Biomedical subjects
S G Smith
Publications and source records attributed to S G Smith.
The response of human dendritic cells to recombinant adenovirus, recombinant Mycobacterium bovis Bacillus Calmette Guerin and biolistic methods of antigen delivery: different induction of contact-dependant and soluble signals.
Dendritic cells (DCs) are the most potent antigen presenting cells for inducing T-cell immune responses. The ability to grow human DCs from monocyte precursors provides an abundant source of these cells, which can be modified in vitro to present antigens. Re-administration of modified DCs to patients as vaccines has been shown in some cases to induce immune responses against cancer and infectious disease. Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading of major histocompatibility complex (MHC) class I molecules. The aim of this study was to use monocyte-derived DCs (MD-DCs) from healthy donors to compare in vitro gene transfer, mediated by adenovirus, M. bovis Bacillus Calmette Guerin (BCG) and biolistic delivery. Efficiency of transfection and effect on DC phenotype, allostimulatory capacity and cytokine secretion was investigated. Adenovirus and BCG both showed a comparable ability to transfect MD-DCs, whereas the biolistic delivery by gene gun was unsuccessful in the reporter gene delivery. BCG transfection promoted MD-DC maturation as is apparent in the surface phenotype, allostimulatory capacity and cytokine secretion from cells. In comparison, adenovirus and biolistic delivery had a reduced effect on MD-DCs although enhancement of co-stimulatory and MHC molecule expression occurred in the cells of some donors. Both BCG and adenovirus represent useful vectors for gene transfer to human DCs. The effect of BCG on DC maturation may provide additional signals for the induction of antigen-specific T-cell responses.
The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA.
The TrkA receptor is activated primarily by nerve growth factor (NGF), but it can also be activated by high concentrations of neurotrophin 3 (NT-3). The pan-neurotrophin receptor p75(NTR) strongly inhibits activation of TrkA by NT-3 but not by NGF. To examine the role of p75(NTR) in regulating the specificity of TrkA signaling, we expressed both receptors in Xenopus oocytes. Application of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of (45)Ca(2+) by a phospholipase C-gamma-dependent pathway. Coexpression of p75(NTR) with TrkA inhibited (45)Ca(2+) efflux in response to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA increased with increasing expression of p75(NTR). Coexpression of a truncated p75(NTR) receptor lacking all but the first 9 amino acids of the cytoplasmic domain inhibited NT-3 stimulation of (45)Ca(2+) efflux, whereas coexpression of an epidermal growth factor receptor/p75(NTR) chimera (extracellular domain of epidermal growth factor receptor with transmembrane and cytoplasmic domains of p75(NTR)) did not inhibit NT-3 signaling through TrkA. These studies demonstrated that the extracellular domain of p75(NTR) was necessary to inhibit NT-3 signaling through TrkA. Remarkably, p75(NTR) binding to NT-3 was not required to prevent signaling through TrkA, since occupying p75(NTR) with brain-derived neurotrophic factor or anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate (45)Ca(2+) efflux. These data suggested a physical association between TrkA and p75(NTR). Documenting this physical interaction, we showed that p75(NTR) and TrkA could be coimmunoprecipitated from Xenopus oocytes. Our results suggest that the interaction of these two receptors on the cell surface mediated the inhibition of NT-3-activated signaling through TrkA.
The role of the basic surgical skills course in the acquisition and retention of laparoscopic skill.
BACKGROUND: This study assesses the transfer of laparoscopic skills to a group of Basic Surgical Trainees (BST) attending the Basic Surgical Skills (BSS) course. METHODS: The virtual reality simulator MIST-VR was used to assess 13 trainees before and after the course and again 3 weeks and 3 months later. Analysis of kinematic data using the Imperial College Surgical Assessment Device gave measures of distance traveled, distance efficiency ratio, time taken, number of errors made, and number of movements made in completing a virtual laparoscopic task. The performance of the group was compared to a control group who underwent no training. RESULTS: All parameters improved significantly after the course, with the exception of distance traveled by the instruments. All outcome measures were significantly improved at 3 weeks. The control group showed a nonsignificant trend toward improvement in all parameters. CONCLUSIONS: The Basic Surgical Skills course produces quantifiable improvements in laparoscopic skill that are measurable by MIST-VR. There is a learning effect associated with using MIST-VR alone.
Skill transfer from virtual reality to a real laparoscopic task.
BACKGROUND: To validate the usefulness of virtual reality surgical simulators, we investigated the transfer of skills achieved by their use to real tasks. METHODS: Thirty medical students underwent a pretest using a real laparoscopic trainer. They were then randomized to the following three groups: group I received no training; group II received training using the Minimal Invasive Surgical Trainer in Virtual Reality (MIST-VR); and group III received training using conventional training exercises. Each group then underwent a posttest. Using the Imperial College Surgical Assessment Device (ICSAD), scores were generated for time taken, distance traveled, number of movements made, and speed of instrument movement. RESULTS: Significant changes between the MIST-VR group (group II) and the conventionally trained group (group III), were observed in the speed of movement of the left hand and the numbers of movements taken by each hand, when compared to the untrained group (group I). CONCLUSION: The training of novices using MIST-VR yields quantifiable changes in skill that are transferable to a simple real task and are similar to the results achieved with conventional training.
