carlyle, James R

2011-01-01

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Allergic aspergillosis and the antigens of Aspergillus fumigatus.

Science.gov (United States)

Singh, Bharat; Singh, Seema; Asif, Abdul R; Oellerich, Michael; Sharma, Gainda L

2014-01-01

Incidence of fungal infections has increased alarmingly in past few decades. Of the fungal pathogens, the Aspergillus fumigatus has been a major cause of allergic bronchopulmonary aspergillosis (ABPA) which has five main stages--the acute, remission, exacerbation, glucocorticoid dependent and fibrotic stage. The diagnosis of ABPA remains difficult due to its overlapping clinical and radiological features with tuberculosis and cystic fibrosis. From past few decades, the crude fractions of A. fumigatus have been used for immunodiagnosis of ABPA. Most of the detection kits based on crude fractions of A. fumigatus are quite sensitive but have low specificity. Till date 21 known and 25 predicted allergens of A. fumigatus have been identified. Of these allergens, only five recombinants (rAsp f1-f4 and f6) are commercially used for diagnosis of allergic aspergillosis. Remaining allergens of A. fumigatus have been restricted for use in specific diagnosis of ABPA, due to sharing of common antigenic epitopes with other allergens. Complete sequencing of A. fumigatus genome identified 9926 genes and the reports on the proteome of A. fumigatus have shown the presence of large number of their corresponding proteins in the pathogen. The analysis of immunoproteomes developed from crude fractions of A. fumigatus by IgG/IgE reactivity with ABPA patients and animal sera have identified the panel of new antigens. A brief description on the current status of A. fumigatus antigens is provided in this review. The implementation of advance recombinant expression and peptidomic approaches on the A. fumigatus antigens may help in the selection of appropriate molecules for the development of tools for more specific early diagnosis of ABPA, and desensitization therapies for patients of allergic disorders.

Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

International Nuclear Information System (INIS)

Myers, C.D.; Vitetta, E.S.

1989-01-01

B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells

Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

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Andrew R Williams

Full Text Available No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

Developmental expression of membrane type 4-matrix metalloproteinase (Mt4-mmp/Mmp17) in the mouse embryo

Science.gov (United States)

Clemente, Cristina; Montalvo, María Gregoria; Seiki, Motoharu; Arroyo, Alicia G.

2017-01-01

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development. PMID:28926609

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

International Nuclear Information System (INIS)

Chao, S.; Chao, L.; Chao, J.

1989-01-01

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

Review of Mycobacteriumavium subsp. paratuberculosis antigen candidates with diagnostic potential

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2011-01-01

antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified...... to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen...... faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior...

Docking of B-cell epitope antigen to specific hepatitis B antibody

Indian Academy of Sciences (India)

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

Prostate specific antigen levels after definitive irradiation for carcinoma of the prostate

International Nuclear Information System (INIS)

Schellhammer, P.F.; Schlossberg, S.M.; El-Mahdi, A.M.; Wright, G.L.; Brassil, D.N.

1991-01-01

Prostate specific antigen (PSA) levels were determined in 78 patients judged clinically to be free of disease at intervals of 36 or more months (range 38 to 186 months, median 87 months) after completion of irradiation therapy by 125-iodine implantation or external beam radiation. Of this select group of patients 38% had undetectable serum PSA levels (0.5 ng./ml. or less) and 38% had PSA levels that were within normal limits (4.0 ng./ml. or less). All stages and grades were represented. Undetectable PSA levels were only rarely found (3%) in patients with carcinoma of the prostate before treatment. In 24 of these 78 patients a negative biopsy of the irradiated prostate had been obtained 18 to 42 months after treatment. When the PSA level was drawn, which ranged from 7 to 16 years after treatment, an equal percentage of these biopsied patients had either an undetectable, normal or elevated level. Irradiation is able to decrease PSA to undetectable levels in some patients with prostatic carcinoma. Whether this reflects suppression of marker production alone or, more importantly, ablation of prostate cancer producing that marker remains to be determined

Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice

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Ying Wang

2014-12-01

Conclusions: Immunization with CL097-conjugated HBV-Ag reversed immune tolerance in HBV-Tg mice and induced antigen-specific immune responses. TLR7/8 agonists appear to be potent adjuvants for the induction of antigen-specific Th1 responses in an immune tolerant state.

Use of a solid-phase radioimmunoassay and formalin-fixed whole bacterial antigen in the detection of antigen-specific immunoglobulin in prostatic fluid.

OpenAIRE

Shortliffe, L M; Wehner, N; Stamey, T A

1981-01-01

The prostatic fluid of two patients with Escherichia coli bacterial prostatitis was analyzed for evidence of a local immune response to bacterial infection. A solid-phase radioimmunoassay was modified to measure the immunoglobulin (Ig)A and IgG antigen-specific antibody responses to infecting bacteria in serum and prostatic fluid from patient. Formalin-fixed whole E. coli were used as antigen. In one patient with acute E. coli prostatic infection, measurements of antigen-specific antibody con...

Prostate-specific antigen density values among patients with symptomatic prostatic enlargement in Nigeria.

Science.gov (United States)

Udeh, Emeka I; Nnabugwu, Ikenna I; Ozoemena, Francis O; Ugwumba, Fred O; Aderibigbe, Adesina S O; Ohayi, Samuel R; Echetabu, Kevin N

2016-06-29

This study aims to estimate the prostate-specific antigen density (PSAD) cutoff level for detecting prostate cancer (CAP) in Nigerian men with "grey zone PSA" (4-10 ng/ml) and normal digital rectal examination findings. We addressed this research question: Is the international PSAD cutoff of 0.15 ideal for detecting CAP in our symptomatic patients with "grey zone PSA?" To estimate the prostate-specific antigen density (PSAD) cutoff level for detecting CAP in Nigerian men with "grey zone PSA" (4-10 ng/ml) and normal digital rectal examination findings. Prospective. A tertiary medical center in Enugu, Nigeria. Two hundred and fifty-four men with either benign prostatic hyperplasia (BPH) or CAP were recruited. Patients with PSA above 4 ng/ml or abnormal digital rectal examination or hypoechoic lesion in the prostate were biopsied. PSAD and histology report of BPH or CAP. Ninety-seven patients had CAP while 157 had benign prostatic hyperplasia (BPH). Seventy-two patients had their serum PSA value within the range of 4.0 and 10 ng/ml. PSAD cutoff level to detect CAP was 0.04 (sensitivity 95.88 %; specificity 28.7 %). The PSAD cutoff level generated for Nigerian men in this study is 0.04 which is relatively different from international consensus. This PSAD cutoff level has a positive correlation with histology and could detect patients with CAP who have "grey zone PSA."

Identification of threshold prostate specific antigen levels to optimize the detection of clinically significant prostate cancer by magnetic resonance imaging/ultrasound fusion guided biopsy.

Science.gov (United States)

Shakir, Nabeel A; George, Arvin K; Siddiqui, M Minhaj; Rothwax, Jason T; Rais-Bahrami, Soroush; Stamatakis, Lambros; Su, Daniel; Okoro, Chinonyerem; Raskolnikov, Dima; Walton-Diaz, Annerleim; Simon, Richard; Turkbey, Baris; Choyke, Peter L; Merino, Maria J; Wood, Bradford J; Pinto, Peter A

2014-12-01

Prostate specific antigen sensitivity increases with lower threshold values but with a corresponding decrease in specificity. Magnetic resonance imaging/ultrasound targeted biopsy detects prostate cancer more efficiently and of higher grade than standard 12-core transrectal ultrasound biopsy but the optimal population for its use is not well defined. We evaluated the performance of magnetic resonance imaging/ultrasound targeted biopsy vs 12-core biopsy across a prostate specific antigen continuum. We reviewed the records of all patients enrolled in a prospective trial who underwent 12-core transrectal ultrasound and magnetic resonance imaging/ultrasound targeted biopsies from August 2007 through February 2014. Patients were stratified by each of 4 prostate specific antigen cutoffs. The greatest Gleason score using either biopsy method was compared in and across groups as well as across the population prostate specific antigen range. Clinically significant prostate cancer was defined as Gleason 7 (4 + 3) or greater. Univariate and multivariate analyses were performed. A total of 1,003 targeted and 12-core transrectal ultrasound biopsies were performed, of which 564 diagnosed prostate cancer for a 56.2% detection rate. Targeted biopsy led to significantly more upgrading to clinically significant disease compared to 12-core biopsy. This trend increased more with increasing prostate specific antigen, specifically in patients with prostate specific antigen 4 to 10 and greater than 10 ng/ml. Prostate specific antigen 5.2 ng/ml or greater captured 90% of upgrading by targeted biopsy, corresponding to 64% of patients who underwent multiparametric magnetic resonance imaging and subsequent fusion biopsy. Conversely a greater proportion of clinically insignificant disease was detected by 12-core vs targeted biopsy overall. These differences persisted when controlling for potential confounders on multivariate analysis. Prostate cancer upgrading with targeted biopsy increases

Characterization of a 14,000 dalton antigen of Dirofilaria immitis infective third stage larvae

International Nuclear Information System (INIS)

Fuller, S.A.; Cachia, P.J.; Wong, M.M.; Hurrell, J.G.R.

1986-01-01

Immunogenic proteins of Dirofilaria immitis (canine heartworm) were identified by probing extracts of adult worms or their excretory-secretory proteins (ESP) blotted to nitrocellulose following SDS-PAGE with control or infected dog sera. A 14,000 dalton antigen (a prominent component of ESP by protein staining) was consistently recognized both in extracts and ESP by dog sera as early as three months post infection. This indicates a larval origin for the antigen since no adult worms are present until approximately five months post infection. Monoclonal antibodies (MAbs) prepared against the 14,000 dalton antigen confirmed by immunoblotting that this antigen is expressed by infective third stage larvae, adults and microfilariae and is present intact in the sera of infected dogs. Surface-labelling of whole adult D. immitis with Na 125 I produced radiolabelled antigens closely corresponding to those of ESP. An anti-14,000 dalton MAb was able to immunoprecipitate radiolabelled antigen which strongly suggest a surface or membrane location in the intact organism. Gel filtration data suggests that the protein is a native monomer. A MAb-affinity column has been used to purify the 14,000 dalton antigen to at least 98% homogeneity in one step from crude worm extracts. Further fractionation by HPLC yields a homogeneous preparation. Amino acid analysis and the N-terminal amino acid sequence data will be presented

Antigenic specificity of serum antibodies in mice fed soy protein

DEFF Research Database (Denmark)

Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

2003-01-01

Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

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Linxi Qian

Full Text Available Alkylglycerols (AKGs are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg. Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL transcription of IgG (γ1 mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1/GATA-3 (Th2 flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2. It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1 and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10 were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

Prostate-specific antigen-positive extramammary Paget's disease--association with prostate cancer

DEFF Research Database (Denmark)

Hammer, Anne; Hager, Henrik; Steiniche, Torben

2008-01-01

Extramammary Paget's disease (EMPD) is a rare intraepidermal adenocarcinoma that primarily affects the anogenital region. Cases of EMPD reacting with PSA (prostate-specific antigen) have previously been associated with underlying prostate cancer. However, a recent case of EMPD in our department has...... led us to question the value of PSA as an indicator of underlying prostate cancer. Clinical and pathological data were obtained for 16 cases of EMPD. Formalin-fixed, paraffin-embedded tissue blocks from the primary skin lesions were investigated using PSA and other immunohistochemical markers. 5...... of the 16 cases of EMPD stained positive for PSA (2 women and 3 men). However, no reactivity was seen for the prostatic marker P501S. Three of the five patients had been diagnosed with internal malignant disease-two with prostate cancer, stage 1. Immunohistochemical investigations of the tumour specimens...

DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT

Directory of Open Access Journals (Sweden)

EMOKE PALL

2008-05-01

Full Text Available Embryonic stem (ES cells, the undifferentiated cells of early embryos are established as permanent lines and are characterised by their self-renewal capacity and the ability to retain their developmental capacity in vivo and in vitro. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains with inactivated genes by homologous recombination. There are several methods to induce the formation of EBs. One of them the formation by aggregating ES cells in hanging drops, using gravity as an aggregation force. This method presents the advantage of obtaining well-calibrated EBs almost identical in size. We used at our experiment the mouse ES cell line KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical examination was aimed to identify tissue-restricted proteins for the two differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A (TROMA for the presence of mesenchymal progenitor cells, Oct-4 for the presence of the undifferentiated ES cells. The beating cardiac muscle clumps showed more synchronous rhythm than those seen in EBs obtained from suspension culture method, where the beating cardiac muscle clumps appeared later, had a lower frequency and were uneven. The synaptic networks of neuronal cells were best developed in EBs from suspension, compared to those observed in EBs from hanging-drop method.

Immunoradiometric assay of a novel proliferation marker: tumour polypeptide specific antigen in breast cancer management in north India

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Phanna-Hazra, P.; Sharma, U.; Gupta, K.K.; Bhatnagar, V.; Idnani, R.; Gangwar, P.K.; Hazra, D.K.

2004-01-01

Tissue Polypeptide Specific Antigen (TPS) is a novel tumour marker defined by the monoclonal antibody M3 ,discovered by Bjorklund,which is claimed to be a proliferation marker, and belonging to the cytokeratin 8-18 family. M3 defines this antigen in particular out of a group of Tissue Polypeptide antigens (TPA) charecterised by the same Swedish group and was claimed to be a pancarcinoma antigen reportedly being elevated in several different cancers in the European literature.Pancarcinoma markers are of interest in relation to cancer detection as well as for assessing therapy and prognosis.Pancarcinoma antigens are also of interest for radiobioconjugate immunotargetting both for diagnosis as well as for therapy. There were no reports on this marker in an Indian population and therefore this study was initiated at two institutions in Meerut and Agra, both in the northern state of Uttar Pradesh. In addition to 250 healthy controls, 288 cases of breast cancer were studied .In addituion benign disorders were studied: breast fibroadenosis 16, breast fibro adenoma 5, breast abscess 4. TPS levels were determined by an immunoradiometric assay. In controls all but 4 had values less than 80 Units/1,the upper normal level quoted by Bjorklund.The relationship of the serum levels of the markers to histological grade and anatomical stage of the tumour were studied. In addition several of the cases underwent therapy with chemotherapy /radiotherapy/surgery or combinations thereof. The response to treatment during follow up was categorized as Complete emission(CR),Partial Remission (PR)Stable Disease(SD), Progressive Disease(PD)and recurrence (R) by the respective clinicians using standard criteria. In the cancer subjects there was a close correlation of TPS elevation with anatomical stage. 10 cases belonged to stage 1 with TPS levels 176.1+-103.94 Units/L ,61 to stage II(TPS levels 206.45 +-168.23).,79 to stage Ilia (TPS 251.5+-168.53),83 to stage IIIB (TPS 537.35+- 691.71),and 55

18F-DCFBC Prostate-Specific Membrane Antigen-Targeted PET/CT Imaging in Localized Prostate Cancer: Correlation With Multiparametric MRI and Histopathology.

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Turkbey, Baris; Mena, Esther; Lindenberg, Liza; Adler, Stephen; Bednarova, Sandra; Berman, Rose; Ton, Anita T; McKinney, Yolanda; Eclarinal, Philip; Hill, Craig; Afari, George; Bhattacharyya, Sibaprasad; Mease, Ronnie C; Merino, Maria J; Jacobs, Paula M; Wood, Bradford J; Pinto, Peter A; Pomper, Martin G; Choyke, Peter L

2017-10-01

To assess the ability of (N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-F-fluorobenzyl-L-cysteine) (F-DCFBC), a prostate-specific membrane antigen-targeted PET agent, to detect localized prostate cancer lesions in correlation with multiparametric MRI (mpMRI) and histopathology. This Health Insurance Portability and Accountability Act of 1996-compliant, prospective, institutional review board-approved study included 13 evaluable patients with localized prostate cancer (median age, 62.8 years [range, 51-74 years]; median prostate-specific antigen, 37.5 ng/dL [range, 3.26-216 ng/dL]). Patients underwent mpMRI and F-DCFBC PET/CT within a 3 months' window. Lesions seen on mpMRI were biopsied under transrectal ultrasound/MRI fusion-guided biopsy, or a radical prostatectomy was performed. F-DCFBC PET/CT and mpMRI were evaluated blinded and separately for tumor detection on a lesion basis. For PET image analysis, MRI and F-DCFBC PET images were fused by using software registration; imaging findings were correlated with histology, and uptake of F-DCFBC in tumors was compared with uptake in benign prostatic hyperplasia nodules and normal peripheral zone tissue using the 80% threshold SUVmax. A total of 25 tumor foci (mean size, 1.8 cm; median size, 1.5 cm; range, 0.6-4.7 cm) were histopathologically identified in 13 patients. Sensitivity rates of F-DCFBC PET/CT and mpMRI were 36% and 96%, respectively, for all tumors. For index lesions, the largest tumor with highest Gleason score, sensitivity rates of F-DCFBC PET/CT and mpMRI were 61.5% and 92%, respectively. The average SUVmax for primary prostate cancer was higher (5.8 ± 4.4) than that of benign prostatic hyperplasia nodules (2.1 ± 0.3) or that of normal prostate tissue (2.1 ± 0.4) at 1 hour postinjection (P = 0.0033). The majority of index prostate cancers are detected with F-DCFBC PET/CT, and this may be a prognostic indicator based on uptake and staging. However, for detecting prostate cancer with high sensitivity, it

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

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Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

microRNA expression during trophectoderm specification.

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Srinivas R Viswanathan

2009-07-01

Full Text Available Segregation of the trophectoderm from the inner cell mass of the embryo represents the first cell-fate decision of mammalian development. Transcription factors essential for specifying trophectoderm have been identified, but the role of microRNAs (miRNAs in modulating this fate-choice has been largely unexplored. We have compared miRNA expression in embryonic stem cell (ESC-derived trophectoderm and in staged murine embryos to identify a set of candidate miRNAs likely to be involved in trophectoderm specification.We profiled embryonic stem cells (ESCs as they were induced to differentiate into trophectodermal cells by ectopic expression of HRas/Q61L. We also profiled murine embryos at progressive stages of preimplantation development (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst, which includes the time window in which the trophectoderm is specified in vivo Q61L/H.We describe miRNA expression changes that occur during trophectoderm specification and validate that our in vitro system faithfully recapitulates trophectoderm specification in vivo. By comparing our in vitro and in vivo datasets, we have identified a minimal set of candidate miRNAs likely to play a role in trophectoderm specification. These miRNAs are predicted to regulate a host of development-associated target genes, and many of these miRNAs have previously reported roles in development and differentiation. Additionally, we highlight a number of miRNAs whose tight developmental regulation may reflect a functional role in other stages of embryogenesis. Our embryo profiling data may be useful to investigators studying trophectoderm specification and other stages of preimplantation development.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

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Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

PSA Nadir of <0.5 ng/mL Following Brachytherapy for Early-Stage Prostate Adenocarcinoma is Associated With Freedom From Prostate-Specific Antigen Failure

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Ko, Eric C. [Department of Radiation Oncology, Mount Sinai Medical Center, New York, NY (United States); Stone, Nelson N. [Department of Radiation Oncology, Mount Sinai Medical Center, New York, NY (United States); Department of Urology, Mount Sinai Medical Center, New York, NY (United States); Stock, Richard G., E-mail: Richard.Stock@mountsinai.org [Department of Radiation Oncology, Mount Sinai Medical Center, New York, NY (United States)

2012-06-01

Purpose: Because limited information exists regarding whether the rate or magnitude of PSA decline following brachytherapy predicts long-term clinical outcomes, we evaluated whether achieving a prostate-specific antigen (PSA) nadir (nPSA) <0.5 ng/mL following brachytherapy is associated with decreased PSA failure and/or distant metastasis. Methods and Materials: We retrospectively analyzed our database of early-stage prostate adenocarcinoma patients who underwent brachytherapy, excluding those receiving androgen-deprivation therapy and those with <2 years follow-up. Median and mean pretreatment PSA were 6 ng/mL and 7.16 ng/mL, respectively. By clinical stage, 775 were low risk ({<=}T2a), 126 were intermediate risk (T2b), and 20 were high risk (>T2b). By Gleason score, 840 were low risk ({<=}6), 71 were intermediate risk (7), and 10 were high risk (>7). Patients were treated with brachytherapy only (I-125, n = 779, or Pd-103, n = 47), or brachytherapy + external-beam radiation therapy (n = 95). Median follow-up was 6.3 years. We noted whether nPSA <0.5 ng/mL was achieved and the time to achieve this nadir and tested for associations with pretreatment risk factors. We also determined whether this PSA endpoint was associated with decreased PSA failure or distant metastasis. Results: Absence of high-risk factors in clinical stage ({<=}T2b), Gleason score ({<=}7), and pretreatment PSA ({<=}20 ng/mL) was significantly associated with achieving nPSA <0.5 ng/mL. By Kaplan-Meier analysis, patients achieving nPSA <0.5 ng/mL had significantly higher long-term freedom from biochemical failure (FFBF) than nonresponders (5-year FFBF: 95.2 {+-} 0.8% vs. 71.5 {+-} 6.7%; p < 0.0005). Among responders, those who achieved nPSA <0.5 ng/mL in {<=}5 years had higher FFBF than those requiring >5 years (5-year FFBF: 96.7 {+-} 0.7% vs. 80.8 {+-} 4.6%; p < 0.0005). On multivariate analysis, patients who achieved nPSA <0.5 ng/mL in {<=}5 years had significantly higher FFBF than other

Surgery confounds biology: the predictive value of stage-, grade- and prostate-specific antigen for recurrence after radical prostatectomy as a function of surgeon experience.

