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.

Effective clinical and translational research is an essential component of improving human health as delineated in the National Institutes of Health (NIH) roadmap,1, 2 yet efficiently conducting that research remains problematic.3, 4 Challenges include the high costs of conducting research, a shortage of qualified investigators,5 low subject enrollment rates,6, 7 slow dissemination of results,8 reduced funding,3 lagging IT systems,9 and increased regulatory burden.10, 11 A priority for the National Center for the Advancement of Translational Science (NCATS), and its Clinical Translational Science Award (CTSA) Consortium, is to address this last concern regarding regulatory burden. Specifically the focus is on the improvement of Institutional Review Board (IRB) processes; especially novel models for regulatory review of multicenter clinical and translational research studies. The three Ohio CTSA sites, housed at Case Western Reserve University (CWRU) in Cleveland, The Ohio State University (OSU) in Columbus, and the University of Cincinnati (UC), have collaborated to create a novel IRB review concept with the hopes of addressing this concern and achieving a more effective and efficient IRB review of multicentered clinical research studies. To date a number of different methods have been used to streamline IRB review of multisite studies. The central IRB model, where a “for-profit,” independent IRB (e.g., Western or Chesapeake) acts as the IRB of record for regulatory review processes, is commonly used for a large number of industry initiated and sponsored clinical trials. Federated IRBs are another type of review model in which one existing IRB within a network of sites serves as the IRB of Record for studies conducted within that network (e.g., Harvard Catalyst is the IRB of Record for all studies within the National Institute of Neurological Disorders and Stroke (NINDS) NeuroNext network). A simpler and less formalized model of reciprocal review involves the use of individual “Institutional IRB Authorizations” (IIA) between collaborating organizations whereby one organization can “authorize” a second organization's IRB to act as the IRB of Record for one or more studies conducted at their organization. In an effort to create a “hybrid” model, the IRB Administration Offices within the Clinical Translational Science Collaborative (CTSC) based at CWRU and including University Hospitals Case Medical Center, MetroHealth Medical Center and the Cleveland Clinic Foundation, moved from a type of bilateral, inter-institutional agreement to a broader agreement amongst partners allowing the lead CTSC site to act as the IRB of Record for a multisite clinical research protocol conducted at any of the partner CTSC sites in late 2009. The CTSC named this process “facilitated review” as this model allowed for any participating IRB to serve as the IRB of Record for protocols conducted by any combination of participating sites while at the same time allowing each site to retain local context review and oversight. More importantly, to address the fact of disparate electronic IRB review systems, the CTSC IRB Advisory Group developed a centralized electronic IRB hub as the main point of interface for each application to further promotes efficiency in IRB review processes while facilitating document storage and communication across research sites amongst CTSC regulatory administrators and investigators alike. In June 2011, 18 months after implementation of the initial facilitated review process and IRB electronic “hub,” reports of the CTSC facilitated review model, and information related to use and successes within the CWRU CTSC were presented at the annual Clinical Research Management Workshop sponsored by the CTSA consortium. Three months later, use of this type of model received significant support by the political leadership in the State of Ohio when the Governor of Ohio and the leader of “JobsOhio” (an economic growth engine for job creation) convened a meeting focused on leveraging healthcare and medical strengths in Ohio to catalyze industry interest and spur economic growth. One result of that meeting was a three-prong charge from the governor to the three Ohio CTSA sites to collaboratively develop Ohio as a recognized destination for clinical research, to foster more efficiencies within the translational research process, and to attract more pharmaceutical and device industries with the goal being improved human health.12, 13 The partner organizations that comprise the Ohio CTSA consortium (Table 1) discussed ways to collaborate shortly after initial discussions with the governor and conceptualized full IRB collaboration. The group immediately followed up with a face-to-face meeting (October 2011) where the CTSC “facilitated review” process and IRB electronic “hub” platform were demonstrated. Following that discussion, the CTSAs across Ohio agreed to move towards the reliant model of IRB review. Members of the Ohio CTSA Consortium IRB Working Group agreed that each institution within the collaborative would participate and exercise authority of the proposed “reliant” IRB review model when investigator collaboration existed among one or more of the Ohio CTSA institutions (i.e., investigator synergy is the starting point). Using current partnerships within the group (the Cleveland CTSC, the IAA between Cincinnati Children's Hospital Medical Center and the University of Cincinnati, and the IRB reciprocity agreement between The Ohio State University and Nationwide Children's Hospital.) as