1.3. Sangro Valley and the Five (Paperless) Seasons: Lessons on Building Effective Digital Recording Workflows for Archaeological Fieldwork

Since 2011 the Sangro Valley Project (Italy) has employed a custom-built paperless recording system with iPads and FileMaker at its core. This paper summarizes the evolution of the project’s paperless system and presents lessons learned during five seasons of use (2011–2015) and during the author’s work with two other projects: the Pompeii Archaeological Research Project: Porta Stabia (Italy), and the Say Kah Archaeological Project (Belize). It identifies problems commonly encountered during the implementation of paperless systems and offers recommendations for avoiding or fixing them. Many of these problems are not unique to projects with digital recording systems, and most difficulties were not technical in nature. Rather, many of the most significant problems arose from integrating workflows. Digital recording systems can streamline fieldwork, improve the quality of data collected in the field, significantly reduce errors and misunderstandings, and facilitate new interpretive approaches, but they require thoughtful preparation and implementation.https://dc.uwm.edu/arthist_mobilizingthepast/1004/thumbnail.jp

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Panel on Paperless Recording and Data Management

Chair: Sebastian Heath, Institute for the Study of the Ancient World Next Steps in Paperless Recording: An Update from the Sangro Valley Project; Christopher F. Motz, University of Cincinnati (Department of Classics) Taking Survey Digital: Implementing a Paperless Workflow on the Eastern Vani Survey; Ryan Hughes, University of Michigan iArchaeology: Explorations in In-Field Digital Data Collection; Kathryn E. DeTore and Rebecca E. Bria, Proyecto de Investigacion Arqueologico Regional Ancash Going Big. Data management strategies for the large scale excavations at Gabii (Italy) Rachel Opitz, Center for Advanced Spatial Technologies, et al

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

<div><p><i>Trypanosoma cruzi</i> infection is controlled but not eliminated by host immunity. The <i>T</i>. <i>cruzi</i> trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against <i>T</i>. <i>cruzi</i> challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for <i>T</i>. <i>cruzi</i> vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.</p></div

CD4⁺ TSaa57-74 (p7)/IA^d- and CD8⁺ TSaa359-367 (TSKd1)/K^d-specific T cell responses are minimally sufficient for induction of protective <i>T</i>. <i>cruzi</i> immunity.

<p>Panel A shows a schematic of the TS consensus protein (all 12–15 active TS subfamily members have at least 90% homology within their catalytic domains), and immunodominant TS CD4 and CD8 epitopes. In panels B and C, BALB/c mice were vaccinated with dendritic cells (DC) pulsed (or not) with this pair of CD4 and CD8 epitopes and later challenged with <i>T</i>. <i>cruzi</i>. BALB/c CD11c⁺ splenic DC were purified from BALB/c mice 2 weeks after injection of 5x10⁶ B16-Flt3L-producing cells and 1 day after i.v. injection of 1μg LPS. 1x10⁶ DC pulsed (or left unpulsed) with 50 μg/ml of the indicated peptides were injected i.v. into groups of naïve BALB/c mice 3 times, 2 weeks apart. Mice were challenged 1 month later with <i>T</i>. <i>cruzi</i> (N = 5 in each of the two control groups and N = 10 in DC+TS peptide group). Both parasitemia (B; 2 weeks post-infection) and mortality (C) were significantly reduced in mice given DC pulsed with both TSaa57-74 (p7) and TSaa359-367 (TSKd1) [*p<0.001 by Mann-Whitney U test (B) and *p<0.01 by 2-tailed Fisher exact and Log-Rank [Mantel-Cox] tests(C)]. Survival results are representative of 3 independent experiments.</p

The WT DNA vaccine induces CD8⁺ T cell responses directed against both immunodominant and subdominant T cell epitopes.