Allen's test: fact or myth?
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Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses.
PURPOSE: To assess the therapeutic potential of a melanoma polyepitope vaccine in human cells. Polyepitope DNA vaccines encoding T-cell epitopes have been demonstrated in murine systems to generate multiple cytotoxic T-cell responses to different antigens. Here, for the first time we demonstrate the ability of a melanoma polyepitope to stimulate lymphocytes from normal human donors to simultaneously generate multiple antigen-specific responses. EXPERIMENTAL DESIGN: Human dendritic cells (DC), transduced with a melanoma-polyepitope cDNA, were used to activate autologous lymphocytes from naïve donors as an in vitro model of DNA vaccination. Lymphocytes were primed with polyepitope or mock-transduced DC, boosted with peptide, then measured for antigen-specific cytotoxicity. RESULTS: Lymphocytes primed with polyepitope-transduced DC and boosted with peptide generated multiple cytotoxic responses. By contrast lymphocytes primed with mock-transfected DCs and boosted with peptide gave no specific cytotoxicity. However, when lymphocytes were repeatedly stimulated with polyepitope-transduced DCs immunodominance was seen with CTLs being generated to only one epitope, MART(27-35). CONCLUSIONS: We show in a human system that a melanoma polyepitope primes CTL to multiple epitopes. However, repeated stimulation by the polyepitope restricts the response to only the MART1 epitope. Thus, although polyepitope vaccines are an effective way of priming multiple naïve T-cell responses, continual boosting with polyepitope vaccines may, as a result of immunodominance, restrict the CTL. These findings have important implications for the use of DNA polyepitope vaccines in cancer immunotherapy.
Interaction of the FimB integrase with the fimS invertible DNA element in Escherichia coli in vivo and in vitro.
The FimB protein is a site-specific recombinase that inverts the fimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fim switch.
The role of simulation in surgical training.
Surgical training has undergone many changes in the last decade. One outcome of these changes is the interest that has been generated in the possibility of training surgical skills outside the operating theatre. Simulation of surgical procedures and human tissue, if perfect, would allow complete transfer of techniques learnt in a skills laboratory directly to the operating theatre. Several techniques of simulation are available including artificial tissues, animal models and virtual reality computer simulation. Each is discussed in this article and their advantages and disadvantages considered.
Erythrodermic CTCL: a case study of treatment with extracorporeal photopheresis.
Erythrodermic cutaneous t-cell lymphoma (CTCL) is an aggressive variant of CTCL with multiple skin manifestations and symptoms. A brief overview of CTCL and a case study of one patient with erythrodermic CTCL who underwent successful treatment in a research protocol with extracorporeal photopheresis and UVADEX are presented. Nursing care, critical for patients with erythrodermic CTCL, is also described.
Dichromatic staining of electrophoretically separated extracellular matrix macromolecules.
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The effect of a second-generation 3D endoscope on the laparoscopic precision of novices and experienced surgeons.
BACKGROUND: Endoscopic surgeons rely on visual feedback to control their movements but lack stereopsis, an important depth cue. Previous three-dimensional (3D) systems alternated images on a two-dimensional (2D) screen, which was uncomfortable for surgeons. A second-generation 3D system provides continuous stereoscopic images on a monitor suspended at arm's length. We studied its effect on the laparoscopic precision of novices and experienced surgeons. METHODS: Experienced laparoscopic surgeons (n = 12) and novices (n = 16) performed a total of 672 tasks in 2D, 3D, and under direct vision. Precision was assessed using the Imperial College Surgical Assessment Device (ICSAD), which generates objective scores of performance by analyzing the movements of surgical instruments. RESULTS: We found that 2D endoscopic vision impaired performance by 35-100% when compared with direct vision, whereas 3D reduced this endoscopic handicap by 41-53% in novices and experienced surgeons (p < 0.03). No side effects were reported with the new 3D system. Even in 2D, novices performed better with an image at arm's length (p < 0. 03). CONCLUSIONS: Second-generation 3D significantly improved the laparoscopic precision of novices and experienced surgeons, without the side effects reported from previous systems. This technology is expected to improve the ease and safety of laparoscopic surgery.
Functional analysis of the FimE integrase of Escherichia coli K-12: isolation of mutant derivatives with altered DNA inversion preferences.
