Incentives for recruiting trainee participants in medical education research.

INTRODUCTION: In the growing field of medical education research, participant recruitment can be challenging. Incentives, either tangible or intangible, may be offered to encourage participation. This study aimed to understand these incentives and explore the relationship between study quality and incentives in medical education research. METHODS: We reviewed research studies examining medical trainees published in five major journals in 2008. Tangible and intangible incentives used in recruitment were extracted by two researchers. For each quantitative article, medical education research quality instrument (MERSQI) score was calculated and citation counts for all articles were compiled. RESULTS: Of 215 included articles, 8% explicitly reported incentives. Tangible incentives (value range $15-$60 USD) were offered in 7.9% of studies. Intangible incentives were identified in 30% of studies but only one specifically discussed their use. Tangible incentives correlated with a higher MERSQI score (p < 0.001) and with citations (p < 0.001). CONCLUSION: Most studies in medical education did not describe incentives for participation. Information regarding incentives should be reported in all studies to help inform future recruitment efforts and also to understand the study context including factors that may influence participants motivation.

Presumed primary immune-mediated neutropenia in 35 dogs: a retrospective study.

OBJECTIVES: To describe, in a cohort of dogs with presumed primary immune-mediated neutropenia, the presenting clinical characteristics, haematology results, bone marrow characteristics, therapies used (drugs and doses), clinical response to treatment, relapse and outcome at six months and one year. METHODS: Multi-institutional recruited retrospective descriptive case series with voluntary submissions. Presumed immune-mediated neutropenia was diagnosed based on a neutrophil concentration <1·5×109 cells/L on a minimum of two complete blood counts, exclusion of other causes of neutropenia based on a diagnostic bone marrow aspirate or biopsy, and exclusion of secondary immune-mediated neutropenia. Dogs meeting these diagnostic criteria between 2006 and 2013, and that had a haematocrit of ≥29% and minimum of two complete blood clounts performed after initiation of therapy, were included. RESULTS: Information on 35 dogs was included. Neutropenia was less than 0·5×109 cells/L in most cases (21 dogs), 0·5 to ·99×109 cells/L in 11, and 1.0 to 1·49×109 cells/L in three. Eight dogs had thrombocytopenia, which was severe (<49·9×109 cells/L) in three. [Correction added on 23 May 2017, after first online publication: the cell numbers were incorrect due to errors in the conversion of cell measurements to international units. The numbers have been corrected throughout the article and Table 2.] Twenty-three dogs had myeloid hyperplasia, 10 dogs had myeloid hypoplasia and two dogs had normal myelopoiesis. Neutropenia resolved in 32 of 33 dogs within two weeks of starting corticosteroid therapy and in all dogs within one month. Relapse of neutropenia occurred in 12 cases within one year. CLINICAL SIGNIFICANCE: Initial response of presumed primary immune-mediated neutropenia cases to corticosteroid therapy can be excellent. Long-term monitoring for relapse is warranted because 34% of cases relapsed during or after taper of immunosuppressive medications.

RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma.

RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Azaspiracid: first evidence of protein binding in shellfish.

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC-MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures. AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.

A non-class I MHC intestinal epithelial surface glycoprotein, gp180, binds to CD8.

The activation of CD8(+) T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the modulation of mucosal immune responses due to the fact that these T cells appear to have regulatory rather than cytolytic activity. A 180-kDa glycoprotein (gp180) has been identified and shown to be important in CD8(+) T cell activation by intestinal epithelial cells. In this study, we examine, in further detail, the role that the CD8 molecule plays in this interaction. It has been previously shown that monoclonal antibodies against gp180 inhibited the activation of CD8-associated p56(lck) in T cells. Although indirectly suggested by these data, there was no evidence that the activation of this protein tyrosine kinase was a direct result of gp180 interacting with the CD8 molecule. In this study, we document that soluble gp180 is able to bind to CD8-Fc fusion proteins and is absorbed by human CD8 alpha but not CD4 transfected murine T cells and that this interaction is dependent upon carbohydrate on the gp180 molecule. Furthermore, the sites used for binding by gp180 are distinct from those used by the conventional CD8 ligand, class I MHC. Thus, gp180 appears to be a novel CD8 ligand that plays an important role in the activation of CD8-associated kinases and of CD8(+) T cells.

CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation.

CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8's ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8's ability to interact with class I MHC is not fixed and is developmentally regulated through the T cell's glycosylation state.

Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers.

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8(+) T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01-0.20% of CD8(+) T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8(+) T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8(+) CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.

Human CD8 beta, but not mouse CD8 beta, can be expressed in the absence of CD8 alpha as a beta beta homodimer.

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8 alpha polypeptide chains and heterodimers of CD8 alpha- and CD8 beta-chains. The function of the CD8 alpha-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8 alpha alpha homodimers. CD8 alpha beta functions as a better coreceptor, but the actual function of CD8 beta is less clear. Addressing this issue has been hampered by the apparent inability of CD8 beta to be expressed without CD8 alpha. This study demonstrates that human, but not mouse, CD8 beta can be expressed on the cell surface without CD8 alpha in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8 beta is responsible for the lack of expression of murine CD8 beta beta dimers. In contrast to CD8 alpha alpha, CD8 beta beta is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 beta-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8 beta beta Ig domain. In addition, we demonstrate that the Ig domains of CD8 alpha are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8 alpha reduced the ability of the protein to fold properly and, therefore, to be expressed.