well as other models of collaborative review (Harvard Catalyst Reciprocity Agreement) as a foundation for legal agreement discussions, a decision was made to execute a single a single IIA signed by all eight participating institutions to clarify and solidify expectations. The total timeframe for the Ohio CTSA Consortium IRB Working Group to achieve a fully executed agreement was approximately 9 months. Much was accomplished in this timeframe without wavering from the ultimate goal of reduction of regulatory burden for investigators through reliant IRB review. Specific requirements of the reliance model include that “each institution agrees to maintain a registered IRB and an OHRP approved Federalwide Assurance (FWA) for human subject research.” In order to serve as the “designated IRB of Record” for State of Ohio consortium-wide projects, the designated IRB of Record's institution must have achieved AAHRPP or other accepted accreditation.” That IRB will then perform the ”initial and continuing review and review of amendments; unanticipated problems that may involve risks to subjects or others; and other documents/information related to the approval and continuing oversight of the research (as applicable).” The quotations emanate from the executed IIA. The relying institutions within the Ohio CTSC accept the IRB of Record's review, approval, and continuing oversight of research covered by the IAA, but retain primary and ultimate responsibility for the protection of human subjects with respect to the conduct of the research covered by this agreement and agree to comply with applicable federal, state and local laws and applicable FWA requirements; as well as to review and manage appropriate education and conflict of interest requirements and any resolutions thereof, and to communicate any events or actions affecting that institution's compliance to the designated IRB of Record. A “reliant IRB” workflow, initially adopted from the CTSC's “facilitated review model” initiative has now been extensively modified and access expanded for more broadly to the Ohio CTSA Consortium (Figure 1). Each participating institution in this model is part of an Academic Medical Center (AMC) and a key driver of such organizations is investigator-initiated clinical research resulting in the focus on executing that part of the institutional mission for each entity in this model. The agreements and the process flow can also be used when and where applicable for industry, foundation, and other sponsored project research activity in order to reduce redundant reviews or improve efficiencies. The strength of the agreement is in its simplicity, as it is strictly focused on IRB activities and responsibilities as defined by Human Subject Protection Regulations and promulgated by the Department of Health and Human Services and the US Food and Drug Administration described in 45 CFR 46 and 45 CFR 50 and 56. It does not extend into additional institutional specific human research protection plan requirements beyond those required for FWA and/or AAHRPP accreditation, it does not address grants administration, nor does it delve into HIPAA requirements and conflicts of interest. The purpose of focusing on only IRB related matters was to ensure that other institutional processes were not altered by IRB collaboration. Each institution is also responsible for managing budgets and contracts as such differs between public and private institutions. The process flow of this model is being refined, but this is an encouraging example that each of these AMCs has been able to agree on basic principles and simplified legal approaches to solve long standing regulatory challenges for conducting multisite clinical research. The model will continue to be refined and tested throughout Ohio. With the continued input and collaboration of its originators, our goal is to expand across the national CTSA consortium, and to also include non-CTSA sites. We anticipate that the economic impact of this regulatory initiative will be positive, accelerating the initiation of clinical and translational research at participating institutions. Metrics are currently being gathered that will quantify protocol review and approval times; and whether use of this approach and electronic system has shortened study timelines. These improvements will likely result in ancillary successes including increased study revenues, increased job opportunities for research coordinators, study nurses and other allied research professionals in regions where institutions participate, increased subject enrollment as patients travel to regulatory efficient sites for access to innovative trials not open for enrollment (or not available) elsewhere; increased clinical volumes due to stimulated clinical trial activity, and finally the reduction of IRB regulatory burden will move innovative treatments more efficiently to the practice of medicine. The authors wish to acknowledge the collaborative efforts of all of the members of the Ohio CTSA Consortium's IRB working group: Jeannie Bailey, Daniel Beyer, Jeremy Corsmo, Debbie Fine, Anthony Gardner, Karen Hale, Missi Hart-Kothari, Kathleen Lawry, Sandra Meadows, Karya Ottey, Ginger Pomiecko, Isabel Sanchez, Kim Volarcik, and Erin Zaletel; without which this model would not exist. Finally, thanks to the Harvard Catalyst for providing their Reciprocity Agreement for review during this process. Efforts of the authors were supported by the National Center for the Advancement of Translational Science (NCATS) through the following grants UL1 TR000077, 8UL1TR000090-05 and UL1TR000439.