Phase variable expression of type 1 fimbriae in Escherichia coli arises from a site-specific recombination event that inverts a short segment of chromosomal DNA carrying the promoter for transcription of the gene encoding the fimbrial subunit protein. Two integrase-like recombinases are involved in switching. The FimB recombinase inverts the DNA segment in either orientation, whereas the FimE protein inverts it predominantly in the ON-to-OFF direction. In this paper, we report the isolation of a FimE mutant protein that has enhanced bidirectional switching activity. This protein has an arginine-to-lysine substitution at position 59, and this confers a FimB-like switching character on FimE without altering its ability to bind to DNA. The arginine was not a member of the arginine-histidine-arginine-tyrosine catalytic tetrad that is common to all integrase-like recombinases. The catalytic tetrad members of FimE were identified at positions 41, 136, 139 and 171 and shown to be essential for FimE function. In addition, other amino acid residues that make important contributions to the DNA binding activity of FimE or its ON-to-OFF inversion efficiency were identified.
Objective assessment of surgical dexterity using simulators.
Surgical skill, like training for any craft, has traditionally been learnt as an apprenticeship. However, whereas carpenters learn on wood that is never displayed, operative training is done on real clinical cases. Over recent years surgical skills training laboratories have in part, replaced the apprenticeship. This article discusses some of the tools used within such laboratories to ensure optimal surgical performance.
The polyepitope approach to DNA vaccination.
The identification of the epitope as the smallest immunogenic subunit derived from antigenic proteins has promoted the development of epitope-based vaccines. These prevent the danger of administering whole proteins or genes that have unknown and possibly dangerous properties. Recent data suggest that DNA encoded epitopes in synthetic constructs can be processed and presented to CD8+ T-lymphocytes despite unnatural flanking amino acid sequences. This has allowed the development of polyepitope vaccines for cancer and infectious disease which induce multiple cytotoxic T-lymphocyte responses. The resultant immunity may be restricted to various HLA alleles and targeted to numerous antigens to avoid escape from immune detection by antigen loss variants. This review will describe the background of epitope and polyepitope-based vaccination strategies and the more recent results that confirm the potential of DNA encoded polyepitope vaccines. Future directions that may aid the design of more effective polyepitopes will also be discussed.
Hepatocellular toxicosis associated with administration of carprofen in 21 dogs.
A diagnosis of hepatocellular toxicosis attributable to carprofen administration was made in 21 dogs on the basis of development of clinical signs and clinicopathologic abnormalities associated with hepatic disease and histopathologic documentation of hepatic necrosis. Clinical signs of toxicosis were anorexia, vomiting, and icterus. Hyperbilirubinemia and high serum activities of alanine transaminase, alkaline phosphatase, and aspartate transaminase were the most notable clinicopathologic abnormalities. In 7 of 9 dogs in which urinalyses were performed, abnormalities suggestive of renal tubular disease were detected. Clinical course of toxicosis was variable; however, most dogs had resolution of clinical signs and improvement or resolution of biochemical abnormalities with discontinuation of the drug and administration of supportive care. As with any medication, clients should be informed of possible adverse effects and reactions associated with administration of carprofen. In the event of those signs, clients should be instructed to immediately discontinue administration of carprofen to their dog and contact their veterinarian.
Two highly related regulatory proteins, Shigella flexneri VirF and enterotoxigenic Escherichia coli Rns, have common and distinct regulatory properties.
The Rns protein of enterotoxigenic Escherichia coli (ETEC) and the VirF protein of Shigella flexneri are members of the AraC family of transcription regulators. Rns is required for positive activation of the CS1 fimbrial genes, while VirF is a positive regulator of an invasion gene regulon. The amino acid sequences of the proteins are 36% identical, and both proteins activate transcription in response to increases in temperature. Here, we show that Rns is capable of complementing fully a null mutation in the S. flexneri virF gene. However, the VirF protein cannot replace Rns as an activator of CS1 gene expression in ETEC. This failure is not due to the absence from ETEC of a co-factor required by VirF since it also occurs when the CS1 system is moved into an S. flexneri genetic background. Nor is it a function of growth medium composition or a failure in virF gene expression. Instead, these findings point to important differences in the mechanisms by which these related transcription factors regulate gene expression in Gram-negative pathogens.
Control of Escherichia coli type 1 fimbrial gene expression in stationary phase: a negative role for RpoS.
Expression of type 1 fimbriae in Escherichia coli is subject to phase-dependent control, with many regulatory inputs from proteins which organise or rearrange the structure of DNA. Inversion of a DNA segment carrying the promoter for expression of fimA, the gene encoding the fimbrial subunit protein, makes a key contribution to the switching of cells between fimbriate and afimbriate states. We have discovered that transcription of fimA is repressed as cells enter stationary phase. This repression is not seen in isogenic strains deficient in rpoS, the gene coding for the stationary phase-specific sigma factor RpoS. RpoS-deficient strains are also altered in the frequency of inversion of the fimA promoter segment. In the strains used in this study, inversion is catalysed by an integrase-like site-specific recombinase encoded by the fimB gene. We report that fimB transcription is repressed strongly as wild-type cells enter stationary phase but that this repression is alleviated in cells deficient in RpoS. These data suggest that RpoS has a negative regulatory role which may be indirect, at both the fimA and the fimB promoters.