Molecular analysis of protein interactions mediating the function of the cell surface protein CD8.

The T cell coreceptor CD8 is a cell-surface glycoprotein expressed either as a disulfide-linked homodimer of two CD8alpha monomers, or a heterodimer of CD8alpha and CD8beta. These receptors interact with ligands, such as major histocompatibility complex (MHC) class I, on the outside of the cell, with proteins inside the cell, such as the tyrosine kinase p56lck, and possibly with proteins on the same cell-surface. The molecular details describing such protein interactions can shed light on how the proteins function and the functional differences between the two forms of CD8. Crystal structures, mutational analysis, affinity measurements, and other approaches are providing those details.

Orientation of the Ig domains of CD8 alpha beta relative to MHC class I.

The cell surface glycoprotein CD8 functions as a coreceptor with the TCR for interaction with MHC class I. The cocrystal structure of the CD8 alpha alpha-MHC complex showed that one CD8 Ig domain provided the majority of the contact with MHC class I and that residue R4 of that domain contacted the alpha2 domain of MHC class I. We previously showed by mutational analysis that this residue was critical for binding to MHC class I. To determine which of the Ig domains for the CD8 alpha beta heterodimer would make the most contact with class I MHC, we expressed single-chain or dimeric forms of CD8 on COS-7 cells and measured the adhesion of MHC class I positive cells. We found that when one of the R4 residues was mutated in a CD8 alpha alpha homodimer binding comparable to that of wild type was observed, whereas a double R4 mutant severely impaired binding. However, when mutant CD8 alpha (R4K) was coexpressed with wild-type CD8 beta, binding was not observed. These results support the model in which it is CD8 alpha, not CD8 beta, that is making the most of the contact with MHC class I, including the alpha 2 domain. In addition, they demonstrate that a single-chain form of CD8 alpha alpha can bind to MHC class I.

Changes in human plasma melatonin profiles in response to 50 Hz magnetic field exposure.

The effects of power-frequency magnetic fields on nighttime plasma melatonin were studied in a group of 30 adult male human subjects. Exposure consisted of 20 microT (200 mG) at 50 Hz (circularly polarized) at certain times in relation to the predicted time of onset of rise in melatonin concentration for a particular individual (the time of onset was predicted from a previous screening night). Response to this exposure was compared to sham-exposure (in random order). When exposure preceded onset of rise, a significant delay in onset time relative to sham-exposure of approximately half an hour was observed, with indications (marginally significant) of a reduction in maximum melatonin level. Analysis of distribution of time-delays is consistent with two populations: those individuals who respond (around 20%) and those that do not. Magnetic fields generated by square-wave currents produce more marked reductions in the maximum level when compared to sinusoidal waveforms, but there was no significant difference in onset time.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Role of LFA-1, ICAM-1, VLA-4 and VCAM-1 in lymphocyte migration across retinal pigment epithelial monolayers in vitro.

The blood-retinal barrier (BRB), which is composed of the retinal pigment epithelium (RPE) and retinal vascular endothelium, normally restricts the traffic of lymphocytes into the retina. During ocular inflammatory conditions such as posterior uveitis there is a large increase in lymphocyte migration across the BRB. The differential role played by the two barrier sites, however, remains unclear. To evaluate the role of the posterior BRB, the migration of CD4+ antigen-specific T-cell line through rat RPE cell monolayers was investigated in vitro using time-lapse videomicroscopy. The adhesion molecules involved in controlling transepithelial migration across normal and interferon-gamma (IFN-gamma)-activated RPE was assessed with monoclonal antibodies directed against cell adhesion molecules. Lymphocytes were treated with antibodies specific for CD11a (alpha L subunit of LFA-1), CD18 (beta 2 subnit of the leucam family) and CD49 d (alpha 4 subnit of very late activation antigen-4, VLA-4), and the RPE with antibodies specific for CD54 (intracellular adhesion molecule-1, ICAM-1) and CD 106 (vascular cell adhesion molecule-1, VCAM-1). Migration across unstimulated RPE was inhibited by antibodies to ICAM-1 (48.6 +/- 3.5% reduction), leucocyte functional antigen-1 (LFA-1) alpha (61 +/- 5.2%) and LFA-1 beta (63.2 +/- 4.7%), but not by antibodies to VLA-4. VCAM-1 was not expressed on untreated RPE. Following activation of the RPE monolayers for 72 hr with IFN-gamma, antibodies to LFA-1 alpha, LFA-1 beta and ICAM-1 inhibited migration by 49.9 +/- 9.4%, 63.6 +/- 5.5% and 47.7 +/- 4.2% respectively. Antibodies to VLA-4 and VCAM-1 blocked migration by 21.5 +/- 8.4% and 32.3 +/- 6.2%, respectively, which correlated with the induction of VCAM-1 expression on RPE and increased migration. Under these conditions blocking both VCAM-1 and ICAM-1 reduced migration by 70.9 +/- 2.3%, which was greater than the effect of blocking either of these molecules alone. These results demonstrate that the posterior barrier of the BRB utilizes the same principle receptor-ligand pairings in controlling lymphocyte traffic into the retina as the vascular endothelium of the anterior BRB.

