Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.

Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.

The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.

Sentinel node biopsy for breast cancer: Using local results for estimation of risk to the patient

Background: Sentinel node biopsy (SNB) is being increasingly used but its place outside randomized trials has not yet been established. Methods: The first 114 sentinel node (SN) biopsies performed for breast cancer at the Princess Alexandra Hospital from March 1999 to June 2001 are presented. In 111 cases axillary dissection was also performed, allowing the accuracy of the technique to be assessed. A standard combination of preoperative lymphoscintigraphy, intraoperative gamma probe and injection of blue dye was used in most cases. Results are discussed in relation to the risk and potential consequences of understaging. Results: Where both probe and dye were used, the SN was identified in 90% of patients. A significant number of patients were treated in two stages and the technique was no less effective in patients who had SNB performed at a second operation after the primary tumour had already been removed. The interval from radioisotope injection to operation was very wide (between 2 and 22 h) and did not affect the outcome. Nodal metastases were present in 42 patients in whom an SN was found, and in 40 of these the SN was positive, giving a false negative rate of 4.8% (2/42), with the overall percentage of patients understaged being 2%. For this particular group as a whole, the increased risk of death due to systemic therapy being withheld as a consequence of understaging (if SNB alone had been employed) is estimated at less than 1/500. The risk for individuals will vary depending on other features of the particular primary tumour. Conclusion: For patients who elect to have the axilla staged using SNB alone, the risk and consequences of understaging need to be discussed. These risks can be estimated by allowing for the specific surgeon's false negative rate for the technique, and considering the likelihood of nodal metastases for a given tumour. There appears to be no disadvantage with performing SNB at a second operation after the primary tumour has already been removed. Clearly, for a large number of patients, SNB alone will be safe, but ideally participation in randomized trials should continue to be encouraged.

Audit of postoperative chemoradiotherapy as adjuvant therapy for resected gastroesophageal adenocarcinoma: An Australian multicentre experience

Background: Improved disease free and overall survivals were seen in curatively resected patients with gastric and gastroesophageal adenocarcinoma treated with the Intergroup 0116 (INT 0116) protocol of postoperative adjuvant chemoradiotherapy compared to surgery alone. This protocol has not been widely adopted in Australian centres because of perceived risks of toxicity. Methods: We reviewed the case records from 45 consecutive patients treated between May 1998 and August 2003 with the INT 0116 protocol and variations at five Australian institutions. The median age was 61.5 years (range 38-79). Twenty-nine patients had gastric and 12 had gastroesophageal junction primaries. All patients had attempted curative resection, however, seven had involved microscopic margins (R1 resection). Thirty-five had regional node involvement and none had evidence of distant metastasis. Results: The overall National Cancer Institute - Common Toxicity Criteria (NCI-CTC) version 2.0 grade 3 and grade 4 toxicity rates for all patients were 37.8% and 4.4%, respectively. There were no treatment related deaths. Gastrointestinal grade 3 toxicity was observed in 20% of patients, while haematologic grade 3 and 4 toxicity was observed in 17.8%. Toxicities experienced led to chemotherapy dose reductions in 22 patients and dose delay in 11 patients. Seven patients had a delay in radiotherapy and two did not proceed with radiotherapy. At a median follow up of 16 months (range 5-35) from surgery, 28 patients have relapsed (six with local recurrence alone) with 22 deaths occurring, all but one caused by cancer. Conclusion: The INT 0116 protocol is a safe and feasible schedule in a multicentre setting with an acceptable rate of toxicity and is an appropriate adjuvant treatment option for high-risk resected gastroesophageal adenocarcinoma.

Nociceptive scores and endorphin-containing cells reduced by low-level laser therapy (LLLT) in inflamed paws of Wistar rat

Objective: This study aimed to investigate how local pain relief is mediated by laser therapy and how dose affects the relationship. Methods: Inflammation was induced in the hind-paws of Wistar rats. Two groups of rats received 780-nm laser therapy (Spectra-Medics Pty Ltd.) at one of two doses (2.5 and 1 J/cm(2)). One group acted as a control. Scores of nociceptive threshold were recorded using paw pressure and paw thermal threshold measures. Results: A dose of 1 J/cm(2) had no statistically significant effect on antinociceptive responses. A dose of 2.5 J/cm(2) demonstrated a statistically significant effect on paw pressure threshold (p < 0.029) compared to controls. There was no difference in paw thermal threshold responses and paw volumes at either dose. Immunohistochemistry in control animals demonstrated normal beta-endorphin containing lymphocytes in control inflamed paws but no beta-endorphin containing lymphocytes in rats that received laser at 2.5 J/cm(2). Conclusion: The results confirm previous findings that the effect of laser therapy is dose-related. The mechanism of effect may occur via a differentiated pressure-sensitive neural pathway rather than a thermal-sensitive neural pathway. The significance of the immunohistochemistry findings remains unknown.