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Vickers, Andrew J; Savage, Caroline J; Bianco, Fernando J; Klein, Eric A; Kattan, Michael W; Secin, Fernando P; Guilloneau, Bertrand D; Scardino, Peter T

2011-04-01

Statistical models predicting cancer recurrence after surgery are based on biologic variables. We have shown previously that prostate cancer recurrence is related to both tumor biology and to surgical technique. Here, we evaluate the association between several biological predictors and biochemical recurrence across varying surgical experience. The study included two separate cohorts: 6,091 patients treated by open radical prostatectomy and an independent replication set of 2,298 patients treated laparoscopically. We calculated the odds ratios for biological predictors of biochemical recurrence-stage, Gleason grade and prostate-specific antigen (PSA)-and also the predictive accuracy (area under the curve, AUC) of a multivariable model, for subgroups of patients defined by the experience of their surgeon. In the open cohort, the odds ratio for Gleason score 8+ and advanced pathologic stage, though not PSA or Gleason score 7, increased dramatically when patients treated by surgeons with lower levels of experience were excluded (Gleason 8+: odds ratios 5.6 overall vs. 13.0 for patients treated by surgeons with 1,000+ prior cases; locally advanced disease: odds ratios of 6.6 vs. 12.2, respectively). The AUC of the multivariable model was 0.750 for patients treated by surgeons with 50 or fewer cases compared to 0.849 for patients treated by surgeons with 500 or more. Although predictiveness was lower overall for the independent replication set cohort, the main findings were replicated. Surgery confounds biology. Although our findings have no direct clinical implications, studies investigating biological variables as predictors of outcome after curative resection of cancer should consider the impact of surgeon-specific factors. Copyright © 2010 UICC.

Sensitivity, Specificity, and Positivity Predictors of the Pneumococcal Urinary Antigen Test in Community-Acquired Pneumonia.

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Molinos, Luis; Zalacain, Rafael; Menéndez, Rosario; Reyes, Soledad; Capelastegui, Alberto; Cillóniz, Catia; Rajas, Olga; Borderías, Luis; Martín-Villasclaras, Juan J; Bello, Salvador; Alfageme, Inmaculada; Rodríguez de Castro, Felipe; Rello, Jordi; Ruiz-Manzano, Juan; Gabarrús, Albert; Musher, Daniel M; Torres, Antoni

2015-10-01

Detection of the C-polysaccharide of Streptococcus pneumoniae in urine by an immune-chromatographic test is increasingly used to evaluate patients with community-acquired pneumonia. We assessed the sensitivity and specificity of this test in the largest series of cases to date and used logistic regression models to determine predictors of positivity in patients hospitalized with community-acquired pneumonia. We performed a multicenter, prospective, observational study of 4,374 patients hospitalized with community-acquired pneumonia. The urinary antigen test was done in 3,874 cases. Pneumococcal infection was diagnosed in 916 cases (21%); 653 (71%) of these cases were diagnosed exclusively by the urinary antigen test. Sensitivity and specificity were 60 and 99.7%, respectively. Predictors of urinary antigen positivity were female sex; heart rate≥125 bpm, systolic blood pressureantibiotic treatment; pleuritic chest pain; chills; pleural effusion; and blood urea nitrogen≥30 mg/dl. With at least six of all these predictors present, the probability of positivity was 52%. With only one factor present, the probability was only 12%. The urinary antigen test is a method with good sensitivity and excellent specificity in diagnosing pneumococcal pneumonia, and its use greatly increased the recognition of community-acquired pneumonia due to S. pneumoniae. With a specificity of 99.7%, this test could be used to direct simplified antibiotic therapy, thereby avoiding excess costs and risk for bacterial resistance that result from broad-spectrum antibiotics. We also identified predictors of positivity that could increase suspicion for pneumococcal infection or avoid the unnecessary use of this test.

Protection of Rhesus Monkeys by a DNA Prime/Poxvirus Boost Malaria Vaccine Depends on Optimal DNA Priming and Inclusion of Blood Stage Antigens

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Weiss, Walter R.; Kumar, Anita; Jiang, George; Williams, Jackie; Bostick, Anthony; Conteh, Solomon; Fryauff, David; Aguiar, Joao; Singh, Manmohan; O'Hagan, Derek T.; Ulmer, Jeffery B.; Richie, Thomas L.

2007-01-01

Background We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine. Methodology In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given iv 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-γ, and by ELISA. Conclusions 1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher

Age determination enhanced by embryonic foot bud and foot plate measurements in relation to Carnegie stages, and the influence of maternal cigarette smoking.

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Lutterodt, M C; Rosendahl, M; Yding Andersen, C; Skouby, S O; Byskov, A G

2009-08-01

Reliable age determination of first-trimester human embryos and fetuses is an important parameter for clinical use and basic science. Age determination by ultrasound or morphometric parameters of embryos 4-6 weeks post conception (p.c.) have been questioned, and more accurate methods are required. Data on whether and how maternal smoking and alcohol consumption influence embryonic and fetal foot growth is also lacking. Embryonic tissue from 102 first-trimester legal abortions (aged 35-69 days p.c.) were collected. All women answered a questionnaire concerning smoking and drinking habits, and delivered a urine sample for cotinine analysis. Embryonic age was evaluated by vaginal ultrasound measurements and by post-termination foot length and compared with the Carnegie stages. Foot bud and foot plate were defined and measured as foot length in embryos aged 35-47 days p.c. (range 0.8-2.1 mm). In embryos and fetuses aged 41-69 days p.c., heel-toe length was measured (range 2.5-7.5 mm). We found a significant linear correlation between foot length and age. Morphology of the feet was compared visually with the Carnegie collection, and we found that the mean ages of the two collections correlated well. Foot length was independent of gender, Environmental Tobacco Smoke, maternal smoking and alcohol consumption. Foot length correlated linearly to embryonic and foetal age, and was unaffected by gender, ETS, maternal smoking and alcohol consumption.

African-American Men with Gleason Score 3+3=6 Prostate Cancer Produce Less Prostate Specific Antigen than Caucasian Men: A Potential Impact on Active Surveillance.

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Kryvenko, Oleksandr N; Balise, Raymond; Soodana Prakash, Nachiketh; Epstein, Jonathan I

2016-02-01

We assess the difference in prostate specific antigen production between African-American and Caucasian men with Gleason score 3+3=6 prostate cancer. We measured tumor volume in 414 consecutive radical prostatectomies from men with National Comprehensive Cancer Network(®) low risk prostate cancer (348 Caucasian, 66 African-American) who had Gleason score 3+3=6 disease at radical prostatectomy. We then compared clinical presentation, pathological findings, prostate specific antigen, prostate specific antigen density and prostate specific antigen mass (an absolute amount of prostate specific antigen in patient's circulation) between African-American and Caucasian men. The t-test and Wilcoxon rank sum were used for comparison of means. African-American and Caucasian men had similar clinical findings based on age, body mass index and prostate specific antigen. There were no statistically significant differences between the dominant tumor nodule volume and total tumor volume (mean 0.712 vs 0.665 cm(3), p=0.695) between African-American and Caucasian men. Prostates were heavier in African-American men (mean 55.4 vs 46.3 gm, p prostate tissue contributing to prostate specific antigen in African-American men, prostate specific antigen mass was not different from that of Caucasian men (mean 0.55 vs 0.558 μg, p=0.95). Prostate specific antigen density was significantly less in African-American men due to larger prostates (mean 0.09 vs 0.105, p prostate cancer produce less prostate specific antigen than Caucasian men. African-American and Caucasian men had equal serum prostate specific antigen and prostate specific antigen mass despite significantly larger prostates in African-American men with all other parameters, particularly total tumor volume, being the same. This finding has practical implications in T1c cases diagnosed with prostate cancer due to prostate specific antigen screening. Lowering the prostate specific antigen density threshold in African-American men may

TISSUE POLYPEPTIDE-SPECIFIC ANTIGEN - A DISCRIMINATIVE PARAMETER BETWEEN PROSTATE-CANCER AND BENIGN PROSTATIC HYPERTROPHY

NARCIS (Netherlands)

MARRINK, J; OOSTEROM, R; BONFRER, HMG; SCHRODER, FH; MENSINK, HJA

1993-01-01

The serum concentration of the cell proliferation marker TPS (tissue polypeptide-specific antigen) was compared with the tumour marker PSA (prostate specific antigen). PSA was found elevated in 50% of the benign prostatic hypertrophy (BPH) patients, in 88% of the patients with active prostate cancer

The sensitivity and the specifity of rapid antigen test in streptococcal upper respiratory tract infections.

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Gurol, Yesim; Akan, Hulya; Izbirak, Guldal; Tekkanat, Zuhal Tazegun; Gunduz, Tehlile Silem; Hayran, Osman; Yilmaz, Gulden

2010-06-01

It is aimed to detect the sensitivity and specificity of rapid antigen detection of group A beta hemolytic streptococci from throat specimen compared with throat culture. The other goal of the study is to help in giving clinical decisions in upper respiratory tract infections according to the age group, by detection of sensitivity and positive predictive values of the rapid tests and throat cultures. Rapid antigen detection and throat culture results for group A beta hemolytic streptococci from outpatients attending to our university hospital between the first of November 2005 and 31st of December 2008 were evaluated retrospectively. Throat samples were obtained by swabs from the throat and transported in the Stuart medium and Quickvue Strep A [Quidel, San Diego, USA] cassette test was applied and for culture, specimen was inoculated on 5% blood sheep agar and identified according to bacitracin and trimethoprim-sulphametaxazole susceptibility from beta hemolytic colonies. During the dates between the first of November 2005 and 31st of December 2008, from 453 patients both rapid antigen detection and throat culture were evaluated. Rapid antigen detection sensitivity and specificity were found to be 64.6% and 96.79%, respectively. The positive predictive value was 80.95% whereas negative predictive value was 92.82%. Kappa index was 0.91. When the results were evaluated according to the age groups, the sensitivity and the positive predictive value of rapid antigen detection in children were 70%, 90.3% and in adults 59.4%, 70.4%. When bacterial infection is concerned to prevent unnecessary antibiotic use, rapid streptococcal antigen test (RSAT) is a reliable method to begin immediate treatment. To get the maximum sensitivity of RSAT, the specimen collection technique used and education of the health care workers is important. While giving clinical decision, it must be taken into consideration that the sensitivity and the positive predictive value of the RSAT is quite

Serological cross-reaction between O-antigens of Shigella dysenteriae type 4 and an environmental Escherichia albertii isolate.

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Rahman, Mohammed Ziaur; Akter, Selina; Azmuda, Nafisa; Sultana, Munawar; Weill, François-Xavier; Khan, Sirajul Islam; Grimont, Patrick A D; Birkeland, Nils-Kåre

2013-11-01

An environmental freshwater bacterial isolate, DM104, appearing as Shigella-like colonies on selective agar plates was found to show strong and specific serological cross-reactivity with Shigella dysenteriae type 4. Biochemical identification according to the analytical profile index, molecular serotyping by restriction of the amplified O-antigen gene cluster (rfb-RFLP), together with phylogenetic analysis of the 16S rRNA gene and multi-locus sequence analysis, identified the isolate as Escherichia albertii. rfb-RFLP of DM104, revealed a profile different from that of S. dysenteriae type 4. However, western blot analysis of extracted lipopolysaccharides demonstrated strong cross-reactivity with S. dysenteriae type 4 using specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. dysenteriae type 4. The observed O-antigen cross-reaction between an E. albertii isolate and S. dysenteriae extends our knowledge of the extent of O-antigen cross-reaction within the Escherichia/Shigella group of organisms, and offers the possibility of using DM104 and similar cross-reacting strains as shigellosis vaccine candidates.

