Quantitative and Qualitative Analysis of the Impact of Adoption of a Mobile Application for the Assessment of Professionalism in Medical Trainees.

PURPOSE: Capturing either lapses or excellence in behaviors related to medical professionalism is difficult. The authors report a mixed-methods analysis of a novel mobile platform for assessing medical professionalism in a training environment. METHOD: A mobile Web-based platform to facilitate professionalism assessment in a situated clinical setting (Professional Mobile Monitoring of Behaviors [PROMOBES]) was developed. A professionalism framework consisting of six domains (reliability, adaptability, peer relationships, upholding principles, team relationships, and scholarship) encompassing 25 subelements underpins the reporting structure. This pilot study involved 26 faculty supervising 93 medical trainees at two sites from January 12 to August 8, 2016. Notable professionalism behaviors were linked to the framework domains and elements; narrative details about incidences were captured on mobile devices. Surveys gauged the technological functionality and impact of PROMOBES on faculty assessment of professionalism. Qualitative focus groups were employed to elucidate user experience. RESULTS: Although users anticipated PROMOBES's utility would be for reporting lapses in professionalism, 94.7% of reports were for commendation. Comfort assessing professionalism (P = .04) and recognition of the reporting procedures for professionalism-related concerns (P = .01) improved. PROMOBES attained high acceptance ratings. Focus group analysis revealed that the explicit connection to the professionalism framework was powerful; similarly, the near real-time reporting capability, multiple observer inputs, and positive feedback facilitation were strengths. CONCLUSIONS: Making the professionalism framework visible and accessible via a mobile platform significantly strengthens faculty knowledge and behaviors regarding assessment. The strong desire to capture positive behaviors was an unexpected finding.

Skin infections among US high school athletes: A national survey.

BACKGROUND: Skin infections have long been a reported problem among high school athletes, particularly wrestlers. There has yet to be a national study describing the epidemiology of skin infections across multiple high school sports. OBJECTIVE: We sought to report the epidemiology of skin infections among US high school athletes. METHODS: High school sports-related skin infections resulting in time loss were reported by a convenience sample of US high schools from 2009/2010 through 2013/2014 via High School Reporting Information Online. RESULTS: During the study, 474 skin infections were reported among 20,858,781 athlete exposures, a rate of 2.27 per 100,000 athlete exposures. The largest number of skin infections occurred in wrestling (73.6%) followed by football (17.9%). The most common infections were bacterial (60.6%) and tinea (28.4%) infections. Body parts most often affected were the head/face (25.3%) followed by the forearm (12.7%). LIMITATIONS: The study included only high schools with National Athletic Trainers' Association-affiliated athletic trainers, which may limit generalizability. However, using athletic trainers as data reporters improved data quality. CONCLUSIONS: Skin infections are an important subset of high school sports-related adverse events. An understanding of the epidemiology of sports-related skin infections should promote awareness and drive evidence-based prevention efforts.

Vaccination with human papillomavirus pseudovirus-encapsidated plasmids targeted to skin using microneedles.

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.

Use of the NBME Comprehensive Basic Science Examination as a progress test in the preclerkship curriculum of a new medical school.

In the present study, we describe the innovative use of the National Board of Medical Examiners (NBME) Comprehensive Basic Science Examination (CBSE) as a progress test during the preclerkship medical curriculum. The main aim of this study was to provide external validation of internally developed multiple-choice assessments in a new medical school. The CBSE is a practice exam for the United States Medical Licensing Examination (USMLE) Step 1 and is purchased directly from the NBME. We administered the CBSE five times during the first 2 yr of medical school. Student scores were compared with scores on newly created internal summative exams and to the USMLE Step 1. Significant correlations were observed between almost all our internal exams and CBSE scores over time as well as with USMLE Step 1 scores. The strength of correlations of internal exams to the CBSE and USMLE Step 1 broadly increased over time during the curriculum. Student scores on courses that have strong emphasis on physiology and pathophysiology correlated particularly well with USMLE Step 1 scores. Student progress, as measured by the CBSE, was found to be linear across time, and test performance fell behind the anticipated level by the end of the formal curriculum. These findings are discussed with respect to student learning behaviors. In conclusion, the CBSE was found to have good utility as a progress test and provided external validation of our new internally developed multiple-choice assessments. The data also provide performance benchmarks both for our future students to formatively assess their own progress and for other medical schools to compare learning progression patterns in different curricular models.

Sustained impact of short-term international medical mission trips: resident perspectives.

