Using a Journal Club Series to Introduce Paramedic Students to Research Fundamentals and Critical Appraisal of Medical Literature.

INTRODUCTION: Despite United States national learning objectives referencing research fundamentals and the critical appraisal of medical literature, many paramedic programs are not meeting these objectives with substantive content. PROBLEM: The objective was to develop and implement a journal club educational module for paramedic training programs, which is all-inclusive and could be distributed to Emergency Medical Services (EMS) educators and EMS medical directors to use as a framework to adapt to their program. METHODS: Four two-hour long journal club sessions were designed. First, the educator provided students with four types of articles on a student-chosen topic and discussed differences in methodology and structures. Next, after a lecture about peer-review, students used search engines to verify references of a trade magazine article. Third, the educator gave a statistics lecture and critiqued the results section of several articles found by students on a topic. Finally, students found an article on a topic of personal interest and presented it to their classmates, as if telling their paramedic partner about it at work. Before and after the series, students from two cohorts (2017, 2018) completed a survey with questions about demographics and perceptions of research. Students from one cohort (2017) received a follow-up survey one year later. RESULTS: For the 2016 cohort, 13 students participated and provided qualitative feedback. For the 2017 and 2018 cohorts, 33 students participated. After the series, there was an increased self-reported ability to find, evaluate, and apply medical research articles, as well as overall positive trending opinions of participating in and the importance of prehospital research. This ability was demonstrated by every student during the final journal club session. McNemar's and Related-Samples Cochran's Q testing of questionnaire responses suggested a statistically significant improvement in student approval of exceptions from informed consent. CONCLUSION: The framework for this paramedic journal club series could be adapted by EMS educators and medical directors to enable paramedics to search for, critically appraise, and discuss the findings of medical literature.

Immune Responses in U.S. Military Personnel Who Received Meningococcal Conjugate Vaccine (MenACWY) Concomitantly with Other Vaccines Were Higher than in Personnel Who Received MenACWY Alone.

Immunological responses to vaccination can differ depending on whether the vaccine is given alone or with other vaccines. This study was a retrospective evaluation of the immunogenicity of a tetravalent meningococcal conjugate vaccine for serogroups A, C, W, and Y (MenACWY) administered alone (n = 41) or concomitantly with other vaccines (n = 279) to U.S. military personnel (mean age, 21.6 years) entering the military between 2006 and 2008. Concomitant vaccines included tetanus/diphtheria (Td), inactivated polio vaccine (IPV), hepatitis vaccines, and various influenza vaccines, among others; two vaccine groups excluded Tdap and IPV. Immune responses were evaluated in baseline and postvaccination sera for Neisseria meningitidis serogroups C and Y 1 to 12 months (mean, 4.96 months) following vaccination. Functional antibodies were measured by using a serum bactericidal antibody assay with rabbit complement (rSBA) and by measurement of serogroup-specific immunoglobulin G (IgG) antibodies. The percentage of vaccinees reaching threshold levels (IgG concentration in serum, ≥2 µg/ml; rSBA titer, ≥8) corresponding to an immunologic response was higher postvaccination than at baseline (P < 0.001). Administration of MenACWY along with other vaccines was associated with higher geometric means of IgG concentrations and rSBA titers than those measured 4.60 months after a single dose of MenACWY. In addition, higher percentages of vaccinees reached the immunological threshold (range of odds ratios [ORs], 1.5 to 21.7) and more of them seroconverted (OR range, 1.8 to 4.8) when MenACWY was administered with any other vaccine than when administered alone. Additional prospective randomized clinical trials are needed to confirm the observed differences among groups in the immune response to MenACWY when given concomitantly with other vaccines to U.S. military personnel.

Effects of Linker Length and Transient Secondary Structure Elements in the Intrinsically Disordered Notch RAM Region on Notch Signaling.

Formation of the bivalent interaction between the Notch intracellular domain (NICD) and the transcription factor CBF-1/RBP-j, Su(H), Lag-1 (CSL) is a key event in Notch signaling because it switches Notch-responsive genes from a repressed state to an activated state. Interaction of the intrinsically disordered RBP-j-associated molecule (RAM) region of NICD with CSL is thought to both disrupt binding of corepressor proteins to CSL and anchor NICD to CSL, promoting interaction of the ankyrin domain of NICD with CSL through an effective concentration mechanism. To quantify the role of disorder in the RAM linker region on the effective concentration enhancement of Notch transcriptional activation, we measured the effects of linker length variation on activation. The resulting activation profile has general features of a worm-like chain model for effective concentration. However, deviations from the model for short sequence deletions suggest that RAM contains sequence-specific structural elements that may be important for activation. Structural characterization of the RAM linker with sedimentation velocity analytical ultracentrifugation and NMR spectroscopy reveals that the linker is compact and contains three transient helices and two extended and dynamic regions. To test if these secondary structure elements are important for activation, we made sequence substitutions to change the secondary structure propensities of these elements and measured transcriptional activation of the resulting variants. Substitutions to two of these nonrandom elements (helix 2, extended region 1) have effects on activation, but these effects do not depend on the nature of the substituting residues. Thus, the primary sequences of these elements, but not their secondary structures, are influencing signaling.