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

Directory of Open Access Journals (Sweden)

Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Dual effect of fetal bovine serum on early development depends on stage-specific reactive oxygen species demands in pigs.

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Seong-Eun Mun

Full Text Available Despite the application of numerous supplements to improve in vitro culture (IVC conditions of mammalian cells, studies regarding the effect of fetal bovine serum (FBS on mammalian early embryogenesis, particularly in relation to redox homeostasis, are lacking. Herein, we demonstrated that early development of in vitro-produced (IVP porcine embryos highly depends on the combination of FBS supplementation timing and embryonic reactive oxygen species (ROS requirements. Interestingly, FBS significantly reduced intracellular ROS levels in parthenogenetically activated (PA embryos regardless of the developmental stage. However, the beneficial effect of FBS on early embryogenesis was found only during the late phase (IVC 4-6 days treatment group. In particular, developmental competence parameters, such as blastocyst formation rate, cellular survival, total cell number and trophectoderm proportion, were markedly increased by FBS supplementation during the late IVC phase. In addition, treatment with FBS elevated antioxidant transcript levels during the late IVC phase. In contrast, supplementation with FBS during the entire period (1-6 days or during the early IVC phase (1-2 days greatly impaired the developmental parameters. Consistent with the results from PA embryos, the developmental competence of in vitro fertilization (IVF or somatic cell nuclear transfer (SCNT embryos were markedly improved by treatment with FBS during the late IVC phase. Moreover, the embryonic stage-specific effects of FBS were reversed by the addition of an oxidant and were mimicked by treatment with an antioxidant. These findings may increase our understanding of redox-dependent early embryogenesis and contribute to the large-scale production of high-quality IVP embryos.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

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Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Usefulness of SCC-antigen for diagnosis and monitoring recurrence and effectiveness of therapies of squamous cell carcinoma of the lung

International Nuclear Information System (INIS)

Mino, Naoko; Iio, Atsushi; Ata, Mariko; Murase, Kenya; Kataoka, Masaaki; Ito, Hisao; Ishine, Masahiro; Kawamura, Masashi; Hamamoto, Ken

1987-01-01

The serum levels of SCC antigen (squamous cell carcinoma related antigen) were measured in 111 patients with primary lung cancer to assess its clinical usefulness for diagnosis of squamous cell carcinoma and for monitoring recurrence and effectiveness of therapies. Serum SCC antigen level in patients with squamous cell carcinoma of the lung was 5.9 ± 10.4 ng/ml, which was high (p < 0.05) compared with those in normal controls (1.6 ± 0.5 ng/ml), patients with other types of lung cancer (2.4 ± 2.9 ng/ml) or benign disease (1.8 ± 1.1 ng/ml). Studies at various clinical stages of squamous cell carcinoma of the lung showed, however, that the SCC antigen levels were high only in the advanced stages (III and IV), whereas not so high in the earlier stages. These results confirmed that SCC antigen is a relatively specific marker to squamous cell carcinoma in the lung, as reported in the uterine cervix and the esophagus. The SCC antigen levels decreased after operation and more markedly after radiotherapy in dose-dependent manner, corresponding to the reduction of the tumor size. On the other hand, the SCC antigen levels were extremely high in the recurrence. It was concluded that SCC antigen is a useful marker for monitoring recurrence or effectiveness of the therapies of SCC of the lung, although not so for its early diagnosis. (author)

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

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Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

2015-09-01

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Serum prostate-specific antigen as a predictor of prostate volume in the community: the Krimpen study.

NARCIS (Netherlands)

Bohnen, A.M.; Groeneveld, F.P.; Bosch, J.L.H.R.

2007-01-01

OBJECTIVES: Serum prostate-specific antigen (PSA) is considered a proxy for prostate volume (PV). This study investigates which range of PSA values has the best utility in the determination of PV (4. Low PSA ranges (0-2 and 2.1-4.0) discriminate better for a PV of 30 cc (eg, in men with a PSA range

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

Science.gov (United States)

Chapuis, Aude G; Roberts, Ilana M; Thompson, John A; Margolin, Kim A; Bhatia, Shailender; Lee, Sylvia M; Sloan, Heather L; Lai, Ivy P; Farrar, Erik A; Wagener, Felecia; Shibuya, Kendall C; Cao, Jianhong; Wolchok, Jedd D; Greenberg, Philip D; Yee, Cassian

2016-11-01

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8 + T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses

Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Domber, E.A.

1986-01-01

These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

Energy Technology Data Exchange (ETDEWEB)

Kleinhenz, M.E.; Ellner, J.J.

1985-07-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

International Nuclear Information System (INIS)

Kleinhenz, M.E.; Ellner, J.J.

1985-01-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

Prostate-specific membrane antigen-based imaging in prostate cancer: impact on clinical decision making process.

Science.gov (United States)

Demirkol, Mehmet Onur; Acar, Ömer; Uçar, Burcu; Ramazanoğlu, Sultan Rana; Sağlıcan, Yeşim; Esen, Tarık

2015-05-01

There is an ongoing need for an accurate imaging modality which can be used for staging purposes, metastatic evaluation, predicting biologic aggresiveness and investigating recurrent disease in prostate cancer. Prostate specific membrane antigen, given its favorable molecular characteristics, holds a promise as an ideal target for prostate cancer-specific nuclear imaging. In this study, we evaluated our initial results of PSMA based PET/CT imaging in prostate cancer. A total of 22 patients with a median age and serum PSA level of 68 years and 4.15 ng/ml, respectively underwent Ga-68 PSMA PET/CT in our hospital between Februrary and August 2014. Their charts were retrospectively reviewed in order to document the clinical characteristics, the indications for and the results of PSMA based imaging and the impact of Ga-68 PSMA PET/CT findings on disease management. The most common indications were rising PSA after local ± adjuvant treatment followed by staging and metastatic evaluation before definitive or salvage treatment. All except 2 patients had prostatic ± extraprostatic PSMA positive lesions. For those who had a positive result; treatment strategies were tailored accordingly. Above the PSA level of 2 ng/ml, none of the PSMA based nuclear imaging studies revealed negative results. PSMA based nuclear imaging has significantly impacted our way of handling patients with prostate cancer. Its preliminary performance in different clinical scenarios and ability to detect lesions even in low PSA values seems fairly promising and deserves to be supplemented with further clinical studies. © 2015 Wiley Periodicals, Inc.

CD4+ T-cell epitope prediction using antigen processing constraints.

Science.gov (United States)

Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

2016-05-01

T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

Science.gov (United States)

Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

2011-02-01

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

Human seminal proteinase and prostate-specific antigen are the ...

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/033/02/0195-0207. Keywords. Kallikrein; prostate cancer biomarker; proteinase activity; seminal plasma; tumour proliferation and metastasis; therapeutic target. Abstract. Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and ...

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques

Directory of Open Access Journals (Sweden)

Bryan C. Au

2016-02-01

Full Text Available Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag-specific responses through direct injections of recombinant lentivectors (LVs that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months—the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an “off-the-shelf” anti-cancer vaccine that could be made at large scale and injected into patients—even on an out-patient basis.

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques.

Science.gov (United States)

Au, Bryan C; Lee, Chyan-Jang; Lopez-Perez, Orlay; Foltz, Warren; Felizardo, Tania C; Wang, James C M; Huang, Ju; Fan, Xin; Madden, Melissa; Goldstein, Alyssa; Jaffray, David A; Moloo, Badru; McCart, J Andrea; Medin, Jeffrey A

2016-02-19

Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag)-specific responses through direct injections of recombinant lentivectors (LVs) that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA)-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months-the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an "off-the-shelf" anti-cancer vaccine that could be made at large scale and injected into patients-even on an out-patient basis.

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

Directory of Open Access Journals (Sweden)

Ryutaro Hirasawa

Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin

Directory of Open Access Journals (Sweden)

Sara eLawrence

2012-11-01

Full Text Available The cholesterol-dependent cytolysins (CDCs attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modelling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

International Nuclear Information System (INIS)

Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru

2013-01-01

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4 + IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4 + IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4 + IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4 + IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4 + LPLs and primed splenic CD4 + T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4 + IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

Effect of antigen on localization of immunologically specific B cells

International Nuclear Information System (INIS)

Ponzio, N.M.; Chapman, J.M.; Thorbecke, G.J.

1976-01-01

Studies were conducted to demonstrate homing of memory B cells to sites of antigen localization in lymph nodes, using functional criteria to detect local presence of memory cells at varying intervals after intravenous injection. Cell suspensions were prepared from spleens of donor mice injected with complete Freund's adjuvant. Recipient mice were injected with Escherichia coli endotoxin and immune or normal spleen cells and were gamma-irradiated. Results indicated that passively transferred unilateral B cell memory was established. The development over a period of several days of this difference between left and right lymph nodes suggests that recirculating memory B cells are being progressively selected by antigen in the lymph node, rather than that this difference is due to a specific exit of cells from the circulation towards the antigen

Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

International Nuclear Information System (INIS)

Kato, H.; Torigoe, T.

1977-01-01

A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with other carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All patients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

Science.gov (United States)

Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

2010-12-15

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

Science.gov (United States)

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

DEFF Research Database (Denmark)

Sørensen, Anja Elaine; Johnsen, Claus R; Dalgaard, Louise Torp

2013-01-01

of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4......). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated...... with pollen extract in vitro and immune factors were evaluated. Results: During SIT, the main changes in the peripheral blood were an increase in CXCR3+CD4+CD25+CD127low/- Tregs and a decrease in CCR4+CD4+CD25+CD127low/- Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first...

Effect of More vs Less Frequent Follow-up Testing on Overall and Colorectal Cancer-Specific Mortality in Patients With Stage II or III Colorectal Cancer: The COLOFOL Randomized Clinical Trial.

