Merkel cell polyomavirus-specific CD8 T cells in Merkel cell carcinoma: T cell receptor diversity & novel immune therapies

Thesis (Ph.D.)--University of Washington, 2016-06

Joining forces - Combining cognition-targeted motor control training with group or individual pain physiology education: A successful treatment for chronic low back pain

Chronic unremittent low back pain (LBP) is characterised by cognitive barriers to treatment. Combining a motor control training approach with individualised education about pain physiology is effective in this group of patients. This randomized comparative trial (i) evaluates an approach to motor control acquisition and training that considers the complexities of the relationship between pain and motor output, and (ii) compares the efficacy and cost of individualized and group pain physiology education. After an "ongoing usual treatment" period, patients participated in a 4-week motor control and pain physiology education program. Patients received four one-hour individualized education sessions (IE) or one 4-hour group lecture (GE). Both groups reduced pain (numerical rating scale) and disability (Roland Morris Disability Questionnaire). IE showed bigger decreases, which were maintained at 12 months (P < 0.05 for all). The combined motor control and education approach is effective. Although group education imparts a lesser effect, it may be more cost-efficient. [ABSTRACT FROM AUTHOR]

Reaching out to promote physical activity in Australia: A statewide randomized controlled trial of a stage-targeted intervention

Purpose. This study examined the broader use of a print-media intervention, which was previously shown to be effective at promoting physical activity to participants recruited from a regional Australian community, as a strategy suitable for a more diverse statewide Population sample. Methods. Participants were randomly selected adults who responded to a telephone interview conducted by the New South Wales Health Department and consented to Participate in a randomized controlled trial. Consenters were allocated to either intervention (n = 361) or control (n = 358) conditions. The intervention, a personalized letter plus stage-targeted booklets, was sent 1 week postbaseline. Data were collected via telephone inter view at baseline and 2 and 8 months and were analyzed using repeated measures analysis of variance (ANOVA) and chi(2) statistics. Results. The groups were similar at baseline (mean age 43 +/- 3 years; 64% women). Process evaluation showed high intervention recall (76% at 2 months) and high follow-up response rules (>85% at 8 months) were achieved. Nonsignificant increases in physical activity were observed (F-1,F-719 = 2.18, p =.14). Discussion. A single mailing of stage-targeted print materials was not effective in promoting increases in physical activity among participants selected from the statewide population. Future research could. examine how the effectiveness of print media might be enhanced, possibly by using supplementary media, community-based Prompts, or other incentives.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Genetic and economic analysis of a targeted marker-assisted wheat breeding strategy

The advent of molecular markers as a tool to aid selection has provided plant breeders with the opportunity to rapidly deliver superior genetic solutions to problems in agricultural production systems. However, a major constraint to the implementation of marker-assisted selection (MAS) in pragmatic breeding programs in the past has been the perceived high relative cost of MAS compared to conventional phenotypic selection. In this paper, computer simulation was used to design a genetically effective and economically efficient marker-assisted breeding strategy aimed at a specific outcome. Under investigation was a strategy involving the integration of both restricted backcrossing and doubled haploid (DH) technology. The point at which molecular markers are applied in a selection strategy can be critical to the effectiveness and cost efficiency of that strategy. The application of molecular markers was considered at three phases in the strategy: allele enrichment in the BC1F1 population, gene selection at the haploid stage and the selection for recurrent parent background of DHs prior to field testing. Overall, incorporating MAS at all three stages was the most effective, in terms of delivering a high frequency of desired outcomes and at combining the selected favourable rust resistance, end use quality and grain yield alleles. However, when costs were included in the model the combination of MAS at the BC1F1 and haploid stage was identified as the optimal strategy. A detailed economic analysis showed that incorporation of marker selection at these two stages not only increased genetic gain over the phenotypic alternative but actually reduced the over all cost by 40%.

Tailoring access to high cost, genetically targeted drugs

Assessment of real cost effectiveness, with data linked to individual health outcomes while protecting patient privacy, is an essential challenge we need to meet

Empiric management of community-acquired pneumonia in Australian emergency departments

Objective: To describe empiric community-acquired pneumonia (CAP) management in Australian hospital emergency departments (EDs) and evaluate this against national guidelines, including use of the pneumonia severity index and antibiotic selection. Design: A multicentre, cross-sectional, retrospective audit, April 2003 to February 2005. Setting: 37 Australian hospitals: 22 principal referral hospitals, six large major city hospitals, four large regional hospitals, four medium hospitals and one private hospital. Participants: Adult patients with a diagnosis of CAP made in the ED. Data on 20 consecutive CAP ED presentations were collected in participating hospitals. Outcome measures: Documented use of the pneumonia severity index, initial antibiotic therapy prescribed in the ED, average length of stay, inpatient mortality, and concordance with national guidelines. Results: 691 CAP presentations were included. Pneumonia severity index use was documented in 5% of cases. Antibiotic therapy covering common bacterial causes of CAP was prescribed in 67% of presentations, although overall concordance with national guidelines was 18%. Antibiotic prescribing was discordant due to inadequate empiric antimicrobial cover, allergy status (including contraindication to penicillin), inappropriate route of administration and/or inappropriate antibiotic choice according to recommendations. There was no significant difference between concordant and discordant antibiotic prescribing episodes in average length of stay (5.0 v 5.7 days; P=0.22) or inpatient mortality (1.6% v 4.1%; chi(2) = 1.82; P=0.18). Conclusions: Antibiotic therapy for CAP prescribed in Australian EDs varied. Concordance with national CAP guidelines was generally low. Targeted interventions are required to improve concordance.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

New insights into growth hormone action

It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.

N-type calcium channel blockers: Novel therapeutics for the treatment of pain

Highly selective Cav2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (ω-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Cav2.2 calcium channel have met with varied success. This review focuses on the properties of the Cav2.2 calcium channel in different pain states, the action of ω-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Cav2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

The HYP program - Targeted motivational interviewing for adolescent violations of school tobacco policy

Objective. To evaluate the efficacy of a short-term tobacco-focused intervention for high school students referred by school administrators because of tobacco use. Method. A sample of 56 adolescents (66% male, mean age 15 years) was recruited through referrals from three state high schools. Participants were randomly assigned to a one-hour motivational interview (MI) session or to standard care (advice/education). The two groups were followed up at one, three, and six-month intervals. Results. The MI intervention resulted in significant short-term reductions in quantity and frequency of smoking relative to standard care, however, effects were not maintained at 3- and 6-month follow-up. Improvements in refusal self-efficacy were significant relative to standard care. Conclusion. For adolescents who are established smokers and at high risk of other problems, motivational interviewing was associated with modest short-term gains relative to standard care. (c) 2006 Elsevier Inc. All rights reserved.