Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

Computational models for improved diagnosis and prognosis of stroke using robot-based biomarkers

Stroke is a leading cause of death and permanent disability worldwide, affecting millions of individuals. Traditional clinical scores for assessment of stroke-related impairments are inherently subjective and limited by inter-rater and intra-rater reliability, as well as floor and ceiling effects. In contrast, robotic technologies provide objective, highly repeatable tools for quantification of neurological impairments following stroke. KINARM is an exoskeleton robotic device that provides objective, reliable tools for assessment of sensorimotor, proprioceptive and cognitive brain function by means of a battery of behavioral tasks. As such, KINARM is particularly useful for assessment of neurological impairments following stroke. This thesis introduces a computational framework for assessment of neurological impairments using the data provided by KINARM. This is done by achieving two main objectives. First, to investigate how robotic measurements can be used to estimate current and future abilities to perform daily activities for subjects with stroke. We are able to predict clinical scores related to activities of daily living at present and future time points using a set of robotic biomarkers. The findings of this analysis provide a proof of principle that robotic evaluation can be an effective tool for clinical decision support and target-based rehabilitation therapy. The second main objective of this thesis is to address the emerging problem of long assessment time, which can potentially lead to fatigue when assessing subjects with stroke. To address this issue, we examine two time reduction strategies. The first strategy focuses on task selection, whereby KINARM tasks are arranged in a hierarchical structure so that an earlier task in the assessment procedure can be used to decide whether or not subsequent tasks should be performed. The second strategy focuses on time reduction on the longest two individual KINARM tasks. Both reduction strategies are shown to provide significant time savings, ranging from 30% to 90% using task selection and 50% using individual task reductions, thereby establishing a framework for reduction of assessment time on a broader set of KINARM tasks. All in all, findings of this thesis establish an improved platform for diagnosis and prognosis of stroke using robot-based biomarkers.

Graded Exercise Therapy Guided Self-Help Trial for Patients with Chronic Fatigue Syndrome (GETSET): Protocol for a Randomized Controlled Trial and Interview Study

Background: Chronic fatigue syndrome, also known as myalgic encephalomyelitis (CFS/ME), is characterized by chronic disabling fatigue and other symptoms, which are not explained by an alternative diagnosis. Previous trials have suggested that graded exercise therapy (GET) is an effective and safe treatment. GET itself is therapist-intensive with limited availability. Objective: While guided self-help based on cognitive behavior therapy appears helpful to patients, Guided graded Exercise Self-help (GES) is yet to be tested. Methods: This pragmatic randomized controlled trial is set within 2 specialist CFS/ME services in the South of England. Adults attending secondary care clinics with National Institute for Health and Clinical Excellence (NICE)-defined CFS/ME (N=218) will be randomly allocated to specialist medical care (SMC) or SMC plus GES while on a waiting list for therapist-delivered rehabilitation. GES will consist of a structured booklet describing a 6-step graded exercise program, supported by up to 4 face-to-face/telephone/Skype™ consultations with a GES-trained physiotherapist (no more than 90 minutes in total) over 8 weeks. The primary outcomes at 12-weeks after randomization will be physical function (SF-36 physical functioning subscale) and fatigue (Chalder Fatigue Questionnaire). Secondary outcomes will include healthcare costs, adverse outcomes, and self-rated global impression change scores. We will follow up all participants until 1 year after randomization. We will also undertake qualitative interviews of a sample of participants who received GES, looking at perceptions and experiences of those who improved and worsened. Results: The project was funded in 2011 and enrolment was completed in December 2014, with follow-up completed in March 2016. Data analysis is currently underway and the first results are expected to be submitted soon. Conclusions: This study will indicate whether adding GES to SMC will benefit patients who often spend many months waiting for rehabilitative therapy with little or no improvement being made during that time. The study will indicate whether this type of guided self-management is cost-effective and safe. If this trial shows GES to be acceptable, safe, and comparatively effective, the GES booklet could be made available on the Internet as a practitioner and therapist resource for clinics to recommend, with the caveat that patients also be supported with guidance from a trained physiotherapist. The pragmatic approach in this trial means that GES findings will be generalizable to usual National Health Service (NHS) practice.