Science.gov (United States)

Wille-Jørgensen, Peer; Syk, Ingvar; Smedh, Kenneth; Laurberg, Søren; Nielsen, Dennis T; Petersen, Sune H; Renehan, Andrew G; Horváth-Puhó, Erzsébet; Påhlman, Lars; Sørensen, Henrik T

2018-05-22

Intensive follow-up of patients after curative surgery for colorectal cancer is common in clinical practice, but evidence of a survival benefit is limited. To examine overall mortality, colorectal cancer-specific mortality, and colorectal cancer-specific recurrence rates among patients with stage II or III colorectal cancer who were randomized after curative surgery to 2 alternative schedules for follow-up testing with computed tomography and carcinoembryonic antigen. Unblinded randomized trial including 2509 patients with stage II or III colorectal cancer treated at 24 centers in Sweden, Denmark, and Uruguay from January 2006 through December 2010 and followed up for 5 years; follow-up ended on December 31, 2015. Patients were randomized either to follow-up testing with computed tomography of the thorax and abdomen and serum carcinoembryonic antigen at 6, 12, 18, 24, and 36 months after surgery (high-frequency group; n = 1253 patients) or at 12 and 36 months after surgery (low-frequency group; n = 1256 patients). The primary outcomes were 5-year overall mortality and colorectal cancer-specific mortality rates. The secondary outcome was the colorectal cancer-specific recurrence rate. Both intention-to-treat and per-protocol analyses were performed. Among 2555 patients who were randomized, 2509 were included in the intention-to-treat analysis (mean age, 63.5 years; 1128 women [45%]) and 2365 (94.3%) completed the trial. The 5-year overall patient mortality rate in the high-frequency group was 13.0% (161/1253) compared with 14.1% (174/1256) in the low-frequency group (risk difference, 1.1% [95% CI, -1.6% to 3.8%]; P = .43). The 5-year colorectal cancer-specific mortality rate in the high-frequency group was 10.6% (128/1248) compared with 11.4% (137/1250) in the low-frequency group (risk difference, 0.8% [95% CI, -1.7% to 3.3%]; P = .52). The colorectal cancer-specific recurrence rate was 21.6% (265/1248) in the high-frequency group compared with 19.4

T-Cell Therapy Using Interleukin-21–Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

Science.gov (United States)

Chapuis, Aude G.; Roberts, Ilana M.; Thompson, John A.; Margolin, Kim A.; Bhatia, Shailender; Lee, Sylvia M.; Sloan, Heather L.; Lai, Ivy P.; Farrar, Erik A.; Wagener, Felecia; Shibuya, Kendall C.; Cao, Jianhong; Wolchok, Jedd D.; Greenberg, Philip D.

2016-01-01

Purpose Peripheral blood–derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti–CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses

Ontogeny and localization of γ-crystallin antigen in the developing pigeon (Columba livia) lens

NARCIS (Netherlands)

Brahma, S.K.; Rabaey, M.; Doorenmaalen, W.J. van

Ontogeny and localization of the lens γ-crystallin antigen were investigated in the embryonic and post-embryonic pigeon lenses by the indirect immunofluorescence with antiserum from rabbit immunized with isolated pigeon lens γ-crystallin. The results show that γ-crystallin appears for the first time

Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

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Shigeo Koido

2010-01-01

Full Text Available The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs that can reduce the tumor mass. Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

Science.gov (United States)

Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

2015-05-01

Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level. © 2015 International Federation for Cell Biology.

Prostate-specific membrane antigen and its truncated form PSM'

Czech Academy of Sciences Publication Activity Database

Mlčochová, Petra; Bařinka, Cyril; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

2009-01-01

Roč. 69, č. 5 (2009), s. 471-479 ISSN 0270-4137 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * prostate cancer Subject RIV: CE - Biochemistry Impact factor: 3.081, year: 2009

The radiation protection effects of TMG to the embryonic effects on the organogenesis stage in ICR mice

International Nuclear Information System (INIS)

Santokuya, Takumi; Gu, Yeunhwa; Suzuki, Ikukatsu; Hasegawa, Takeo; Yamamoto, Youichi; Iwasa, Toshihiro; Yanagisawa, Takaharu

1999-01-01

The Organogenesis stage is the most important from the viewpoint of ionizing protection. Many physical and chemical agents in the environment can effect an embryo. Fetal deaths were classified as Preimplantation, Embryonic and Fetal. We studied an excuse as a radio protective agent of the high malformation of the sensitivity most by using TMG to the radiation. This study aimed at obtaining the information to provide it for the fetus' protection to the various environmental radiations. As for Preimplantation death, there was a statistical difference the 2.0Gy Group in comparison with the control group (p<0.05). Embryonic death, a statistical difference was recognized as in all the treatment groups (p<0.001). But, as for the TMG+radiation group, Malformation rate decreased to 1/2. As for Fetal body weight, a statistical difference was recognized in radiation group, the radiation+TMG infusion group chisels for medical use (p<0.05). Therefore, TMG protecting effect to the radiation was made clear by this research

Observations of pretreatment prostate-specific antigen doubling time in 107 patients referred for definitive radiotherapy

International Nuclear Information System (INIS)

Lee, W. Robert; Hanks, Gerald E.; Corn, Benjamin W.; Schultheiss, Timothy E.

1995-01-01

Purpose: To determine pretreatment prostate-specific antigen doubling times (PSADT) in patients referred for definitive radiotherapy. Methods and Materials: One hundred and seven patients with histologically proven nonmetastatic prostate cancer and an elevated prostate-specific antigen (PSA) who were referred for radiation therapy had three serum PSA values obtained prior to the start of definitive therapy. Prostate-specific antigen doubling times were calculated by linear regression. Results: Prostate-specific antigen values increased during the period of observation in 78 patients (73%). Forty-three patients (40%) had calculated PSADT of less than 2 years and of those patients with pretreatment serum PSA values of greater than 10 ng/mL more than 50% has calculated PSADT of less than 2 years. Conclusions: A significant minority of patients referred for radiotherapy have calculated PSADT of less than 2 years. The significance of this relatively fast growth rate is as yet undetermined, but suggests that patients referred for radiotherapy may have aggressive disease prior to treatment

Anti-cytotoxic T lymphocyte antigen-4 antibodies in melanoma

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Tosti G

2013-10-01

Full Text Available Giulio Tosti, Emilia Cocorocchio, Elisabetta PennacchioliDivisione Melanomi e Sarcomi, Istituto Europeo di Oncologia, Milano, ItalyAbstract: Approaches aimed at enhancement of the tumor specific response have provided proof for the rationale of immunotherapy in cancer, both in animal models and in humans. Ipilimumab, an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4 antibody, is a new generation immunotherapeutic agent that has shown activity in terms of disease free and overall survival in metastatic melanoma patients. Its use was approved by the US Food and Drug Administration in March 2011 to treat patients with late stage melanoma that has spread or that cannot be removed by surgery. The mechanism of action of CTLA-4 antibodies in the activation of an antitumor immune response and selected clinical studies of ipilimumab in advanced melanoma patients are discussed. Ipilimumab treatment has been associated with immune related adverse events due to T-cell activation and proliferation. Most of these serious adverse effects are associated with the gastrointestinal tract and include severe diarrhea and colitis. The relationship between immune related adverse events and antitumor activity associated with ipilimumab was explored in clinical studies. Potential biomarkers predictive for clinical response and survival in patients treated with anti-CTLA-4 therapy are presently under investigation. Besides the conventional patterns of response and stable disease as defined by standard Response Evaluation Criteria in Solid Tumors criteria, in subsets of patients, ipilimumab has shown patterns of delayed clinical activity which were associated with an improved overall survival. For this reason a new set of response criteria for tumor immunotherapy has been proposed, which was termed immune related response criteria. These new criteria are presently used to better analyze clinical activity of immunotherapeutic regimens. Ipilimumab is currently under

Prostate-specific antigen and radiation therapy for clinically localized prostate cancer

International Nuclear Information System (INIS)

Zagars, Gunar K.; Pollack, Alan; Kavadi, Vivek S.; Eschenbach, Andrew C. von

1995-01-01

Purpose: This study was undertaken to: (a) define the prognostic significance of pretreatment serum prostate-specific antigen (PSA) levels in localized prostate cancer treated with radiation; (b) define the prognostic usefulness of postradiation PSA levels; (c) evaluate the outcome of radiation using PSA as an endpoint. Methods and Materials: Disease outcome in 707 patients with Stages T1 (205 men), T2 (256 men), T3 (239 men), and T4 (7 men), receiving definitive external radiation as sole therapy, was evaluated using univariate and multivariate techniques. Results: At a mean follow-up of 31 months, 157 patients (22%) developed relapse or a rising PSA. Multivariate analysis revealed pretreatment PSA level to be the most significant prognostic factor, with lesser though significant contributions due to Gleason grade (2-6 vs. 7-10) and transurethral resection in (T3(T4)) disease. The following four prognostic groupings were defined: group I, PSA ≤ 4 ng/ml, any grade; group II, 4 20, any grade. Five-year actuarial relapse rates in these groups were: I, 12%; II, 34%; III, 40%; and IV, 81%. Posttreatment nadir PSA was an independent determinant of outcome and only patients with nadir values < 1 ng/ml fared well (5-year relapse rate 20%). Using rising PSA as an endpoint the 461 patients with (T1(T2)) disease had an actuarial freedom from disease rate of 70% at 5 years, which appeared to plateau, suggesting that many were cured. No plateau was evident for (T3(T4)) disease. Conclusion: Pretreatment serum PSA is the single most important predictor of disease outcome after radiation for local prostate cancer. Tumor grade has a lesser though significant prognostic role. Postirradiation nadir PSA value during the first year is a sensitive indicator of response to treatment. Only nadir values < 1 ng/ml are associated with a favorable outlook. A significant fraction of men with (T1(T2)) disease may be cured with radiation. There was no evidence for a cured fraction among

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

Science.gov (United States)

Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

Science.gov (United States)

Maywald, Martina; Rink, Lothar

2017-08-01

The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. Cell proliferation and pro-inflammatory cytokine production were monitored by [ 3 H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + T cells (p zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

Allopurinol reduces antigen-specific and polyclonal activation of human T cells

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Damián ePérez-Mazliah

2012-09-01

Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

Dissection of T-cell antigen specificity in human melanoma

DEFF Research Database (Denmark)

Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

2012-01-01

Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-as...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined....

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

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Ruixin Hao

Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

Science.gov (United States)

Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

2005-09-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Left-Right Asymmetry of Maturation Rates in Human Embryonic Neural Development.

Science.gov (United States)

de Kovel, Carolien G F; Lisgo, Steven; Karlebach, Guy; Ju, Jia; Cheng, Gang; Fisher, Simon E; Francks, Clyde

2017-08-01

Left-right asymmetry is a fundamental organizing feature of the human brain, and neuropsychiatric disorders such as schizophrenia sometimes involve alterations of brain asymmetry. As early as 8 weeks postconception, the majority of human fetuses move their right arms more than their left arms, but because nerve fiber tracts are still descending from the forebrain at this stage, spinal-muscular asymmetries are likely to play an important developmental role. We used RNA sequencing to measure gene expression levels in the left and right spinal cords, and the left and right hindbrains, of 18 postmortem human embryos aged 4 to 8 weeks postconception. Genes showing embryonic lateralization were tested for an enrichment of signals in genome-wide association data for schizophrenia. The left side of the embryonic spinal cord was found to mature faster than the right side. Both sides transitioned from transcriptional profiles associated with cell division and proliferation at earlier stages to neuronal differentiation and function at later stages, but the two sides were not in synchrony (p = 2.2 E-161). The hindbrain showed a left-right mirrored pattern compared with the spinal cord, consistent with the well-known crossing over of function between these two structures. Genes that showed lateralization in the embryonic spinal cord were enriched for association signals with schizophrenia (p = 4.3 E-05). These are the earliest stage left-right differences of human neural development ever reported. Disruption of the lateralized developmental program may play a role in the genetic susceptibility to schizophrenia. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Immunity to tumour antigens.

