Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a response to burn injury.

Single patient (n-of-1) trials with binary treatment preference

The use of a fully parametric Bayesian method for analysing single patient trials based on the notion of treatment 'preference' is described. This Bayesian hierarchical modelling approach allows for full parameter uncertainty, use of prior information and the modelling of individual and patient sub-group structures. It provides updated probabilistic results for individual patients, and groups of patients with the same medical condition, as they are sequentially enrolled into individualized trials using the same medication alternatives. Two clinically interpretable criteria for determining a patient's response are detailed and illustrated using data from a previously published paper under two different prior information scenarios. Copyright (C) 2005 John Wiley & Sons, Ltd.

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved.

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n = 20) or placebo (n = 4) before and after vaccination. 11 cytokines were measured: IL- 1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, 1L- 10, IL- 12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient toot for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies. (c) 2005 Elsevier Ltd. All rights reserved.

Protection against group A streptococcal infection by vaccination with self-adjuvanting lipid core M protein peptides

We have investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting group A streptococcal (GAS) vaccine delivery approach. LCP constructs were synthesised incorporating peptides from the M protein conserved carboxy terminal C-repeat region, the amino terminal type-specific region and from both of these regions. Immunisation with the constructs without adjuvant led to the induction of peptide-specific serum IgG antibody responses, heterologous opsonic antibodies, and complete protection from GAS infection. These data indicate that protective immunity to GAS infection can be evoked using the self-adjuvanting LCP system, and point to the potential application of this system in human mucosal GAS vaccine development. (c) 2005 Elsevier Ltd. All rights reserved.

Fusing subunit antigens to interleukin-2 and encapsulating them in liposomes improves their antigenicity but not their protective efficacy

Subunit vaccines commonly lack sufficient immunogenicity to stimulate a comprehensive protective immune response in vivo. We have investigated the potential of specific cytokines (interleukin-2) and particulate delivery systems (liposomes) to enhance antigenicity. Here we report that the IgG1 and IFN-gamma responses to a subunit antigen, consisting of a T and B-cell epitope from Influenza haemagglutinin, can be improved when it is both fused to interelukin-2 and encapsulated in liposomes. However, this vaccine formulation was not able to protect animals against a challenge with live Influenza A/PR/8/34 virus. The addition of more potent immune stimulators may be necessary to improve responses. (c) 2005 Elsevier Ltd. All rights reserved.

Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

The effect of exercise on large artery haemodynamics in healthy young men

Background Brachial blood pressure predicts cardiovascular outcome at rest and during exercise. However, because of pulse pressure amplification, there is a marked difference between brachial pressure and central (aortic) pressure. Although central pressure is likely to have greater clinical importance, very little data exist regarding the central haemodynamic response to exercise. The aim of the present study was to determine the central and peripheral haemodynamic response to incremental aerobic exercise. Materials and methods Twelve healthy men aged 31 +/- 1 years (mean +/- SEM) exercised at 50%, 60%, 70% and 80% of their maximal heart rate (HRmax) on a bicycle ergometer. Central blood pressure and estimated aortic pulse wave velocity, assessed by timing of the reflected wave (T-R), were obtained noninvasively using pulse wave analysis. Pulse pressure amplification was defined as the ratio of peripheral to central pulse pressure and, to assess the influence of wave reflection on amplification, the ratio of peripheral pulse pressure to nonaugmented central pulse pressure (PPP : CDBP-P-1) was also calculated. Results During exercise, there was a significant, intensity-related, increase in mean arterial pressure and heart rate (P < 0.001). There was also a significant increase in pulse pressure amplification and in PPP : CDBP-P-1 (P < 0.001), but both were independent of exercise intensity. Estimated aortic pulse wave velocity increased during exercise (P < 0.001), indicating increased aortic stiffness. There was also a positive association between aortic pulse wave velocity and mean arterial pressure (r = 0.54; P < 0.001). Conclusions Exercise significantly increases pulse pressure amplification and estimated aortic stiffness.