PURPOSE: To assess perspectives of residents: (1) who participated in short-term international medical mission trips (STIMMTs) as medical students regarding impact of the experiences on their professional development; and (2) who did not participate in STIMMTs regarding barriers to participation. METHODS: Three hundred seventy-nine residents from 16 programs at two Florida institutions completed surveys requesting Participant and Trip Details and Impact of Participation (including items rating learning, cultural competency, and social responsibility). RESULTS: One hundred thirty-one residents participated in at least one STIMMT. They identified improved adaptability to new healthcare settings, communication with patients and professionals from different backgrounds, and appreciation for the impact of culture on health as positive outcomes. Leading barriers to STIMMT participation included cost, timing, and lack of availability. CONCLUSIONS: Years after participation in STIMMTs, residents perceived sustained benefits in cultural competency, communication skills, adaptability, and desire for service. Institutions may consider facilitating STIMMTs as one way to address standards specified by accrediting authorities to provide training in cultural competency, social responsibility, altruism, and understanding the importance of caring for underserved populations. Barriers to STIMMT participation may be reduced through availability of institution-sponsored scholarships, identification of external grant and scholarship opportunities, and coordination of fund-raising activities.

The impact of social context on learning and cognitive demands for interactive virtual human simulations.

Interactive virtual human (IVH) simulations offer a novel method for training skills involving person-to-person interactions. This article examines the effectiveness of an IVH simulation for teaching medical students to assess rare cranial nerve abnormalities in both individual and small-group learning contexts. Individual (n = 26) and small-group (n = 30) interaction with the IVH system was manipulated to examine the influence on learning, learner engagement, perceived cognitive demands of the learning task, and instructional efficiency. Results suggested the IVH activity was an equally effective and engaging instructional tool in both learning structures, despite learners in the group learning contexts having to share hands-on access to the simulation interface. Participants in both conditions demonstrated a significant increase in declarative knowledge post-training. Operation of the IVH simulation technology imposed moderate cognitive demand but did not exceed the demands of the task content or appear to impede learning.

Synthesis and biological evaluation of antimetastatic agents predicated upon dihydromotuporamine C and its carbocyclic derivatives.

The motuporamines isolated from the sea sponge Xestospongia exigua are of biological interest because of their unique antimigration and antiangiogenic properties. Key bioactive features were found to be a saturated 15-membered heterocycle and a norspermidine motif. This paper describes new analogues that modulate the cytotoxicity of this compound class and have enhanced antimigration properties. By movement of the polyamine chain outside the ring, new carbocycles were discovered that doubled the antimigration potency and reduced compound toxicity by 133-fold. Mice injected with metastatic human L3.6pl pancreatic cancer cells demonstrated significant reduction in liver metastases when treated with N(1)-(3-aminopropyl)-N(3)-(cyclopentadecylmethyl)propane-1,3-diamine compared with dihydromotuporamine C. Significant changes in specific ceramide populations (N16:0 and N22:1) were noted in L3.6pl cells treated with dihydromotuporamine C but not for the cyclopentadecylmethylnorspermidine derivative, which had lower toxicity. Both compounds gave increased levels of specific low molecular weight sphingomyelins, suggesting that they may act upon sphingomyelin processing enzymes.

Virtual patient simulations and optimal social learning context: a replication of an aptitude-treatment interaction effect.

BACKGROUND: Virtual patients (VPs) offer valuable alternative encounters when live patients with rare conditions, such as cranial nerve (CN) palsies, are unavailable; however, little is known regarding simulation and optimal social learning context. AIM: Compare learning outcomes and perspectives between students interacting with VPs in individual and team contexts. METHODS: Seventy-eight medical students were randomly assigned to interview and examine four VPs with possible CN damage either as individuals or in three-person teams, using Neurological Examination Rehearsal Virtual Environment (NERVE). Learning was measured through diagnosis accuracy and pre-/post-simulation knowledge scores. Perspectives of learning context were collected post-simulation. RESULTS: Students in teams submitted correct diagnoses significantly more often than students as individuals for CN-IV (p = 0.04; team = 86.1%; individual = 65.9%) and CN-VI (p = 0.03; team = 97.2%; individual = 80.5%). Knowledge scores increased significantly in both contexts (p < 0.001); however, a significant aptitude-treatment interaction effect was observed (p = 0.04). At pre-test scores ≤25.8%, students in teams scored significantly higher (66.7%) than students as individuals (43.1%) at post-test (p = 0.03). Students recommended implementing future NERVE exercises in teams over five other modality-timing combinations. CONCLUSION: Results allow us to define best practices for integrating VP simulators into medical education. Implementing NERVE experiences in team environments with medical students in the future may be preferable.

Genetic vaccine for respiratory syncytial virus provides protection without disease potentiation.