Persistence of serogroup C antibody responses following quadrivalent meningococcal conjugate vaccination in United States military personnel.

Serogroup C meningococcal (MenC) disease accounts for one-third of all meningococcal cases and causes meningococcal outbreaks in the U.S. Quadrivalent meningococcal vaccine conjugated to diphtheria toxoid (MenACYWD) was recommended in 2005 for adolescents and high risk groups such as military recruits. We evaluated anti-MenC antibody persistence in U.S. military personnel vaccinated with either MenACYWD or meningococcal polysaccharide vaccine (MPSV4). Twelve hundred subjects vaccinated with MenACYWD from 2006 to 2008 or MPSV4 from 2002 to 2004 were randomly selected from the Defense Medical Surveillance System. Baseline serologic responses to MenC were assessed in all subjects; 100 subjects per vaccine group were tested during one of the following six post-vaccination time-points: 5-7, 11-13, 17-19, 23-25, 29-31, or 35-37 months. Anti-MenC geometric mean titers (GMT) were measured by rabbit complement serum bactericidal assay (rSBA) and geometric mean concentrations (GMC) by enzyme-linked immunosorbent assay (ELISA). Continuous variables were compared using the Wilcoxon rank sum test and the proportion of subjects with an rSBA titer ≥ 8 by chi-square. Pre-vaccination rSBA GMT was <8 for the MenACWYD group. rSBA GMT increased to 703 at 5-7 months post-vaccination and decreased by 94% to 43 at 3 years post-vaccination. GMT was significantly lower in the MenACWYD group at 5-7 months post-vaccination compared to the MPSV4 group. The percentage of MenACWYD recipients achieving an rSBA titer of ≥ 8 decreased from 87% at 5-7 months to 54% at 3 years. There were no significant differences between vaccine groups in the proportion of subjects with a titer of ≥ 8 at any time-point. GMC for the MenACWYD group was 0.14 µg/mL at baseline, 1.07 µg/mL at 5-7 months, and 0.66 µg/mL at 3 years, and significantly lower than the MPSV4 group at all time-points. Anti-MenC responses wane following vaccination with MenACYWD; a booster dose is needed to maintain protective levels of circulating antibody.

Molecular signatures of antibody responses derived from a systems biology study of five human vaccines.

Many vaccines induce protective immunity via antibodies. Systems biology approaches have been used to determine signatures that can be used to predict vaccine-induced immunity in humans, but whether there is a 'universal signature' that can be used to predict antibody responses to any vaccine is unknown. Here we did systems analyses of immune responses to the polysaccharide and conjugate vaccines against meningococcus in healthy adults, in the broader context of published studies of vaccines against yellow fever virus and influenza virus. To achieve this, we did a large-scale network integration of publicly available human blood transcriptomes and systems-scale databases in specific biological contexts and deduced a set of transcription modules in blood. Those modules revealed distinct transcriptional signatures of antibody responses to different classes of vaccines, which provided key insights into primary viral, protein recall and anti-polysaccharide responses. Our results elucidate the early transcriptional programs that orchestrate vaccine immunity in humans and demonstrate the power of integrative network modeling.

Electromyographic analysis of the rotator cuff in postoperative shoulder patients during passive rehabilitation exercises.

BACKGROUND: Numerous rehabilitation protocols exist for postoperative rotator cuff repairs. Because the goal of early rehabilitation is to prevent postoperative adhesions while protecting the repaired tendons, it would be advantageous to know which range-of-motion exercises allow the rotator cuff to remain the most passive in a painful, guarded, postsurgical shoulder. METHODS: Twenty-six subjects who had undergone subacromial decompression, distal clavicle resection, or a combination of both procedures volunteered to participate within the first 4 days after surgery. Fine-wire electrodes were inserted into the subject's supraspinatus (SS) and infraspinatus (IS). Muscle activity was recorded at resting baseline (BL) and during 14 exercises that have been found in the passive phase of rotator cuff protocols and tested in healthy subjects. Each exercise was compared with BL activity as well as with other exercises in the same movement group. RESULTS: The SS remained as passive as BL during therapist- and self-assisted external rotation, therapist-assisted elevation, pendulums, and isometric internal rotation and adduction. The IS was activated greater than BL for all 14 exercises studied. CONCLUSION: Of the 14 exercises studied, 6 allowed the SS and 0 allowed the IS to remain as passive as quiet-stance BL in postsurgical subacromial decompression/distal clavicle resection patients.