<p>BALB/c mice were vaccinated i.m. twice, two weeks apart with 100μg of WT TS DNA or TSKd1 null TS DNA. Four weeks later, CD8⁺ splenic T cells from these vaccinated mice were stimulated overnight in IFN-γ ELISPOT assays with APC (A20 cells) pulsed with TS peptides predicted to bind BALB/c MHC [H2-K^d (A), H2- D^d (B) and H2-L^d(C). Results are representative of 3 experiments. As expected, vaccination of mice with WT but not TSKd1 null TS DNA constructs elicited T cell responses to TSKd1. CD8⁺ T cells from WT TS DNA vaccinated mice also responded to TS peptides Kd2, Kd5, Kd6, Kd7, Kd8, Kd9, Dd2, Dd6, Ld1 and Ld2, while cells from TSKd1 null vaccinate mice responded to only TS Kd8. Mutation of the 2 binding residues of TSKd1 to H2-Kd1 resulted in a marked immunofocusing of CD8⁺ T cell responses. Panel D further shows that WT TS DNA vaccination induced higher levels of TS-specific antibody, compared with TSKd1 null DNA vaccination.</p

TS enzymatic activity is associated with costimulatory effects.

<p>In panel A, enzymatic activities of WT and TSKd1 mutant proteins were determined in lysates and supernatants of 293T cells transfected with WT TS and TSKd1 null DNA using fluorometric TS enzymatic activity assays. Panel B shows the co-stimulatory properties of WT rTS and TSKd1 null rTS on naïve CD8⁺ T cells. CD8⁺ T cells from naïve 4–5 week old BALB/c mice were purified by positive magnetic bead selection and incubated with suboptimal doses of PMA (12.5ng/ml) ± WT or TSKd1 null rTS. After 3 days, proliferation was measured by ³H-Thymidine incorporation. Shown are incorporated ³H-Thymidine counts per minute (CPM) above suboptimal PMA treatment alone. Similar TS costimulation assays using naïve CD8⁺ T cells were conducted with CD45 inhibitor PTP or Src-family kinase inhibitor PP2 added (C). In panel D, increasing concentrations of TSKd1 peptide were added to constant amounts of WT TS in TS enzyme assays. Results shown are representative of 2–4 independent experiments.</p

TSKd1 tolerization during WT TS DNA vaccination reduces CD8⁺ T cell responses directed against both immunodominant and subdominant TS epitopes.

<p>BALB/c mice were injected with tERK-1 control or TSKd1 peptide i.v. on indicated days before and after i.m. vaccination with WT TS DNA followed by <i>T</i>. <i>cruzi</i> challenge as shown in panel A. One month following the final immunization, CD8⁺ splenic T cells from representative mice were stimulated with APC (A20) pulsed with TS peptides in overnight IFN-γ ELISPOT assays (B). Results are representative of 2 independent experiments. Other groups of tolerized TS DNA vaccinated mice were challenged with 5,000 <i>T</i>. <i>cruzi</i> BFT and survival monitored (C; 10–12 mice/group; p<0.05 comparing TS DNA vaccinated tolerized with TSKd1 or the tERK-1 peptide by Fisher exact tests).</p

Identification of <i>trans</i>-sialidase MHC class I-restricted T cell epitope(s).

<p>TSaa359-367 (TSKd1) has been observed to be an immunodominant epitope during both TS vaccination and <i>T</i>. <i>cruzi</i> infection. We utilized a consensus immunoinformatic approach using several MHC prediction tools to identify other TS sequences predicted to bind BALB/c MHC (H2-K^d, H2-D^d, and H2-L^d, shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005896#ppat.1005896.s001" target="_blank">S1 Table</a>). APC (A20) were pulsed with these synthetic peptides and used to stimulate purified splenic CD8⁺ T cells from naïve (A), WT TS DNA-vaccinated (B), and <i>T</i>. <i>cruzi</i> “Infection Memory” mice (C) in overnight IFN-γ ELISPOT assays. Infection Memory (C) was induced by multiple, virulent Tulahuen strain <i>T</i>. <i>cruzi</i> challenges, resulting in mice with potently protective T cell immunity directed against a variety of parasite antigens and epitopes. Nearly all of the predicted peptides elicited memory T cell IFN-γ responses in mice previously infected with <i>T</i>. <i>cruzi</i> and mice vaccinated with WT TS DNA. Results are representative of 3 independent experiments.</p