Respiratory syncytial virus (RSV) is a major cause of infectious lower respiratory disease in infants and the elderly. As there is no vaccine for RSV, we developed a genetic vaccine approach that induced protection of the entire respiratory tract from a single parenteral administration. The approach was based on adenovirus vectors derived from newly isolated nonhuman primate viruses with low seroprevalence. We show for the first time that a single intramuscular (IM) injection of the replication-deficient adenovirus vectors expressing the RSV fusion (F0) glycoprotein induced immune responses that protected both the lungs and noses of cotton rats and mice even at low doses and for several months postimmunization. The immune response included high titers of neutralizing antibody that were maintained ≥ 24 weeks and RSV-specific CD8+ and CD4+ T cells. The vectors were as potently immunogenic as a human adenovirus 5 vector in these two key respiratory pathogen animal models. Importantly, there was minimal alveolitis and granulocytic infiltrates in the lung, and type 2 cytokines were not produced after RSV challenge even under conditions of partial protection. Overall, this genetic vaccine is highly effective without potentiating immunopathology, and the results support development of the vaccine candidate for human testing.

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity.

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4(+) T cells.

Human type I interferons (IFNs) include IFN-ß and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4(+) T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN-α8 and -α10 most potently enhanced expression of IFN-γ, IL-2, and IL-4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL-2 and IL-4 correlated with the time of preincubation with type I IFN, IFN-γ production was enhanced best when IFN-α was added immediately preceding or simultaneously with T-cell stimulation. Comparison of T-cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN-α best enhanced cytokine expression when CD4(+) T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T-cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4(+) T-cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.

Optimal learning in a virtual patient simulation of cranial nerve palsies: the interaction between social learning context and student aptitude.

BACKGROUND: Simulation in medical education provides students with opportunities to practice interviews, examinations, and diagnosis formulation related to complex conditions without risks to patients. AIM: To examine differences between individual and team participation on learning outcomes and student perspectives through use of virtual patients (VPs) for teaching cranial nerve (CN) evaluation. METHODS: Fifty-seven medical students were randomly assigned to complete simulation exercises either as individuals or as members of three-person teams. Students interviewed, examined, and diagnosed VPs with possible CN damage in the neurological exam rehearsal virtual environment (NERVE). Knowledge of CN abnormalities was assessed pre- and post-simulation. Student perspectives of system usability were evaluated post-simulation. RESULTS: An aptitude-treatment interaction (ATI) effect was detected; at pre-test scores ≤ 50%, students in teams scored higher (83%) at post-test than did students as individuals (62%, p = 0.02). Post-simulation, students in teams reported greater confidence in their ability to diagnose CN abnormalities than did students as individuals (p = 0.02; mean rating = 4.0/5.0 and 3.4/5.0, respectively). CONCLUSION: The ATI effect allows us to begin defining best practices for the integration of VP simulators into the medical curriculum. We are persuaded to implement future NERVE exercises with small teams of medical students.

Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2.

Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1ß in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.

Enhancing learning through optimal sequencing of web-based and manikin simulators to teach shock physiology in the medical curriculum.

The Association of American Medical Colleges has encouraged educators to investigate proper linkage of simulation experiences with medical curricula. The authors aimed to determine if student knowledge and satisfaction differ between participation in web-based and manikin simulations for learning shock physiology and treatment and to determine if a specific training sequencing had a differential effect on learning. All 40 second-year medical students participated in a randomized, counterbalanced study with two interventions: group 1 (n = 20) participated in a web-based simulation followed by a manikin simulation and group 2 (n = 20) participated in reverse order. Knowledge and attitudes were documented. Mixed-model ANOVA indicated a significant main effect of time (F(1,38) = 18.6, P < 0.001, η(p)(2) = 0.33). Group 1 scored significantly higher on quiz 2 (81.5%) than on quiz 1 (74.3%, t(19) = 3.9, P = 0.001), for an observed difference of 7.2% (95% confidence interval: 3.3, 11.0). Mean quiz scores of group 2 did not differ significantly (quiz 1: 77.0% and quiz 2: 79.7%). There was no significant main effect of group or a group by time interaction effect. Students rated the simulations as equally effective in teaching shock physiology (P = 0.88); however, the manikin simulation was regarded as more effective in teaching shock treatment (P < 0.001). Most students (73.7%) preferred the manikin simulation. The two simulations may be of similar efficacy for educating students on the physiology of shock; however, the data suggest improved learning when web-based simulation precedes manikin use. This finding warrants further study.

Protein kinase R is a novel mediator of CD40 signaling and plays a critical role in modulating immunoglobulin expression during respiratory syncytial virus infection.