Dissecting and circumventing the requirement for RAM in CSL-dependent Notch signaling.

The Notch signaling pathway is an intercellular communication network vital to metazoan development. Notch activation leads to the nuclear localization of the intracellular portion (NICD) of the Notch receptor. Once in the nucleus, NICD binds the transcription factor CSL through a bivalent interaction involving the high-affinity RAM region and the lower affinity ANK domain, converting CSL from a transcriptionally-repressed to an active state. This interaction is believed to directly displace co-repressor proteins from CSL and recruit co-activator proteins. Here we investigate the consequences of this bivalent organization in converting CSL from the repressed to active form. One proposed function of RAM is to promote the weak ANK:CSL interaction; thus, fusion of CSL-ANK should bypass this function of RAM. We find that a CSL-ANK fusion protein is transcriptionally active in reporter assays, but that the addition of RAM in trans further increases transcriptional activity, suggesting another role of RAM in activation. A single F235L point substitution, which disrupts co-repressor binding to CSL, renders the CSL-ANK fusion fully active and refractory to further stimulation by RAM in trans. These results suggest that in the context of a mammalian CSL-ANK fusion protein, the main role of RAM is to displace co-repressor proteins from CSL.

Relationship of adipokines with immune response and lung function in obese asthmatic and non-asthmatic women.

BACKGROUND: Obesity is a risk factor for asthma. Studies in mice suggest that the adipokines leptin and adiponectin affect asthmatic responses. The purpose of this study was to determine if adipokines associated with obesity are (1) altered in obese women with asthma compared to controls and (2) associated with increased cytokines and chemokines involved in allergic inflammation. METHODS: We performed a cross-sectional study of asthmatic and non-asthmatic obese premenopausal women. Participants answered questionnaires and performed lung function tests. Serum and peripheral blood mononuclear cells (PBMCs) were collected for analysis of cytokines and adipokines. RESULTS: A total of 22 asthmatic (mean body mass index 40.0 ± 5.1 kg/m(2)) and 20 non-asthmatic women (mean body mass index 41.3 ± 5.6 kg/m(2)) participated. We found no difference in serum adipokine concentrations between asthmatics and non-asthmatics. Serum adiponectin correlated positively with PBMC eotaxin (r(s) = 0.55, p = .0003) and RANTES (regulated upon activation, normal T-cell expressed, and secreted) (r(s) = 0.36, p = .03), whereas serum leptin correlated negatively with PBMC eotaxin (r(s) = -0.34, p = .04). There was a negative correlation between serum adiponectin and PBMC interferon-γ (r(s) = -0.41, p = .01). CONCLUSIONS: Perturbations of adipokines that occur in obesity were correlated with decreased cytokine production typically associated with allergic responses in PBMC of obese premenopausal women. This study suggests that although obese asthmatics may have elements of Th2-mediated inflammation, adipokine derangements in obesity are associated with Th1 rather than Th2 bias. Obesity has complex effects on allergic inflammation and is likely to be important modifier of the pathogenesis of airway disease in asthma.

Concomitant administration of recombinant PsaA and PCV7 reduces Streptococcus pneumoniae serotype 19A colonization in a murine model.

A murine colonization model was used to determine the effect of co-administering 7-valent polysaccharide-protein conjugate vaccine and pneumococcal surface adhesin A. Mice were challenged intranasally with either PCV7 serotypes, 4 or 14, or a non-PCV7 serotype, 19A. Post-challenge samples were evaluated for IgG antibody levels, opsonophagocytic activity, and nasopharyngeal colonization. No interference was observed between immune responses from the concomitant and individual immunizations. Concomitant immunizations reduced carriage for tested serotypes; largest reduction was observed for 19A. From these mouse studies, co-administering pneumococcal antigens appear to expand coverage and reduce colonization against a non-PCV7 serotype without inhibiting immunogenicity to other serotypes.

Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2.