Science.gov (United States)

Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

2005-01-01

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

Prostatic biopsy in the prostate specific antigen gray zone; La biopsia prostatica multipla nalla zona grigia dei valori dell'antigene prostatico specifico

Energy Technology Data Exchange (ETDEWEB)

Drudi, F. M.; Ricci, P.; Iannicelli, E.; Di Nardo, R.; Novelli, L.; Laghi, A.; Passariello, R. [Rome Univ. La Sapienza, Rome (Italy). Ist. di Radiologia II Cattedra; Perugia, G. [Rome Univ. La Sapienza, Rome (Italy). Dipt. di Urologia U. Bracci

2000-02-01

The main purpose of this study was to identify cases of undetected prostatic cancer in patients with normal findings at digital examination and transrectal US, and prostate specific antigen (PSA) values ranging 4-10 ng/mL. 290 patients were submitted to transrectal US and random bilateral prostatic biopsy; 3 samples were collected from each side of the gland using 16-Gauge thru-cut needles. Of the 290 patients who gave full informed consent, 34 people were selected whose age range was between 56 to 76 years (mean: 64). Inclusion criteria were PSA 4-10 ng/mL, PSAD cut-off 0.15, free/total PSA ratio 15-25%, and normal findings at digital examination and transrectal US. PSA velocity was calculated collecting 3 blood samples every 30 days for 2 months. 5 of the 34 selected patients (15%) had prostatic cancer, and 2 (6%) Pin (1 Pin 1 and 1 Pin 2). As for the other 27 patients, biopsy demonstrated 4 (12%) cases of prostatitis and 23 (62%) cases of BPH. PSA values increased in all patients with positive histology, versus only 6 (22%) of those with negative histology. Our findings confirm that prostatic biopsy can detect tumors also in areas which appear normal at transrectal US and digital examination, and that PSA rate increases in patients with positive histology. Finally, the actual clinical role of prostatic biopsy relative to all other diagnostic imaging techniques remains to be defined. [Italian] Si intende qui dimostrare la percentuale di neoplasie prostatiche sfuggite all'esplorazione rettale e all'ecografia transrettale nei pazienti convalori di antigene prostatico specifico tra 4 e 10 ng/ml. 290 pazienti sono stati sottoposti a ecografia transrettale e biopsia multipla (6 prelievi, ago da 16 Gauge) dopo consenso informato. Di questi sono stati selezionati 34: eta' tra 56 e 76 anni, eta' media 64 anni. Parametri di selezione: antigene prostatico specifico con valori tra 4 e 10ng/ml; densita' dell'antigene prostatico specifico con

A VAR2CSA:CSP conjugate capable of inducing dual specificity ...

African Journals Online (AJOL)

Background: Vaccine antigens targeting specific P. falciparum parasite stages are under pre-clinical ... against individual antigen components in a dual stage anti-malaria vaccine. ..... (D8428, Sigma Aldrich) was prepared in Dulbecco's PBS.

Virus-specific regulatory T cells ameliorate encephalitis by repressing effector T cell functions from priming to effector stages.

Directory of Open Access Journals (Sweden)

Jingxian Zhao

2014-08-01

Full Text Available Several studies have demonstrated the presence of pathogen-specific Foxp3+ CD4 regulatory T cells (Treg in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg and conventional CD4 T cells (M133 Tconv in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN. Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

Science.gov (United States)

Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

2012-11-01

Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

On the importance of controlling film architecture in detecting prostate specific antigen

Science.gov (United States)

Graça, Juliana Santos; Miyazaki, Celina Massumi; Shimizu, Flavio Makoto; Volpati, Diogo; Mejía-Salazar, J. R.; Oliveira, Osvaldo N., Jr.; Ferreira, Marystela

2018-03-01

Immunosensors made with nanostructured films are promising for detecting cancer biomarkers, even at early stages of the disease, but this requires control of film architecture to preserve the biological activity of immobilized antibodies. In this study, we used electrochemical impedance spectroscopy (EIS) to detect Prostate Specific Antigen (PSA) with immunosensors produced with layer-by-layer (LbL) films containing anti-PSA antibodies in two distinct film architectures. The antibodies were either adsorbed from solutions in which they were free, or from solutions where they were incorporated into liposomes of dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation into DPPG liposomes was confirmed with surface plasmon resonance experiments, while the importance of electrostatic interactions on the electrical response was highlighted using the Finite Difference Time-Domain Method (FDTD). The sensitivity of both architectures was sufficient to detect the threshold value to diagnose prostate cancer (ca. 4 ng mL-1). In contrast to expectation, the sensor with the antibodies incorporated into DPPG liposomes had lower sensitivity, though the range of concentrations amenable to detection increased, according to the fitting of the EIS data using the Langmuir-Freundlich adsorption model. The performance of the two film architectures was compared qualitatively by plotting the data with a multidimensional projection technique, which constitutes a generic approach for optimizing immunosensors and other types of sensors.

Pretreatment antigen-specific immunity and regulation - association with subsequent immune response to anti-tumor DNA vaccination.

Science.gov (United States)

Johnson, Laura E; Olson, Brian M; McNeel, Douglas G

2017-07-18

Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune "responders." The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial.

Case of rhesus antigen weak D type 4.2. (DAR category detection

Directory of Open Access Journals (Sweden)

L. L. Golovkina

2015-01-01

Full Text Available Serological methods of Rhesus antigens identification in humans cannot identify D-antigen variants. In this article the serological characteristics of Rhesus antigen D weak type 4.2. (Category DAR are described.

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

Directory of Open Access Journals (Sweden)

Gomez Bianca P

2012-10-01

Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

Prostate specific antigen - brief update on its clinical use | Heyns ...

African Journals Online (AJOL)

Prostate specific antigen - brief update on its clinical use. ... (45 years in those with a family history of prostate cancer and – possibly – African men); ... PSA doubling time (the period it takes for the PSA to double) correlates with the prognosis ...

Identification and expression analysis of zebrafish glypicans during embryonic development.

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Mansi Gupta

Full Text Available Heparan sulfate Proteoglycans (HSPG are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

Acanthocheilonema viteae: Vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

International Nuclear Information System (INIS)

Lucius, R.; Textor, G.; Kern, A.; Kirsten, C.

1991-01-01

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3

The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb

OpenAIRE

Cotney, Justin; Leng, Jing; Yin, Jun; Reilly, Steven K.; DeMare, Laura E.; Emera, Deena; Ayoub, Albert E.; Rakic, Pasko; Noonan, James P.

2013-01-01

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find...

Predictive value of prostate specific antigen in a European HIV-positive cohort

DEFF Research Database (Denmark)

Shepherd, Leah; Borges, Álvaro H; Ravn, Lene

2016-01-01

BACKGROUND: It is common practice to use prostate specific antigen (PSA) ≥4.0 ng/ml as a clinical indicator for men at risk of prostate cancer (PCa), however, this is unverified in HIV+ men. We aimed to describe kinetics and predictive value of PSA for PCa in HIV+ men. METHODS: A nested case...... control study of 21 men with PCa and 40 matched-controls within EuroSIDA was conducted. Prospectively stored plasma samples before PCa (or matched date in controls) were measured for the following markers: total PSA (tPSA), free PSA (fPSA), testosterone and sex hormone binding globulin (SHBG). Conditional...... logistic regression models investigated associations between markers and PCa. Mixed models were used to describe kinetics. Sensitivity and specificity of using tPSA >4 ng/ml to predict PCa was calculated. Receiver operating characteristic curves were used to identify optimal cutoffs in HIV+ men for total...

Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development

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Halbach Oliver

2008-10-01

Full Text Available Abstract Background Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate. Results We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased. Conclusion BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

Science.gov (United States)

Gunasekaran, Manojkumar; Chatterjee, Prodyot K.; Shih, Andrew; Imperato, Gavin H.; Addorisio, Meghan; Kumar, Gopal; Lee, Annette; Graf, John F.; Meyer, Dan; Marino, Michael; Puleo, Christopher; Ashe, Jeffrey; Cox, Maureen A.; Mak, Tak W.; Bouton, Chad; Sherry, Barbara; Diamond, Betty; Andersson, Ulf; Coleman, Thomas R.; Metz, Christine N.; Tracey, Kevin J.; Chavan, Sangeeta S.

2018-01-01

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses. PMID:29755449

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

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Manojkumar Gunasekaran

2018-04-01

Full Text Available The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR, dorsal root ganglion (DRG sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM into wild-type (WT mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate.

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Karen G Dowell

Full Text Available Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.

Parent-of-origin dependent gene-specific knock down in mouse embryos

International Nuclear Information System (INIS)

Iqbal, Khursheed; Kues, Wilfried A.; Niemann, Heiner

2007-01-01

In mice hemizygous for the Oct4-GFP transgene, the F1 embryos show parent-of-origin dependent expression of the marker gene. F1 embryos with a maternally derived OG2 allele (OG2 mat /-) express GFP in the oocyte and during preimplantation development until the blastocyst stage indicating a maternal and embryonic expression pattern. F1-embryos with a paternally inherited OG2 allele (OG2 pat /-) express GFP from the 4- to 8-cell stage onwards showing only embryonic expression. This allows to study allele specific knock down of GFP expression. RNA interference (RNAi) was highly efficient in embryos with the paternally inherited GFP allele, whereas embryos with the maternally inherited GFP allele showed a delayed and less stringent suppression, indicating that the initial levels of the target transcript and the half life of the protein affect RNAi efficacy. RT-PCR analysis revealed only minimum of GFP mRNA. These results have implications for studies of gene silencing in mammalian embryos

New developments in the standardization of total prostate-specific antigen.

Science.gov (United States)

Blijenberg, B G; Storm, B N; Van Zelst, B D; Kruger, A E; Schröder, F H

1999-11-01

Analytical evaluation of the calibration of three recently launched assays for the measurement of total prostate-specific antigen, i.e., IMx Total PSA (Abbott), Elecsys PSA (Roche), and IMMULITE 3rd Generation PSA (DPC). For accuracy assessment two reference materials were applied namely, Stanford 90:10 PSA Calibrator and Certified Reference Material 613 Prostate-Specific Antigen. Dilutions of these preparations were analyzed with all assays. In addition, clinical specimens from known prostate cancer or benign prostate hyperplasia patients and samples taken from an ongoing prostate cancer screening study were used for comparison. Application of the Stanford Calibrator revealed results well within 10% of the calculated values for all assays. Regarding the CRM Calibrator only the IMx Total PSA proved to approach the line of identity. The IMMULITE results differed about 40% and the Elecsys about 18% from the calculated values. The comparison with clinical specimens showed statistically different results for the combination IMMULITE-IMx and for IMMULITE-Elecsys. The regression lines for both collections were: y(IMx) = 0.86x(IMMULITE) +0.12 (n = 104, r = 0.970, Sy/x = 0.883 microg/L) and y(Elecsys) = 0.98x(IMMULITE) +0.38 (n = 97, r = 0.976, Sy/x = 0.733 microg/L). In the lower measuring range (PSA differences were less pronounced. In analytical sense a difference was found for both reference preparations in the assays studied. Clinically, despite improvements in methodology, results for total prostate-specific antigen are still not interchangeable. The possible consequences need to be elaborated.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

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Martin Kreutz

Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Pluripotent Stem Cell Studies Elucidate the Underlying Mechanisms of Early Embryonic Development

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Lingyu Li

2011-03-01

Full Text Available Early embryonic development is a multi-step process that is intensively regulated by various signaling pathways. Because of the complexity of the embryo and the interactions between the germ layers, it is very difficult to fully understand how these signals regulate embryo patterning. Recently, pluripotent stem cell lines derived from different developmental stages have provided an in vitro system for investigating molecular mechanisms regulating cell fate decisions. In this review, we summarize the major functions of the BMP, FGF, Nodal and Wnt signaling pathways, which have well-established roles in vertebrate embryogenesis. Then, we highlight recent studies in pluripotent stem cells that have revealed the stage-specific roles of BMP，FGF and Nodal pathways during neural differentiation. These findings enhance our understanding of the stepwise regulation of embryo patterning by particular signaling pathways and provide new insight into the mechanisms underlying early embryonic development.