Effects of a positive emotion-based adjuvant psychological therapy in colorectal cancer patients: a pilot study

Objective: To examine the effectiveness of an “Enhancing Positive Emotions Procedure” (EPEP) based on positive psychology and cognitive behavioral therapy in relieving distress at the time of adjuvant chemotherapy treatment in colorectal cancer patients (CRC). It is expected that EPEP will increase quality of life and positive affect in CRC patients during chemotherapy treatment intervention and at 1 month follow-up.Method: A group of 24 CRC patients received the EPEP procedure (intervention group), whereas another group of 20 CRC patients did not receive the EPEP (control group). Quality of life (EORTC-QLQC30), and mood (PANAS) were assessed in three moments: prior to enter the study (T1), at the end of the time required to apply the EPEP (T2, 6 weeks after T1), and, at follow-up (T3, one-month after T2). Patient’s assessments of the EPEP (improving in mood states, and significance of the attention received) were assessed with Lickert scales.Results: Insomnia was reduced in the intervention group. Treatment group had better scores on positive affect although there were no significantly differences between groups and over time. There was a trend to better scores at T2 and T3 for the intervention group on global health status, physical, role, and social functioning scales. Patients stated that positive mood was enhanced and that EPEP was an important resource.Conclusions: CRC patients receiving EPEP during chemotherapy believed that this intervention was important. Furthermore, EPEP seems to improve positive affect and quality of life. EPEP has potential benefits, and its implementation to CRC patients should be considered.

Virtual Clinical Trials in PET Imaging for Improved Diagnosis of and Evaluation of Therapies for Cancer

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

Small bowel pseudomelanosis and oral iron therapy

Small bowel pseudomelanosis is a rarely reported clinical entity characterized by brown pigmentation of small bowel mucosa. The authors describe two cases, both with iron deficiency anemia, one of an 81-year-old female patient submitted for capsule endoscopy that revealed a brown pigmentation of all small bowel mucosa and another of an 81-year-old male whose retrograde double-balloon enteroscopy revealed a diffuse brown pattern of small bowel mucosa. Ileal biopsies confirmed intense iron deposition in the macrophages of the lamina propria. Both patients were on oral iron therapy and the second one had a previous double-balloon enteroscopy, 2 years earlier, which revealed only ileal angiodysplasias. These two cases demonstrate the importance of two new endoscopic methods for diagnosis of small bowel pseudomelanosis, the rarity of such an entity and its close relation with oral iron therapy.

Long Term Therapy with Lenalidomide in a patient with POEMS Syndrome

Lenalidomide is an effective therapy against malignant plasma cells and a potent agent against proinflammatory and proangiogenic cytokines. The use of lenalidomide in POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein with plasma cells, skin changes) has been reported, but its benefit in long-term use is not well established. A 55-year-old man with POEMS and debilitating polyneuropathy was treated with lenalidomide and dexamethasone followed by maintenance lenalidomide. He remains in haematologic remission and in complete recovery of functional status 3.5 years after diagnosis. This case supports the long-term use of lenalidomide in patients with POEMS syndrome.

Cutaneous primary B-cell lymphomas: from diagnosis to treatment

Primary cutaneous B-cell lymphomas are a heterogeneous group of mature B-cells neoplasms with tropism for the skin, whose biology and clinical course differ significantly from the equivalent nodal lymphomas. The most indolent forms comprise the primary cutaneous marginal zone and follicle center B-cell lymphomas that despite the excellent prognosis have cutaneous recurrences very commonly. The most aggressive forms include the primary cutaneous large B-cell lymphomas, consisting in two major groups: the leg type, with poor prognosis, and others, the latter representing a heterogeneous group of lymphomas from which specific entities are supposed to be individualized over time, such as intravascular large B-cell lymphomas. Treatment may include surgical excision, radiotherapy, antibiotics, corticosteroids, interferon, monoclonal antibodies and chemotherapy, depending on the type of lymphoma and on the type and location of the skin lesions. In subtypes with good prognosis is contraindicated overtreatment and in those associated with a worse prognosis the recommended therapy relies on CHOP-like regimens associated with rituximab, assisted or not with local radiotherapy. We review the primary cutaneous B-cell lymphomas, remembering the diagnostic criteria, differential diagnosis, classification, and prognostic factors and presenting the available therapies.

