Evaluating various areas of process improvement in an effort to improve clinical research: discussions from the 2012 Clinical Translational Science Award (CTSA) Clinical Research Management workshop.

Emphasis has been placed on assessing the efficiency of clinical and translational research as part of the National Institutes of Health (NIH) goal to "improve human health." Improvements identified and implemented by individual organizations cannot address the research infrastructure needs of all clinical and translational research conducted. NIH's National Center for Advancing Translational Sciences (NCATS) has brought together 61 Clinical and Translational Science Award (CTSA) sites creating a virtual national laboratory that reflects the diversity and breadth of academic medical centers to collectively improve clinical and translational science. The annual Clinical Research Management workshop is organized by the CTSA consortium with participation from CTSA awardees, NIH, and others with an interest in clinical research management. The primary objective of the workshop is to disseminate information that improves clinical research management although the specific objectives of each workshop evolve within the consortium. The fifth annual workshop entitled "Learning by doing; applying evidence-based tools to re-engineer clinical research management" took place in June 2012. The primary objective of the 2012 workshop was to utilize data to evaluate, modify, and improve clinical research management. This report provides a brief summary of the workshop proceedings and the major themes discussed among the participants.

Factors that Affect Integrity of Authorship of Scientific Meeting Abstracts.

BACKGROUND: Strict criteria for manuscript authorship exist to guide decisions on who should be considered an author. Less is known about how authorship for scientific meetings is determined. Our goal was to explore factors that influence decisions about authorship of conference abstracts. METHODS: In 2010, we conducted qualitative focus groups with a stratified sample of 36 trainees, 19 junior faculty, and 11 senior faculty. Focus group transcripts were coded using a coding scheme derived from an initial review of the transcripts and a preliminary theoretical framework, which was based on the literature, anecdotes, and personal experience. RESULTS: We identified 6 themes related to abstract authorship: comparisons with manuscripts; collaboration dynamics; time; experience and professional development; standards for authorship; and funding. We found that: views of abstracts as a lesser form of publication lead to diminished integrity of authorship; trainee inexperience and the dynamics of collaboration adversely influence the integrity of authorship independently of the perceived difference between an abstract and an article; and early communication about authorship appears to increase the integrity of authorship decisions. CONCLUSIONS: Authors do not hold abstracts to the same standard as manuscripts. As such, authorship decisions are frequently inconsistent with authorship criteria pertaining to manuscripts. Such inconsistencies might be improved with stricter institutional rules, clear and consistent authorship guidelines for abstracts submitted to conferences, a requirement that all authors verify their contributions to the abstract, and additional training in the responsible conduct of research.

Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism.

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

Shiga toxin subtypes display dramatic differences in potency.

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

Potent adjuvant activity of cationic liposome-DNA complexes for genital herpes vaccines.

Development of a herpes simplex virus (HSV) vaccine is a priority because these infections are common. It appears that potent adjuvants will be required to augment the immune response to subunit HSV vaccines. Therefore, we evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant in a mouse model of genital herpes. Using a whole-virus vaccine (HVAC), we showed that the addition of CLDC improved antibody responses compared to vaccine alone. Most important, CLDC increased survival, reduced symptoms, and decreased vaginal virus replication compared to vaccine alone or vaccine administered with monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) following intravaginal challenge of mice. When CLDC was added to an HSV gD2 vaccine, it increased the amount of gamma interferon that was produced from splenocytes stimulated with gD2 compared to the amount produced with gD2 alone or with MPL-alum. The addition of CLDC to the gD2 vaccine also improved the outcome following vaginal HSV type 2 challenge compared to vaccine alone and was equivalent to vaccination with an MPL-alum adjuvant. CLDC appears to be a potent adjuvant for HSV vaccines and should be evaluated further.

Antibody-mediated protection against genital herpes simplex virus type 2 disease in mice by Fc gamma receptor-dependent and -independent mechanisms.

The ability of antibody (Ab) to modulate HSV pathogenesis is well recognized but the mechanisms by which HSV-specific IgG antibodies protect against genital HSV-2 disease are not well understood. The requirement for Ab interactions with Fcgamma receptors (FcgammaR) in protection was examined using a murine model of genital HSV-2 infection. IgG antibodies isolated from the serum of HSV-immune mice protected normal mice against HSV-2 disease when administered prior to genital HSV-2 inoculation. However, protection was significantly diminished in recipient mice lacking the gamma chain subunit utilized in FcgammaRI, FcgammaRIII, FcgammaRIV and FcepsilonRI receptors and in normal mice depleted of Gr-1(+) immune cell populations known to express FcgammaR, suggesting protection was largely mediated by an FcgammaR-dependent mechanism. To test whether neutralizing Ab might provide superior protection, a highly neutralizing HSV glycoprotein D (gD)-specific monoclonal antibody (mAb) was utilized. Similar to results with HSV-specific polyclonal IgG, administration of the gD-specific mAb did not prevent initial infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia; however, this protection was independent of host FcgammaR expression and was manifest in mice depleted of Gr-1(+) immune cells. Together, these data demonstrate that substantial Ab-mediated protection against genital HSV-2 disease could be achieved by either FcgammaR-dependent or -independent mechanisms. These studies suggest that HSV vaccines might need to elicit multiple, diverse antibody effector mechanisms to achieve optimal protection.