The Notch signaling pathway is a cell-cell communication network giving rise to cell differentiation during metazoan development. Activation of the pathway releases the intracellular portion of the Notch receptor to translocate to the nucleus, where it is able to interact with the effector transcription factor CSL, converting CSL from a transcriptional repressor to an activator. This conversion is dependent upon the high affinity binding of the RAM region of the Notch receptor to the beta-trefoil domain (BTD) of CSL. Here we probe the energetics of binding to BTD of each conserved residue of RAM through the use of isothermal titration calorimetry and single residue substitution. We find that although the highly conserved PhiW PhiP motif is the largest determinant of binding, energetically significant interactions are contributed by N-terminal residues, including a conserved Arg/Lys-rich region. Additionally, we present a thermodynamic analysis of the interaction between the Epstein-Barr virus protein EBNA2 with BTD and explore the extent to which the EBNA2- and RAM-binding sites on BTD are nonoverlapping, as proposed by Fuchs et al. (Fuchs, K. P., Bommer, G., Dumont, E., Christoph, B., Vidal, M., Kremmer, E., and Kempkes, B. (2001) Eur. J. Biochem. 268, 4639-4646). Combining these results with displacement isothermal titration calorimetry, we propose a mechanism by which the PhiW PhiP motif of RAM and EBNA2 compete with one another for binding at the hydrophobic pocket of BTD using overlapping but specific interactions that are unique to each BTD ligand.

Low-temperature preparation of anatase thin films on tantalum.

Titanium dioxide thin films were grown on oxidized Ta surfaces using a cyclic layer-by-layer wet chemistry method: successive-ionic-layer-adsorption-and-reaction (SILAR). Film thicknesses varied monotonically and approximately linearly with the number of cycles. As-grown (AG) films were amorphous and rougher (16.2 nm root-mean-square (rms)) than the Ta substrate (10.2 nm rms). After hydrothermal annealing (AN) at a remarkably low temperature of 393 K, the films exhibited anatase crystallites (10 nm dimensions) and reduced roughness (11.8 nm rms). The atomic composition of both AG and AN films was consistent with that of TiO2 containing no more than 4 atom % carbon. A small Si impurity (<1 atom %) was eliminated by using polypropylene beakers and sample holders in the SILAR steps.

Adherence of recombinant pneumococcal surface adhesin A (rPsaA)-coated particles to human nasopharyngeal epithelial cells for the evaluation of anti-PsaA functional antibodies.

Pneumococcal surface adhesin A (PsaA) is a pneumococcal vaccine candidate. In this study, we detect functional antibodies to PsaA by using carboxylate-modified fluospheres coated with either recombinant non-lipidated PsaA (rPsaA) or synthetic peptides with relevant epitopes of PsaA. Peptides P1-P3 were derived from phage display sequences; peptides P4-P7 were homologous to rPsaA. P1- and P4-coated fluospheres had similar adherence to Detroit 562 nasopharyngeal cells when compared to rPsaA-coated fluospheres. Homologous and heterologous competitive inhibitions with peptides in solution determined the specificity of the adherence. There was no significant difference (P=0.25) between the inhibition of adherence of rPsaA- and P4-coated fluospheres. This study indicates that P1 and P4 contain a functional epitope(s) for the adherence of PsaA to nasopharyngeal cells making them suitable targets for the measurement of functional antibodies to PsaA.

Inhibition of pneumococcal adherence to human nasopharyngeal epithelial cells by anti-PsaA antibodies.

The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 10(4) to 10(5) bacteria/well, the mean +/- standard deviation count in controls was 163 +/- 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 micro g/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Inhibition of pneumococcal carriage in mice by subcutaneous immunization with peptides from the common surface protein pneumococcal surface adhesin a.

Pneumococcal surface adhesin A (PsaA), a common protein expressed on all 90 pneumococcal serotypes, is a vaccine candidate. Three anti-PsaA monoclonal antibody phage display-expressed monopeptides (15 mers), in various formulations as lipidated or nonlipidated multiantigenic peptides or as bi- or tripeptide constructs, were studied in a mouse nasopharyngeal carriage model to determine the inhibitory effect of induced antibodies on carriage of pneumococcal serotypes 2, 4, and 6B. Antibodies to each of the various peptides tested reduced carriage of the 3 serotypes. Reduction in carriage by nonlipidated multiantigenic peptide antibodies was highly variable (39%-94%; mean, 59%; standard deviation [SD], 20.2%); however, more-consistent results were observed in mice immunized with lipidated (56%-98%; mean, 69%; SD, 13.6%) and combination or bipeptide (55%-91%; mean, 76%; SD, 13.1%) formulations. These peptides are immunogenic, and their induced antibodies reduce carriage in mice. PsaA peptides demonstrate potential for being important new vaccines against pneumococcal carriage, otitis media, and invasive pneumococcal disease.