Strength and endurance training lead to different post exercise glucose profiles in diabetic participants using a continuous subcutaneous glucose monitoring system

Background Although both strength training (ST) and endurance training (ET) seem to be beneficial in type 2 diabetes mellitus (T2D), little is known about post-exercise glucose profiles. The objective of the study was to report changes in blood glucose (BG) values after a 4-month ET and ST programme now that a device for continuous glucose monitoring has become available. Materials and methods Fifteen participants, comprising four men age 56.5 +/- 0.9 years and 11 women age 57.4 +/- 0.9 years with T2D, were monitored with the MiniMed (Northridge, CA, USA) continuous glucose monitoring system (CGMS) for 48 h before and after 4 months of ET or ST. The ST consisted of three sets at the beginning, increasing to six sets per week at the end of the training period, including all major muscle groups and ET performed with an intensity of maximal oxygen uptake of 60% and a volume beginning at 15 min and advancing to a maximum of 30 min three times a week. Results A total of 17 549 single BG measurements pretraining (619.7 +/- 39.8) and post-training (550.3 +/- 30.1) were recorded, correlating to an average of 585 +/- 25.3 potential measurements per participant at the beginning and at the end of the study. The change in BG-value between the beginning (132 mg dL(-1)) and the end (118 mg dL(-1)) for all participants was significant (P = 0.028). The improvement in BG-value for the ST programme was significant (P = 0.02) but for the ET no significant change was measured (P = 0.48). Glycaemic control improved in the ST group and the mean BG was reduced by 15.6% (Cl 3-25%). Conclusion In conclusion, the CGMS may be a useful tool in monitoring improvements in glycaemic control after different exercise programmes. Additionally, the CGMS may help to identify asymptomatic hypoglycaemia or hyperglycaemia after training programmes.

Total pharyngolaryngectomy for squamous cell carcinoma of the hypopharynx: A review

Objectives. To evaluate our experience with total pharyngolaryngectomy in the treatment of hypopharyngeal squamous cell carcinoma. Study Design: Retrospective analysis of consecutively treated patients in an academic otolaryngology, head and neck department. Methods. One hundred eighty patients who had total pharyngolaryngectomy performed for hypopharyngeal carcinoma were included in this study. Patients with a history of previous head and neck cancer were excluded. Clinicopathologic parameters were recorded and survival calculated using the Kaplan-Meier method. Results. One hundred sixty-two (90%) of the patients were male, and the patients had a mean age of 62 years. The majority (91%) of patients had advanced overall clinical stage disease (stage 3,4). Thirty-one (17.8%) and 43 (24%) patients developed locoregional and metastatic disease recurrence, respectively. The 2- and 5-year disease-specific survival rates were 72% and 52%, respectively. Advanced nodal stage, perineural invasion, lymphovascular invasion, and positive margins were predictors of poor survival on univariate analysis, and lymphovascular invasion was an independent prognostic factor on multivariate analysis. Conclusion: Surgery and postoperative radiotherapy remains the treatment against which other modalities should be compared for advanced stage hypopharyngeal squamous cell carcinoma.

Alpha B-crystallin, a new independent marker for poor prognosis in head and neck cancer

Objectives. Gene expression profiling has provided many insights into tumor progression but translation to clinical practice has been limited. We have previously identified a list of potential markers by the differences of expression profiling of seven matched head and neck cancer (HNSCC) tumors with autologous normal oral mucosa (NOM). Alpha B-crystallin (CRYAB) was in the top 5% of genes identified with statistically significant differences in expression between tumor and NOM at the mRNA level. The objective was to confirm this in routine paraffin sections at the protein level. Study Design: The level of alpha B-crystallin was determined in tumors of 62 HNSCC patients whose prognosis was known for 5 years. Methods. Immunohistochemical detection of alpha B-crystallin expression was performed on HNSCC paraffin sections. Results. Univariate survival analysis identified lack of alpha B-crystallin staining as an independent prognostic marker for disease-free interval (P < 0.001) and overall survival (P < 0.002) of HNSCC patients over the 5-year observation period. Notably, all 13 patients (100%), including 5 patients with nodal disease whose tumors lacked alpha B-crystallin had no recurrences (P < 0.001). Nineteen of 27 node-negative patients stained positive for alpha B-crystallin and seven of the 19 (36.8%) had recurrences. Conclusion: Presence or absence of expression of alpha B-crystallin was a powerful marker for prognosis in this series of patients.

Vaccination against fowl cholera with acapsular Pasteurella multocida A : 1

We have previously constructed an acapsular Pasteurella multocida X-73 (serogroup A) mutant strain which was attenuated in virulence for chickens (Chung JY, Wilkie IW, Boyce JD, Townsend KM, Frost AJ, Ghodussi M, Adler B. Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect. Immun. 2001;69:2487-2492). In this study, we have assessed the ability of this acapsular strain (PBA930) to induce protection against wild-type challenge in mice and the natural host chickens. Intramuscular administration of PBA930 to mice stimulated significant protection against X-73 and the heterologous strain P-1059 (A:3), but not against challenge with P-1662 (A:4). No protection was observed when PBA930 was introduced by the intraperitoneal or subcutaneous routes in mice. Significantly, the acapsular strain PBA930 was able to induce protection against challenge with wild type X-73 in chickens. (c) 2004 Elsevier Ltd. All rights reserved.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.