Comparative analysis of the abilities of Shiga toxins 1 and 2 to bind to and influence neutrophil apoptosis.

Hemolytic-uremic syndrome (HUS), the life-threatening complication following infection by the intestinal pathogen Escherichia coli O157:H7, is due to the ability of the pathogen to produce toxins in the Shiga toxin (Stx) family. Activated neutrophils are observed in HUS patients, yet it is unclear whether Stx exerts a direct effect on neutrophils or whether the toxin acts indirectly. The effect of Stx1 and Stx2 on human neutrophils was examined. Neither Stx1 nor Stx2 altered the rate of neutrophil apoptosis. Minimal binding of either toxin to neutrophils was observed, and the toxin was easily eluted from the cells. Stx1 and Stx2 were found to circulate in the plasma of mice following intravenous injection, and both toxins were cleared rapidly from the blood. Together these results suggest that neither Stx1 nor Stx2 interacts directly with neutrophils.

Commensal bacteria influence Escherichia coli O157:H7 persistence and Shiga toxin production in the mouse intestine.

The presence of commensal flora reduced colonization of Escherichia coli O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with E. coli that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible E. coli.

Clearance of herpes simplex virus type 2 by CD8+ T cells requires gamma interferon and either perforin- or Fas-mediated cytolytic mechanisms.

The T-cell-mediated resolution of herpes simplex virus type 2 (HSV-2) genital infections is not fully understood. In these studies, the mechanisms by which CD8+ T cells clear virus from the genital epithelium were examined. Ovalbumin (OVA)-specific CD8+ T cells from OT-I transgenic mice cleared a thymidine kinase-deficient, ovalbumin-expressing HSV-2 virus (HSV-2 tk- OVA) from the genital epithelium of recipient mice, and clearance was abrogated by in vivo neutralization of gamma interferon (IFN-gamma). Further, CD8+ OT-I T cells deficient in IFN-gamma were unable to clear HSV-2 tk- OVA from the vaginal epithelium. The requirement for cytolytic mechanisms in HSV-2 tk- OVA clearance was tested in radiation chimeras by adoptive transfer of wild-type or perforin-deficient OT-I T cells to irradiated Fas-defective or wild-type recipients. Although a dramatic decrease in viral load was observed early after challenge with HSV-2 tk- OVA, full resolution of the infection was not achieved in recipients lacking both perforin- and Fas-mediated cytolytic pathways. These results suggest that IFN-gamma was responsible for an early rapid decrease in HSV-2 virus titer. However, either perforin- or Fas-mediated cytolytic mechanisms were required to achieve complete clearance of HSV-2 from the genital epithelium.

A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo.

CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to beta2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered.

Nonpathogenic Escherichia coli can contribute to the production of Shiga toxin.

The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

Synaptotagmin I-DeltaC2B. A novel synaptotagmin isoform with a single C2 domain in the bovine adrenal medulla.

Synaptotagmin I is a 65 kDa type 1 membrane glycoprotein found in secretory organelles that plays a key role in regulated exocytosis. We have characterised two forms (long and short) of synaptotagmin I that are present in the bovine adrenal medulla. The long form is a type I integral membrane protein which has two cytoplasmic C2 domains and corresponds to the previously characterised full-length synaptotagmin I isoform. The short-form synaptotagmin I-DeltaC2B has the same structure in the lumenal and transmembrane sequences, but synaptotagmin I-DeltaC2B is truncated such that it only has a single cytoplasmic C2 domain. Analysis of synaptotagmin I-DeltaC2B expression indicates that synaptotagmin I-DeltaC2B is preferentially expressed in the bovine adrenal medulla. However, it is absent from the dense core chromaffin granules. Furthermore, when expressed in the rat pheochromocytoma cell line PC12 bovine synaptotagmin I-DeltaC2B is largely absent from dense core granules and synaptic-like microvesicles. Instead, indirect immunofluorescence microscopy reveals the intracellular location of synaptotagmin I-DeltaC2B to be the plasma membrane.