Gene therapy strategies for the treatment of spinal cord injury

Spinal cord injury is a complex pathology often resulting in functional impairment and paralysis. Gene therapy has emerged as a possible solution to the problems of limited neural tissue regeneration through the administration of factors promoting axonal growth, while also offering long-term local delivery of therapeutic molecules at the injury site. Of note, gene therapy is our response to the requirements of neural and glial cells following spinal cord injury, providing, in a time-dependent manner, growth substances for axonal regeneration and eliminating axonal growth inhibitors. Herein, we explore different gene therapy strategies, including targeting gene expression to modulate the presence of neurotrophic growth or survival factors and increase neural tissue plasticity. Special attention is given to describing advances in viral and non-viral gene delivery systems, as well as the available routes of gene delivery. Finally, we discuss the future of combinatorial gene therapies and give consideration to the implementation of gene therapy in humans. © 2014 Future Science Ltd.

Cognitive behaviour therapy for mothers of children with food allergy:a case series

Background: Food allergy affects quality of life in patients and parents and mothers report high levels of anxiety and stress. Cognitive Behaviour Therapy (CBT) may be helpful in reducing the psychological impact of food allergy. The aim of this study was to examine the appropriateness and effectiveness of CBT to improve psychological outcomes in parents of children with food allergy. Methods: Five parents (all mothers) from a local allergy clinic requested to have CBT; six mothers acted as controls and completed questionnaires only. CBT was individual and face-to face and lasted 12 weeks. All participants completed measures of anxiety and depression, worry, stress, general mental health, generic and food allergy specific quality of life at baseline and at 12 weeks. Results: Anxiety, depression and worry in the CBT group significantly reduced and overall mental health and QoL significantly improved from baseline to 12 weeks (all p < 0.05) in mothers in the CBT group; control group scores remained stable. Conclusions: CBT appears to be appropriate and effective in mothers of children with food allergy and a larger randomised control trial now needs to be conducted. Ways in which aspects of CBT can be incorporated into allergy clinic visits need investigation.

Role of Hormonal Treatment in Prostate Cancer Patients with Nonmetastatic Disease Recurrence After Local Curative Treatment : A Systematic Review

Peer reviewed

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Restoring function after neuromuscular trauma: novel paradigms for enhancing volitional control of neural activity in untethered environments and vHAB, a gamified therapy platform

Thesis (Ph.D.)--University of Washington, 2016-08

The use of Acceptance and Commitment Therapy to address psychological distress experienced by caregivers: a randomised controlled feasibility trial

Background: An extensive research literature has documented the impact of caring for an individual with acquired brain injury (ABI) on caregivers and family members, including role adjustment, psychological distress, social isolation, family tension and coping with the cognitive and behavioural difficulties of the injured person. Given these findings it is important this population have access to services and supports. Acceptance and Commitment Therapy (ACT) is an intervention that helps individuals to accept difficult experiences and commit to behaviour that is consistent with their values. Research into the effectiveness of ACT to support caregivers is at a preliminary stage. Aim: To investigate the feasibility of using ACT to reduce psychological distress and increase psychological flexibility in ABI caregivers. A secondary aim was to gain an understanding of the experience of caregivers in this context and how this can inform the development and delivery of interventions for this population. Method: Phase one was a randomised controlled feasibility trial of an ACT intervention for use with ABI caregivers. The parameters of this study were formulated around the PICO (population, intervention, control, and outcome) framework. Eighteen carers were recruited and randomised to ACT or an enhanced treatment as usual (ETAU) group. ACT was implemented over 3 sessions; and ETAU was implemented over 2 sessions. The General Health Questionnaire, Valuing Questionnaire, Acceptance and Action Questionnaire, Experiential Avoidance of Caregiving Questionnaire and the Flexibility of Responses to Self-Critical Thoughts Scale were administered to both groups at baseline and following the final session. Phase two used a retrospective qualitative design that involved conducting semi-structured interviews with four participants from phase one. Results: ACT and control participants were successfully recruited. Positive feedback was obtained from ACT participants suggesting that the intervention was acceptable. There were no significant differences between the ACT and ETAU groups on outcome measures. However, there were challenges retaining participants and the overall attrition rate was high (44.44%). Therefore a number of participants did not complete the full complement of sessions, which may have impacted on this result. Qualitative results illustrated the challenges this population face including significant adjustments in their life, the emotional impact of having a loved one with a brain injury and trying to adapt to the changes in the injured person. In addition, findings elucidated the types of support that this population would find helpful and the barriers to accessing same. Conclusions: Findings from this study highlight factors that will help the development of this intervention further for a caring population. Recommendations for future implementation include completing some preparatory work with carers before beginning the intervention, consideration of a larger sample and wider recruitment strategy from local services, barriers to attending interventions and the possibility of holding groups in local venues.

Multifactorial approach to non-viral gene therapy: development of an efficient system for the retina

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016