Rapid construction of a dendritic cell vaccine through physical perturbation and apoptotic malignant T cell loading

We have demonstrated that adherence and release of monocytes from a plastic surface drives their differentiation into immature dendritic cells (DC,) that can mature further during overnight incubation in the presence of apoptotic malignant T cells. Based on these results, we sought to develop a clinically, practical, rapid means for producing DC loaded with malignant cells. A leukapheresis harvest containing the clonal, leukemic expansion of malignant CD4(+ )T cells was obtained from the blood of patients with cutaneous T cell lymphoma (CTCL). CTCL cells were purified with a CD3-magnetic bead column where CD3 engagement rendered the malignant T cells apoptotic. The monocyte fraction was simultaneously activated by column passage, re-added to the apoptotic CTCL cells and co-cultured overnight. CTCL cell apoptosis, DC differentiation and apoptotic malignant T cell ingestion were measured by immunostaining. The results demonstrate that as monocytes passed through the column matrix, they became activated and differentiated into semi-mature DC expressing significantly increased levels of class II, CD83 and CD86 (markers associated with maturing DC) and reduced expression of the monocyte markers CD14 and CD36. Apoptotic malignant T cells were avidly engulfed by the phagocytic transitioning DC. The addition of supportive cytokines further enhanced the number of DC that contained apoptotic malignant T cells. Functional studies confirmed that column passaged DC increased class II expression as shown by significantly enhanced stimulation in mixed leukocyte culture compared to control monocytes. In addition, DC loaded with apoptotic CTCL cells stimulated an increase in the percentage and absolute number of CD8 T cells compared to co-cultivation with non-loaded DC. After CD8 T cells were stimulated by DC loaded with malignant cells, they mediated increased apoptosis of residual CTCL cells and TNF-α secretion indicating development of enhanced cytolytic function. We report a simple one-step procedure where maturing DC containing apoptotic malignant T cells can be prepared rapidly for potential use in vaccine immunotherapy. Ready access to both the DC and apoptotic cells provided by this system will allow extension to other malignancies through the addition of a variety of apoptotic tumor cells and maturation stimuli

Improved generation of anti-tumor immunity by antigen dose limitation

BACKGROUND: The malignant cells of cutaneous T cell lymphoma (CTCL) display immunogenic peptides derived from the clonal T cell receptor (TCR) providing an attractive model for refinement of anti-tumor immunization methodology. To produce a clinically meaningful anti-tumor response, induction of cytotoxic anti-CTCL cells must be maximized while suppressive T regulatory cells (Treg) should be minimized. We have demonstrated that engulfment of apoptotic CTCL cells by dendritic cells (DC) can lead to either CD8 anti-CTCL responses or immunosuppressive Treg induction. Treg generation is favored when the number of apoptotic cells available for ingestion is high. METHODS: In this study, we sought to determine whether the balance between immunity and immunosuppression could be shifted towards a CD8 anti-CTCL response by lowering the ratio of apoptotic CTCL cells available for DC ingestion. CTCL cell apoptosis was produced by engagement of the TCR by anti-CD3 antibody affixed to magnetic beads. RESULTS: The physical perturbation inherent in passage through a separation column induced monocytes to differentiate into DC, demonstrated by increased expression of class II and CD86 and decreased expression of the monocyte marker CD14. The immature DC internalized and processed apoptotic CTCL cells and could potentially present the tumor-derived peptides in the context of MHC class I and II. As the number of apoptotic cells increased, there was a dose-dependent increase in the expression of Treg markers CTLA-4, CD25, and FoxP3, with a ratio of apoptotic cell/DC loading of > 10:1 corresponding to the greatest Treg induction. These inducible phenotypic Treg also functionally inhibited CD8-mediated perforin expression in vitro. At lower levels of apoptotic cell/DC loading of < 5:1, there was an expansion of the CD8 T cell compartment with increased perforin expression and increased CTCL cell death, indicating anti-tumor activity. CONCLUSION: These findings demonstrate that the ratio of apoptotic cells supplied to DC is an important determinant of whether CD8 anti-tumor immunity or immunosuppression is generated