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.

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.

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.

This study is focused on the development and evaluation of transdermal delivery of E. coli-specific T4 bacteriophages both ex-vivo and in-vivo using microemulsion as delivery carrier in eradicating the infection caused by E. coli. Microemulsions were prepared by mixing selected oil, surfactants and aqueous phase containing bacteriophages. The formulations were subjected to physicochemical characterization, ex-vivo and in-vivo permeation, stability studies, histological and immunofluorescence examination. The colloidal system exhibits a uniform size distribution, of finite size (150–320 nm). Transmission electron microscopy revealed the encapsulation of bacteriophage in the aqueous globule. Ex-vivo permeation across skin was successfully achieved as 6 × 106 PFU/mL and 6.7 × 106 PFU/mL of T4 permeated from ME 6% and 10%, respectively. ME 6% was found to be thermodynamically stable and in-vivo permeation resulted in 5.49 × 105 PFU/mL of bacteriophages in the blood of the E. coli challenged rats, while 2.48 × 105 PFU/mL was detected in germ free rats, at the end of the study. Infected rats that were treated with bacteriophage were survived while significant mortality was observed in others. Histological and IL-6 immunofluorescence examination of the tissues revealed the efficacy/safety of the therapy. The microemulsion-based transdermal delivery of bacteriophage could be a promising approach to treat the infections caused by antibiotic-resistant bacteria.

Abstract The requirement for Ab -Fcγ receptor (FcγR) interactions or for virus neutralization in protection against genital HSV-2 challenge was examined. Serum IgG Ab isolated from HSV-immune mice protected normal mice against HSV-2 disease when administered prior to challenge. However, protection was significantly diminished in mice lacking the γ chain subunit utilized in FcγRI, FcγRIII, FcγRIV, and FcεRI and in normal mice depleted of FcγR+, Gr-1+ immune cells suggesting protection was largely mediated by an FcγR-dependent mechanism. To test if FcγR-independent antibody-mediated mechanisms might manifest protection differently, a highly neutralizing, HSV glycoprotein D -specific monoclonal antibody (mAb) was utilized. Administration of IgG1, IgG2a, or IgG2b switch variants of the mAb did not prevent 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 independently of host FcγR expression. Together, these data demonstrate two distinct antibody effector mechanisms capable of providing substantial protection against genital HSV-2 disease. The presence of either FcγR -dependent Ab or strongly neutralizing Ab did not completely prevent HSV-2 infection but limited initial infection of genital and neuronal tissues and diminished HSV-2 disease. NIH Grants AI42815 and AI054444.

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.

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 Fcγ receptors (FcγR) 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 FcγRI, FcγRIII, FcγRIV and FcepsilonRI receptors and in normal mice depleted of Gr-1+ immune cell populations known to express FcγR, suggesting protection was largely mediated by an FcγR-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 FcγR 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 FcγR-dependent or -independent mechanisms. These studies suggest that HSV vaccines might need to elicit multiple, diverse antibody effector mechanisms to achieve optimal protection.

ABSTRACT 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.

ABSTRACTThe 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-γ). Further, CD8+OT-I T cells deficient in IFN-γ 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-γ 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.

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 β2-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.

ABSTRACT 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 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.

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc). Incubation of Hc-infected Mo with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH. In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7. Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy. Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes. Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early "bulk flow" endocytic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.