Identification of immunodominant Leishmania major antigenic markers of the early C57BL/6 and BALB/c mice infection stages.

Science.gov (United States)

Sassi, Atfa; Kaak, Olfa; Elgaaied, Amel Benammar

2015-08-24

The C57BL/6 mouse strain is resistant to Leishmania (L.) major infection and, unlike susceptible BALB/c, develops small self healing cutaneous lesions. The specific antibody responses of C57BL/6 and BALB/c mice were previously characterized by the predominance of IgG2a ("resistant" isotype associated with Th1) and IgG1 ("pathogenic" isotype associated with Th2) antibodies, respectively. In this study, we looked for the presence of antigens able to elicit an exclusive or predominant IgG1 production during the early stages of C57BL/6 lesion development and checked whether they are recognized or not by BALB/c mice. We demonstrate first that IgG2a predominance in C57BL/6 sera occurs only late after infection whereas in BALB/c, IgG1 antibodies dominate mostly in the early stages. Interestingly, soon after inoculation of live amastigotes, C57BL/6 displayed an exclusive IgG1 reactivity against particular L. major antigens but with MWs different from those identified in BALB/c. Furthermore, mice immunized with killed amastigotes displayed striking differences in their immunodetection profiles, particularly for the IgG1 isotype. Taken together, the observed differences in the specific antibody repertoires between infected mice resulted, at least in part, from immunological events independent from those triggered by the replicating parasite, and bring new insights into the selection of future vaccine candidates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Gender-specific differences in cancer-specific survival after radical cystectomy for patients with urothelial carcinoma of the urinary bladder in pathologic tumor stage T4a.

Science.gov (United States)

May, Matthias; Bastian, Patrick J; Brookman-May, Sabine; Fritsche, Hans-Martin; Tilki, Derya; Otto, Wolfgang; Bolenz, Christian; Gilfrich, Christian; Trojan, Lutz; Herrmann, Edwin; Moritz, Rudolf; Tiemann, Arne; Müller, Stefan C; Ellinger, Jörg; Buchner, Alexander; Stief, Christian G; Wieland, Wolf F; Höfner, Thomas; Hohenfellner, Markus; Haferkamp, Axel; Roigas, Jan; Zacharias, Mario; Nuhn, Philipp; Burger, Maximilian

2013-10-01

Bladder cancer (UCB) staged pT4a show heterogeneous outcome after radical cystectomy (RC). No risk model has been established to date. Despite gender-specific differences, no comparative studies exist for this tumor stage. Cancer-specific survival (CSS) of 245 UCB patients without neoadjuvant chemotherapy staged pT4a, pN0-2, M0 after RC were analyzed in a retrospective multi-center study. Seventeen patients were excluded from further analysis due to carcinoma in situ (CIS) of the prostatic urethra and/or positive surgical margins. Average follow-up period was 30 months (IQR: 14-45). The influence of different clinical and histopathologic variables on CSS was determined through uni- and multivariate Cox regression analyses. Two risk groups were generated using factors with independent effect in multivariate models. Internal validity of the prediction model was evaluated by bootstrapping. Eighty-four percent of the patients (n = 192) were male; 72% (n = 165) showed lymphovascular invasion (LVI). The 5-year CSS rate was 31%, and significantly different between male and female (35% vs. 15%, P = 0.003). Multivariate Cox regression modeling, female gender (HR = 1.83, P = 0.008), LVI (HR = 1.92, P = 0.005), and absence of adjuvant chemotherapy (HR = 0.61, P = 0.020) significantly worsened CSS. Two risk groups were generated using these 3 criteria, which differed significantly between each other in CSS (5-year-CSS: 46% vs. 12%, P < 0.001). The c-index value of the risk model was 0.61 (95% CI: 0.53-0.68, P < 0.001). Prognosis in UCB staged pT4a is heterogeneous. Female gender and LVI are adverse factors. Adjuvant chemotherapy seems to improve outcome. The present analysis establishes the first risk model for this demanding tumor stage. Copyright © 2013 Elsevier Inc. All rights reserved.

Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

DEFF Research Database (Denmark)

Ravn, P; Pedersen, B K

1996-01-01

by a defect in the cytotoxic capacity of the individual CD4+ lymphocyte after antigen stimulation, and it could not be explained by a reduction in the total number of CD4+ cells before antigen stimulation. The antigen-specific cytotoxic activity was, however, closely related to the ability of the CD4+ T cells...

Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

Science.gov (United States)

Hus, I; Schmitt, M; Tabarkiewicz, J; Radej, S; Wojas, K; Bojarska-Junak, A; Schmitt, A; Giannopoulos, K; Dmoszyńska, A; Roliński, J

2008-05-01

Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

NARCIS (Netherlands)

Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

2009-01-01

OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

2013-06-14

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

Specificity of antigens on UV radiation-induced antigenic tumor cell variants measured in vitro and in vivo

International Nuclear Information System (INIS)

Hostetler, L.W.; Romerdahl, C.A.; Kripke, M.L.

1989-01-01

The purpose of this study was to determine whether antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants

Generation of Tumor Antigen-Specific iPSC-Derived Thymic Emigrants Using a 3D Thymic Culture System

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Raul Vizcardo

2018-03-01

Full Text Available Summary: Induced pluripotent stem cell (iPSC-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs. iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

International Nuclear Information System (INIS)

Hebishima, Takehisa; Tada, Seiichi; Takeshima, Shin-nosuke; Akaike, Toshihiro; Ito, Yoshihiro; Aida, Yoko

2011-01-01

Highlights: ► To develop effective vaccine, we examined the effects of CO 3 Ap as an antigen carrier. ► OVA contained in CO 3 Ap was taken up by BMDCs more effectively than free OVA. ► OVA-immunized splenocytes was activated by OVA contained in CO 3 Ap effectively. ► OVA contained in CO 3 Ap induced strong OVA-specific immune responses to C57BL/6 mice. ► CO 3 Ap is promising antigen carrier for the achievement of effective vaccine. -- Abstract: The ability of carbonate apatite (CO 3 Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO 3 Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO 3 Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO 3 Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO 3 Ap and OVA-containing alumina salt (Alum), suggesting that CO 3 Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO 3 Ap.

Characterization of the onset of embryonic control and early development in the bovine embryo

International Nuclear Information System (INIS)

Barnes, F.L.

1988-01-01

Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

Ultrasound Backscatter Microscopy Image-Guided Intraventricular Gene Delivery at Murine Embryonic Age 9.5 and 10.5 Produces Distinct Transgene Expression Patterns at the Adult Stage

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Jiwon Jang

2013-11-01

Full Text Available In utero injection of a retroviral vector into the embryonic telencephalon aided by ultrasound backscatter microscopy permits introduction of a gene of interest at an early stage of development. In this study, we compared the tissue distribution of gene expression in adult mice injected with retroviral vectors at different embryonic ages in utero. Following ultrasound image-guided gene delivery (UIGD into the embryonic telencephalon, adult mice were subjected to whole-body luciferase imaging and immunohistochemical analysis at 6 weeks and 1 year postinjection. Luciferase activity was observed in a wide range of tissues in animals injected at embryonic age 9.5 (E9.5, whereas animals injected at E10.5 showed brain-localized reporter gene expression. These results suggest that mouse embryonic brain creates a closed and impermeable structure around E10. Therefore, by injecting a transgene before or after E10, transgene expression can be manipulated to be local or systemic. Our results also provide information that widens the applicability of UIGD beyond neuroscience studies.

Combination of prostate specific antigen and pathological stage regarding to gleason score to predict bone metastasis of newly diagnosed prostate cancer

International Nuclear Information System (INIS)

Wang Zhen; Zhou Liquan; Gao Jiangping; Shi Lixin; Zhao Xiaoyi; Hong Baofa

2004-01-01

To determine the value of tumor grade and serum prostate-specific antigen in predicting skeletal metastases in untreated prostate cancer, the results of bone scans were related retrospectively to levels of serum PSA and tumor Grade based on pathologyical examination in 202 patients with prostate cancer newly diagnosed. Skeletal metastases were present in 7% of patients with serum PSA 100 μg/L. Bone scans are omitted likely in a man newly diagnosed with prostate cancer who has no suggestive clinical features, a serum PSA 100 μg/L. (authors)

Tumor-Associated Antigens for Specific Immunotherapy of Prostate Cancer

Energy Technology Data Exchange (ETDEWEB)

Kiessling, Andrea [Biologics Safety and Disposition, Preclinical Safety, Translational Sciences, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Werk Klybeck, Klybeckstraße 141, Basel CH-4057 (Switzerland); Wehner, Rebekka [Institute of Immunology, Medical Faculty, University of Technology Dresden, Fetscherstraße 74, Dresden 01307 (Germany); Füssel, Susanne [Department of Urology, Medical Faculty, University of Technology Dresden, Fetscherstraße 74, Dresden 01307 (Germany); Bachmann, Michael [Institute of Immunology, Medical Faculty, University of Technology Dresden, Fetscherstraße 74, Dresden 01307 (Germany); Wirth, Manfred P. [Department of Urology, Medical Faculty, University of Technology Dresden, Fetscherstraße 74, Dresden 01307 (Germany); Schmitz, Marc, E-mail: marc.schmitz@tu-dresden.de [Institute of Immunology, Medical Faculty, University of Technology Dresden, Fetscherstraße 74, Dresden 01307 (Germany)

2012-02-22

Prostate cancer (PCa) is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among men in the United States. Effective treatment modalities for advanced metastatic PCa are limited. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches to prevent progression from localized to advanced PCa and to improve survival outcomes for patients with advanced disease. CD8{sup +} cytotoxic T lymphocytes (CTLs) efficiently recognize and destroy tumor cells. CD4{sup +} T cells augment the antigen-presenting capacity of dendritic cells and promote the expansion of tumor-reactive CTLs. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity, activation of the complement system, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Consequently, several tumor-associated antigens (TAAs) have been identified that represent promising targets for T cell- or antibody-based immunotherapy. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. Clinical trials provide evidence that specific immunotherapeutic strategies using such TAAs represent safe and feasible concepts for the induction of immunological and clinical responses in PCa patients. However, further improvement of the current approaches is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy.

Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

Science.gov (United States)

Riedl, Petra; Reiser, Michael; Stifter, Katja; Krieger, Jana; Schirmbeck, Reinhold

2014-07-01

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostate specific antigen and its clinical application

International Nuclear Information System (INIS)

Xu Yang

2000-01-01

Prostate-Specific Antigen (PSA), a serine proteases, is a glycoprotein consisting of a single polypeptide chain. Secreted exclusively by epithelial cells of the prostate gland, PSA is found largely in seminal plasma. Only a small amount of PSA can be found in normal serum. Serum PSA levels are found to be, considerably increased in prostate cancer patients. A number of studies on PSA have made great achievement on its biochemistry, analytical method and clinical application. PSA as one of the most important tumor marker, is used to help diagnosis and monitor the therapeutic efficacy of prostate cancer

Exposure to occupational antigens might predispose to IgG4-related disease

NARCIS (Netherlands)

de Buy Wenniger, Lucas J. Maillette; Culver, Emma L.; Beuers, Ulrich

2014-01-01

Evidence is mounting that the immune system of patients with IgG4-related disease (IgG4-RD) shows indications of chronic antigenic stimulation. Hypothesizing a possible role for occupational antigenic exposure, we observed in two independent cohorts of patients with IgG4-RD that the majority had had

Antigen recognition by IgG4 antibodies in human trichinellosis

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Pinelli E.

2001-06-01

Full Text Available The antibody isotype response to Trichinella spiralis excretory/secretory (ES products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 45 kDa component was strongly recognized by different antibody classes and subclasses. We observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

Prostate-specific antigen and radiation therapy for clinically localized prostate cancer

Energy Technology Data Exchange (ETDEWEB)

Zagars, Gunar K; Pollack, Alan; Kavadi, Vivek S; Eschenbach, Andrew C. von

1995-05-15

Purpose: This study was undertaken to: (a) define the prognostic significance of pretreatment serum prostate-specific antigen (PSA) levels in localized prostate cancer treated with radiation; (b) define the prognostic usefulness of postradiation PSA levels; (c) evaluate the outcome of radiation using PSA as an endpoint. Methods and Materials: Disease outcome in 707 patients with Stages T1 (205 men), T2 (256 men), T3 (239 men), and T4 (7 men), receiving definitive external radiation as sole therapy, was evaluated using univariate and multivariate techniques. Results: At a mean follow-up of 31 months, 157 patients (22%) developed relapse or a rising PSA. Multivariate analysis revealed pretreatment PSA level to be the most significant prognostic factor, with lesser though significant contributions due to Gleason grade (2-6 vs. 7-10) and transurethral resection in (T3(T4)) disease. The following four prognostic groupings were defined: group I, PSA {<=} 4 ng/ml, any grade; group II, 4 < PSA {<=} 20, grades 2-6; group III, 4 < PSA {<=} 20, grades 7-10; group IV, PSA > 20, any grade. Five-year actuarial relapse rates in these groups were: I, 12%; II, 34%; III, 40%; and IV, 81%. Posttreatment nadir PSA was an independent determinant of outcome and only patients with nadir values < 1 ng/ml fared well (5-year relapse rate 20%). Using rising PSA as an endpoint the 461 patients with (T1(T2)) disease had an actuarial freedom from disease rate of 70% at 5 years, which appeared to plateau, suggesting that many were cured. No plateau was evident for (T3(T4)) disease. Conclusion: Pretreatment serum PSA is the single most important predictor of disease outcome after radiation for local prostate cancer. Tumor grade has a lesser though significant prognostic role. Postirradiation nadir PSA value during the first year is a sensitive indicator of response to treatment. Only nadir values < 1 ng/ml are associated with a favorable outlook. A significant fraction of men with (T1(T2

Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

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Yacine Kharraz

Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

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Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

Novel Treponema pallidum Recombinant Antigens for Syphilis Diagnostics: Current Status and Future Prospects

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Aleksey Kubanov

2017-01-01

Full Text Available The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized.

Novel Treponema pallidum Recombinant Antigens for Syphilis Diagnostics: Current Status and Future Prospects.

Science.gov (United States)

Kubanov, Aleksey; Runina, Anastassia; Deryabin, Dmitry

2017-01-01

The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized.

ELISA with double antigen sandwich for screening specific serum anti-TP antibody in blood donors

International Nuclear Information System (INIS)

Wang Yiqing; Shi Zhixu

2002-01-01

Objective: To select a sensitive and specific laboratory examination suitable for screening serum anti-TP antibody in blood donors. Methods: The serum anti-TP antibody in 11271 blood donors were detected using ELISA with double antigen sandwich and the outcomes were compared with those using RPR assay. The conflicting specimen were confirmed by repeating the test with TPHA assay. Results: The positive rates of serum anti-TP antibody by ELISA with double antigen sandwich and RPR was 0.36% (41/11271) and 0.26% (29/11271), respectively. The coincidence of the detecting outcomes by ELISA with double antigen sandwich and RPR with TPHA was 97.5% (40/41) and 63.41%(26/41) respectively. Conclusion: Compared with RPR assay, ELISA with double antigen sandwich has higher sensibility and specificity for screening serum anti-TP antibody in blood donors

Regional differences in expression of specific markers for human embryonic stem cells

DEFF Research Database (Denmark)

Laursen, Steen B; Møllgård, Kjeld; Olesen, Christian

2007-01-01

Characterization of human embryonic stem cell (hESC) lines derived from the inner cell masses of blastocysts generally includes expression analysis of markers such as OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Expression is usually detected by immunocytochemical staining of entire colonies...... of hESC, using one colony for each individual marker. Four newly established hESC lines showed the expected expression pattern and were capable of differentiating into the three germ layers in vitro. Neighbouring sections of entire colonies grown for 4, 11, 21 and 28 days respectively were stained...

An Evaluation of Usefulness of Prostate Specific Antigen and Digital ...

African Journals Online (AJOL)

Objective: To evaluate the usefulness of prostate specific antigen (PSA) and digital rectal examination (DRE) in the diagnosis of cancer of the prostate (CaP) amongst unscreened patients. Patients, Materials ans Methods: A prospective study168 unscreened men who were referred for evaluation for CaP. They all had a ...

Female-biased dimorphism underlies a female-specific role for post-embryonic Ilp7 neurons in Drosophila fertility

Science.gov (United States)

Castellanos, Monica C.; Tang, Jonathan C. Y.; Allan, Douglas W.

2013-01-01

In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila. PMID:23981656

Spatiotemporal expression profile of the Pumilio gene in the embryonic development of silkworm.

Science.gov (United States)

Chen, Liang; You, Zaizhi; Xia, Hengchuan; Tang, Qi; Zhou, Yang; Yao, Qin; Chen, Keping

2014-01-01

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene's involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens.

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Nancy O Duah

2009-10-01

Full Text Available It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types and MSP3 (two allelic types. Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h(2 estimates for 48% (20/42 of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested among the adults, 48% (20/42 among the children in the dry season and 31% (13/42 among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20 of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

DEFF Research Database (Denmark)

Robert-Moreno, Àlex; Robert-Moreno, Àlex; Guiu, Jordi

2008-01-01

Specific deletion of Notch1 and RBPjκ in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult...... to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including...... activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse....

Antibiotics may not decrease prostate-specific antigen levels or prevent unnecessary prostate biopsy in patients with moderately increased prostate-specific antigen levels: A meta-analysis.

Science.gov (United States)

Yang, Lu; Zhu, Yuchun; Tang, Zhuang; Chen, Yongji; Gao, Liang; Liu, Liangren; Han, Ping; Li, Xiang; Wei, Qiang

2015-05-01

To evaluate the effect of empiric antibiotics on decreasing prostate-specific antigen (PSA) levels and the possibility of avoiding unnecessary prostate biopsies (PBs). A systematic search of PubMed, Embase, and the Cochrane Library was performed to identify all randomized controlled trials (RCTs) that compared effects of empiric antibiotics with no treatment or placebo on lowering PSA levels and minimizing unnecessary PBs in patients with moderately increased PSA levels. The Cochrane Collaboration Review Manager software (RevMan 5.1.4) was used for statistical analysis. The inclusion criteria for the study were met by 6 RCTs (1 placebo controlled and 5 no treatment controlled) involving 656 patients. The synthesized data from these RCTs indicated that there were no significant differences between the antibiotic and control groups in the PSA levels after treatment (mean difference [MD] = 0.15, 95% CI:-0.50 to 0.81, P = 0.65], number of patients with decreased PSA levels after treatment (relative risk [RR] = 1.22, 95% CI: 0.90-1.65, P = 0.20], prostate-specific antigen density levels after treatment (MD =-0.04, 95% CI:-0.15 to 0.07, P = 0.47), f/t% PSA after treatment (MD =-1.47, 95% CI:-4.65 to 1.71, P = 0.37), number of patients with responsive PSA (RR = 1.02, 95% CI: 0.58-1.81, P = 0.94), and individual Pca-positiverate in these patients (RR = 1.07, 95% CI: 0.53-2.16, P = 0.86), and Pca-positiverates (RR = 0.85, 95% CI: 0.48-1.50, P = 0.57). However, the antibiotic group had a significant change in the net PSA decrease after treatment compared with the control group (MD = 1.44, 95% CI: 0.70-2.17, P = 0.0001). The use of empiric antibiotics may not significantly decrease PSA levels or avoid unnecessary PBs. Copyright © 2015 Elsevier Inc. All rights reserved.

CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

Science.gov (United States)

Brown, W C; Davis, W C; Dobbelaere, D A; Rice-Ficht, A C

1994-01-01

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

Science.gov (United States)

Baek, Kwang-Hyun; Lee, Heyjin; Yang, Sunmee; Lim, Soo-Bin; Lee, Wonwoo; Lee, Jeoung Eun; Lim, Jung-Jin; Jun, Kisun; Lee, Dong-Ryul; Chung, Young

2012-01-01

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

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Kwang-Hyun Baek

Full Text Available BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Bone scan can be spared in asymptomatic prostate cancer patients with PSA of ≤20 ng/ml and gleason score of ≤6 at the initial stage of diagnosis

International Nuclear Information System (INIS)

Tanaka, Nobumichi; Fujimoto; Kiyohide; Shinkai, Takayuki

2011-01-01

According to several guidelines, it is acceptable to spare a bone scan in the patients who are newly diagnosed with low-risk prostate cancer. Our aim is to clarify a suitable group whereby a bone scan could be spared at the initial staging of prostate cancer. Consecutive 857 patients who were newly diagnosed from 2004 through 2009 and received bone scans using technetium 99m methylene diphosphonate at the initial staging were enrolled. The proportion of positive bone metastases by age distribution, prostate-specific antigen level at diagnosis, Gleason score and clinical T stage were evaluated. Univariate and multivariate logistic regression analyses were performed to identify the predictors of positive bone metastases. Of all 857 patients, 40 patients (4.7%) showed bone metastases. Patients with higher age, prostate-specific antigen level, clinical stage and Gleason score showed significantly higher rate of bone metastases (P 50 ng/ml and the Gleason score ≥4+3 were independent predictors of bone metastases. The incidences of bone metastases in patients with a prostate-specific antigen level of ≤20 ng/ml and Gleason score of ≤6 were reasonably low. Collectively, a bone scan is not necessary as a routine examination for these patients at their initial staging of prostate cancer. (author)

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

Science.gov (United States)

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.