Meta-analysis of studies on biochemical marker tests for the diagnosis of premature rupture of membranes: comparison of performance indexes

BACKGROUND: Premature rupture of the membranes (PROM) is most commonly diagnosed using physical examination; however, accurate decision making in ambiguous cases is a major challenge in current obstetric practice. As this may influence a woman's subsequent management, a number of tests designed to assist with confirming a diagnosis of PROM are commercially available. This study sought to evaluate the published data for the accuracy of two amniotic fluid-specific biomarker tests for PROM: insulin-like growth factor binding protein-1 (IGFBP-1 - Actim® PROM) and placental alpha microglobulin-1 (PAMG-1 - AmniSure®). METHODS: Main analysis included all PubMed referenced studies related to Actim® PROM and AmniSure® with available data to extract performance rates. To compare accuracy, a comparison of pooled indexes of both rapid tests was performed. Studies in which both tests were used in the same clinical population were also analysed. Membrane status, whether it was known or a suspected rupture, and inclusion or not of women with bleeding, were considered. RESULTS: All the available studies published in PubMed up to April 2013 were reviewed. Data were retrieved from 17 studies; 10 for Actim® PROM (n = 1066), four for AmniSure® (n = 1081) and three studies in which both biomarker tests were compared directly. The pooled analysis found that the specificity and positive predictive value were significantly higher for AmniSure® compared with Actim® PROM. However, when 762 and 1385 women with known or suspected rupture of membranes, respectively, were evaluated, AmniSure® only remained significantly superior in the latter group. Furthermore, when the two tests were compared directly in the same study no statistically significant differences were observed. Remarkably, women with a history or evidence of bleeding were excluded in all four studies for AmniSure®, in two Actim® PROM studies and in two of the three studies reporting on both tests. CONCLUSIONS: No differences were observed in the performance of the two tests in studies where they were used under the same clinical conditions or in women with known membrane status. Although AmniSure® performed better in suspected cases of PROM, this may need further analysis as exclusion of bleeding may not be representative of the real clinical presentation of women with suspected PROM

Second Generation Voices: The Pleasures and Afflictions of Inherited Exilic Legacy

The Exile Studies Program In Collaboration with The Betsy-South Beach Hotel The Department of English & The College of Arts, Sciences & Education Presents Panel Discussion Second Generation Voices: The Pleasures and Afflictions of Inherited Exilic Legacyhttps://digitalcommons.fiu.edu/cri_events/1379/thumbnail.jp

Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits

The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly—the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments

Survival of Batrachochytrium dendrobatidis in Water: Quarantine and Disease Control Implications

Amphibian chytridiomycosis is an emerging infectious disease of amphibians thought to be moved between countries by trade in infected amphibians. The causative fungus, Batrachochytrium dendrobatidis, produces aquatic, motile zoospores; infections have been achieved in experiments by exposing amphibians to water containing zoospores. However, the ability of this fungus to survive in the environment in the absence of an amphibian host is unknown. We show that B. dendrobatidis will survive in tap water and in deionized water for 3 and 4 weeks, respectively. In lake water, infectivity was observed for 7 weeks after introduction. The knowledge that water can remain infective for up to 7 weeks is important for the formulation of disease control and quarantine strategies for the management of water that has been in contact with amphibians