Effective immunoglobulin responses play a vital role in protection against most pathogens. However, the molecular mediators and mechanisms responsible for signaling and selective expression of immunoglobulin types remain to be elucidated. Previous studies in our laboratory have demonstrated that protein kinase R (PKR) plays a crucial role in IgE responses to double-stranded RNA (dsRNA) in vitro. In this study, we show that PKR plays a critical role in IgG expression both in vivo and in vitro. PKR(-/-) mice show significantly altered serum IgG levels during respiratory syncytial virus (RSV) infection. IgG2a expression is particularly sensitive to a lack of PKR and is below the detection level in mock- or RSV-infected PKR(-/-) mice. Interestingly, we show that upon activation by anti-CD40 and gamma interferon (IFN-γ), B cells from PKR(-/-) mice show diminished major histocompatibility complex class II (MHC II), CD80, and CD86 levels on the cell surface compared to wild-type (WT) mice. Our data also show that PKR is necessary for optimal expression of adhesion molecules, such as CD11a and ICAM-1, that are necessary for homotypic aggregation of B cells. Furthermore, in this report we demonstrate for the first time that upon CD40 ligation, PKR is rapidly phosphorylated and activated, indicating that PKR is an early and novel downstream mediator of CD40 signaling pathways.

Insights gained from the analysis of performance and participation in online formative assessment.

BACKGROUND: Online quizzes are simple, cost-effective methods to provide formative assessment, but their effectiveness in enhancing learning and performance in medical education is unclear. PURPOSE: The purpose of this article is to determine the extent to which online quiz performance and participation enhances students' performance on summative examinations. METHODS: A retrospective case study investigating relationships between formative and summative assessment in terms of use and outcomes. RESULTS: Online quiz scores and the rates of quiz participation were significantly correlated with corresponding performance on summative examinations. However, correlations were not dependent on the specific quiz content, and changes in patterns of quiz use were not reflected in corresponding changes in summative examination performance. CONCLUSIONS: The voluntary use of online quizzes, as well as the score attained, provides a useful general indicator of student performance but is unlikely to be sensitive enough to direct an individual student's learning plan.

Primary human mDC1, mDC2, and pDC dendritic cells are differentially infected and activated by respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ∼6.8% of mDC1 and ∼0.9% pDCs were infected. We extended these studies to include CD1c(-)CD141(+) mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity.

Epitope-specific regulatory CD4 T cells reduce virus-induced illness while preserving CD8 T-cell effector function at the site of infection.

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA(b)M(209)-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA(b)M(209)-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.

TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity.

Agonists for TLR7, TLR8, and TLR9 have been shown to enhance vaccine immunogenicity. We evaluated the impact of TLR activation on RSV disease in a murine model by administering TLR7/8 and TLR9 agonists during FI-RSV immunization or RSV infection. CpG administered during immunization reduced disease following challenge as evidenced by decreased lung pathology, illness, and cytokines. In marked contrast, TLR7/8 agonist had little impact. To evaluate potential therapeutic use, TLR agonists were administered during primary infection. Although type 2 cytokine responses decreased and type 1 cytokines and MIP-1-alpha/beta increased, both TLR7/8 and TLR9 agonists increased clinical symptoms and pulmonary inflammation when administered during primary infection. Thus, TLR9-induced signaling during FI-RSV immunization reduced vaccine-enhanced disease whereas immunostimulatory properties of TLR agonists enhanced disease severity when used during RSV infection. Immunomodulation elicited by TLR9 agonist confirms the adjuvant potential of TLR agonists during RSV immunization. However, in contrast to work done with HIV-1 vaccines, the inability of TLR7/8 agonist to boost type 1 vaccine-induced RSV immunity demonstrates pathogen-TLR specificity. These data reveal that the timing of administration of immunomodulatory agents is critical. Furthermore, these data underscore that amplification of anti-viral immune responses may result in immunopathology rather than immune-mediated protection.

Characterization of respiratory syncytial virus M- and M2-specific CD4 T cells in a murine model.

CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2 proteins with core sequences M(213-223) (FKYIKPQSQFI) and M2(27-37) (YFEWPPHALLV). Peptides containing the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-gamma), interleukin 2 (IL-2), and other Th1- and Th2-type cytokines in an I-A(b)-restricted pattern. Construction of fluorochrome-conjugated peptide-I-A(b) class II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and had greater IFN-gamma expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of IL-2. In addition, M(209-223) peptide-activated CD4 T cells reduced IFN-gamma and IL-2 production in M- and M2-specific CD8 T-cell responses to D(b)-M(187-195) and K(d)-M2(82-90) peptides more than M2(25-39) peptide-stimulated CD4 T cells. This correlated with the fact that I-A(b)-M(209-223) tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-A(b)-M2(26-39) tetramer-positive CD4 T cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide-I-A(b) tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.