Lymphocyte migration across the anterior and posterior blood-retinal barrier in vitro.

The migration of lymphocytes through monolayers of rat retinal pigment epithelium (RPE) and retinal vascular endothelium, which form the posterior and anterior blood-retinal barrier (BRB) respectively, was investigated in vitro. After a 4-hr assay the migration of untreated peripheral lymph node (PLN) cells through RPE monolayers was negligible (<1%) with only a small increase found after activation of the PLN cells with concanavalin A or by cross-linking CD3. Activation of the RPE with IFN-gamma augmented migration with maximal PLN cell migration being achieved with a combination of CD3 cross-linking and IFN-gamma activation (17% migration). The highest level of lymphocyte migration was observed with three CD4+ antigen-specific T cell lines specific for purified protein derivative (PPD; 33% migration), ovalbumin (OA; 31%), and S-antigen (S-Ag; 57%). Migration of both untreated and Con A-activated PLN cells through retinal endothelial cells (EC) from PVG rats was negligible, whereas the migration of the antigen-specific T cell lines was 23, 29 and 23% for PPD, OA, and S-Ag lines, respectively. Migration of these cell lines through retinal endothelium derived from Lewis rats was significantly greater (44% for PPD, 39% for OA, and 39% for S-Ag) which corresponded with a greater expression of ICAM-1 on the EC.

Aerosolized lipopolysaccharide increases pulmonary clearance of 99mTc-DTPA in rabbits.

Bacterial endotoxins alter the permeability of endothelium, but little is known of their effect on epithelium. We exposed specific pathogen-free rabbits to aerosolized Pseudomonas aeruginosa LPS or saline and performed serial measurements of RL, Cdyn, BP, WBC count and differential, and platelet counts. Pulmonary 99mTc-DTPA half-life was measured 4, 6, or 8 h after exposure. The animals were sacrificed and BAL performed. Background and PMA-stimulated superoxide production was measured from individual AM using electrooptical determination of reduction of NBT. Lung tissue was processed for light microscopy and ratio of wet to dry weight. 99mTc-DTPA half-life was significantly shorter in LPS-exposed animals at 6 h (p < 0.05) and 8 h (p < 0.001). There were no differences in Cdyn, RL, BP, WBC, differential, platelet, or BAL cell count at any time between groups. No histologic changes or differences in lung wet to dry weight ratios were found. PMA-stimulated AM superoxide production was significantly increased (p < 0.01) in LPS-exposed animals. This effect was time dependent and could be duplicated in AM from control animals following a 4-h incubation with LPS, lavage fluid from LPS-exposed animals, or recombinant murine TNF. These results demonstrate that aerosolized Pseudomonas LPS increases pulmonary epithelial permeability and primes AM superoxide production.

Bronchoalveolar lavage fluid cytology reflects airway inflammation in beagle puppies with acute bronchiolitis.

Beagle puppies infected with both canine parainfluenza virus type 2 (CPI2) and Bordetella bronchiseptica (Bb) develop more severe acute bronchiolitis and airways hyperresponsiveness than do those infected with CPI2 or Bb alone. The aim of our study was to characterize the inflammatory response associated with airway hyperresponsiveness, and to determine whether the inflammatory cell response of bronchoalveolar lavage fluid (BALF) reflected changes in the bronchioles in this model. We investigated 25 beagle puppies (ages 76 +/- 5 days, mean +/- SEM) in four groups: controls (n = 6), or puppies inoculated with both CPI2 and Bb (CPI2-Bb) (n = 11), with only CPI2 (n = 4), or only Bb (n = 4). The puppies were killed 3-4 days after inoculation, the lungs excised, the intermediate lobe lavaged, and BALF and the bronchiolar wall tissue examined for neutrophils and other inflammatory cells. Control puppies had no evidence of inflammation. However, the CPI2-Bb puppies had developed cough and rhinitis, positive cultures for CPI2 and Bb, and a neutrophilic cellular response in both the bronchioles and the BALF. Puppies inoculated with only CPI2 or Bb had milder illnesses and no significant bronchiolar and BALF neutrophilic response. For all groups, the severity of bronchiolar wall inflammation correlated with the total number of BALF inflammatory cells, and bronchiolar wall neutrophil counts correlated with the percentage of neutrophils in the BALF. The illness and the airway hyperresponsiveness observed in the CPI2-Bb group were associated with airway neutrophilia. Our studies support the hypothesis that neutrophils are associated with airway dysfunction in this model, and the use of BALF to study the process.

VA develops integrated text and image data system.

A standard PC workstation allows physicians to review a patient's entire record--both text and images--with a new integrated HIS now installed at the Department of Veterans Affairs Medical Center in Washington, D.C. The system uses high-resolution video cameras and the latest in fiberoptic technology.