Lethal Injection, Politics, and the Future of the Death Penalty

“Welcome and Keynote:” Stephen Bright, Harvey Karp Visiting Lecturer at Yale Law School, and President and Senior Counsel with the Southern Center for Human Rights. (9:00 a.m. - 9:45 a.m.) “The Death Penalty Today: Lethal Injection Issues:” Panel 1 featured Deborah W. Denno, Arthur A. McGivney Professor of Law at Fordham University School of Law; Joel Zivot, Assistant Professor of Anesthesiology and Surgery at Emory University School of Medicine, and Medical Director of the Cardiothoracic Intensive Care Unit at Emory University Hospital; Eric Berger, Associate Professor of Law at Nebraska College of Law; and Frank Green, Reporter for the Richmond Times-Dispatch. Jim Gibson, Associate Dean for Student Affairs and Professor of Law at the University of Richmond School of Law, served as moderator. (10:00 a.m. -11:30 a.m.) “The Shifting Politics of the Death Penalty:” Panel 2 featured Mark Earley, former Attorney General of Virginia; Richard B. Roper, Partner with Thompson & Knight LLP, Corinna Barrett Lain, Associate Dean for Faculty Development and Professor of Law at the University of Richmond School of Law; and Stephen Smith, Professor of Law at Notre Dame Law School. Henry L. Chambers, Professor of Law at the University of Richmond School of Law, served as moderator. (1:00 p.m. - 2:30 p.m.) “The Future of the Death Penalty:” Panel 3 featured John Douglass, Professor of Law at the University of Richmond School of Law; Brandon L. Garrett, Professor of Law at the University of Virginia School of Law; and Richard Dieter, Executive Director of the Death Penalty Information Center. Mary Kelly Tate, Professor of Law at the University of Richmond School of Law, served as moderator. (2:45 p.m. - 4:15 p.m.

Radio Emission from SN 1994I in NGC 5194 (M 51) - The Best Studied Type Ib/c Radio Supernova

We present the results of detailed monitoring of the radio emission from the Type Ic supernova SN 1994I from 3 days after optical discovery on 1994 March 31 until eight years later at age 2927 days on 2002 April 05. The data were mainly obtained using the Very Large Array at the five wavelengths, 1.3, 2.0, 3.6, 6.2, and 21 cm, and from the Cambridge 5 km Ryle Telescope at 2.0 cm. Two additional measurements were obtained at millimeter wavelengths. This data set represents the most complete, multifrequency radio observations ever obtained for a Type Ib/c supernova. The radio emission evolves regularly in both time and frequency and is well described by established SN emission/absorption models. It is the first radio supernova with sufficient data to show that it is clearly dominated by the effects of synchrotron self-absorption at early times.Comment: 43 pages, 5 figure

Geochemistry of Carbonates on Mars: Implications for Climate History and Nature of Aqueous Environments

Ongoing research on martian meteorites and a new set of observations of carbonate minerals provided by an unprecedented series of robotic missions to Mars in the past 15 years help define new constraints on the history of martian climate with important crosscutting themes including: the CO_2 budget of Mars, the role of Mg-, Fe-rich fluids on Mars, and the interplay between carbonate formation and acidity. Carbonate minerals have now been identified in a wide range of localities on Mars as well as in several martian meteorites. The martian meteorites contain carbonates in low abundances (<1 vol.%) and with a wide range of chemistries. Carbonates have also been identified by remote sensing instruments on orbiting spacecraft in several surface locations as well as in low concentrations (2–5 wt.%) in the martian dust. The Spirit rover also identified an outcrop with 16 to 34 wt.% carbonate material in the Columbia Hills of Gusev Crater that strongly resembled the composition of carbonate found in martian meteorite ALH 84001. Finally, the Phoenix lander identified concentrations of 3–6 wt.% carbonate in the soils of the northern plains. The carbonates discovered to date do not clearly indicate the past presence of a dense Noachian atmosphere, but instead suggest localized hydrothermal aqueous environments with limited water availability that existed primarily in the early to mid-Noachian followed by low levels of carbonate formation from thin films of transient water from the late Noachian to the present. The prevalence of carbonate along with evidence for active carbonate precipitation suggests that a global acidic chemistry is unlikely and a more complex relationship between acidity and carbonate formation is present

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

SummaryPrimases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. How DnaG associates with nucleotide substrates and how certain naturally prevalent nucleotide analogs impair DnaG function are unknown. We have examined one of the earliest stages in primer synthesis and its control by solving crystal structures of the S. aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates or the nucleotidyl alarmones, pppGpp and ppGpp. These structures, together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, pinpoint the predominant nucleotide-binding site of DnaG and explain how the induction of the stringent response in bacteria interferes with primer synthesis