Cardiac magnetic resonance imaging in the diagnosis and determination of outcome of pulmonary hypertension

Pulmonary hypertension (PH) is a rare but serious condition that causes progressive right ventricular (RV) failure and death. PH may be idiopathic, associated with underlying connective-tissue disease or hypoxic lung disease, and is also increasingly being observed in the setting of heart failure with preserved ejection fraction (HFpEF). The management of PH has been revolutionised by the recent development of new disease-targeted therapies which are beneficial in pulmonary arterial hypertension (PAH), but can be potentially harmful in PH due to left heart disease, so accurate diagnosis and classification of patients is essential. These PAH therapies improve exercise capacity and pulmonary haemodynamics, but their overall effect on the right ventricle remains unclear. Current practice in the UK is to assess treatment response with 6-minute walk test and NYHA functional class, neither of which truly reflects RV function. Cardiac magnetic resonance (CMR) imaging has been established as the gold standard for the evaluation of right ventricular structure and function, but it also allows a non-invasive and accurate study of the left heart. The aims of this thesis were to investigate the use of CMR in the diagnosis of PH, in the assessment of treatment response, and in predicting survival in idiopathic and connective-tissue disease associated PAH. In Chapter 3, a left atrial volume (LAV) threshold of 43 ml/m2 measured with CMR was able to distinguish idiopathic PAH from PH due to HFpEF (sensitivity 97%, specificity 100%). In Chapter 4, disease-targeted PAH therapy resulted in significant improvements in RV and left ventricular ejection fraction (p<0.001 and p=0.0007, respectively), RV stroke volume index (p<0.0001), and left ventricular end-diastolic volume index (p=0.0015). These corresponded to observed improvements in functional class and exercise capacity, although correlation coefficients between Δ 6MWD and Δ RVEF or Δ LVEDV were low. Finally, in Chapter 5, one-year and three-year survival was worse in CTD-PAH (75% and 53%) than in IPAH (83% and 74%), despite similar baseline clinical characteristics, lung function, pulmonary haemodynamics and treatment. Baseline right ventricular stroke volume index was an independent predictor of survival in both conditions. The presence of LV systolic dysfunction was of prognostic significance in CTD-PAH but not IPAH, and a higher LAV was observed in CTD-PAH suggesting a potential contribution from LV diastolic dysfunction in this group.

Does prenatal diagnosis modify neonatal treatment and early outcome of children with esophageal atresia?

International audience

Discovery and validation of serologic biomarkers for the diagnosis and prognosis of invasive candidiasis by computational immunomics

Invasive candidiasis (IC) is an opportunistic systemic mycosis caused by Candida species (commonly Candida albicans) that continues to pose a significant public health problem worldwide. Despite great advances in antifungal therapy and changes in clinical practices, IC remains a major infectious cause of morbidity and mortality in severely immunocompromised or critically ill patients, and further accounts for substantial healthcare costs. Its impact on patient clinical outcome and economic burden could be ameliorated by timely initiation of appropriate antifungal therapy. However, early detection of IC is extremely difficult because of its unspecific clinical signs and symptoms, and the inadequate accuracy and time delay of the currently available diagnostic or risk stratification methods. In consequence, the diagnosis of IC is often attained in advanced stages of infection (leading to delayed therapeutic interventions and ensuing poor clinical outcomes) or, unfortunately, at autopsy. In addition to the difficulties encountered in diagnosing IC at an early stage, the initial therapeutic decision-making process is also hindered by the insufficient accuracy of the currently available tools for predicting clinical outcomes in individual IC patients at presentation. Therefore, it is not surprising that clinicians are generally unable to early detect IC, and identify those IC patients who are most likely to suffer fatal clinical outcomes and may benefit from more personalized therapeutic strategies at presentation. Better diagnostic and prognostic biomarkers for IC are thus needed to improve the clinical management of this life-threatening and costly opportunistic fungal infection...

Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milk

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e Biomédicas

Can Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography Be Used As a Useful Method to Evaluate the Treatment Response to Neoadjuvant Therapy Combined With Sorafenib and Anti-VEGF in Children Diagnosed With Metastatical Bone Sarcoma?

Background: The prognosis is still poor for patients with a metastatic bone tumor and new treatment approaches (anti-VEGF and tyrosine kinase inhibitors vs) are therefore needed. Objectives: The aim of our study was to evaluate how the primary and metastatic lesions of our patients with a bone tumor were affected by these treatments and to determine the importance of the 18F-FDG PET method. Patients and Methods: Twenty metastatic bone tumor cases were included. Sorafenib and anti-VEGF were added to the standard treatment in cases with widespread metastatic disease at diagnosis or after neoadjuvant chemotherapy showing less than 90% tumor necrosis in the surgical sample. Positron emission tomography (PET) imaging was performed at diagnosis, the preoperative period following neoadjuvant chemotherapy, during postoperative follow-up, and when treatment was discontinued. Results: The primary treatment region median SUVmax level decreased from 7.35 to 2.5 in the living patients (n = 16) while there was no significant decrease in the patients who succumbed to the disease (P < 0.001). Comparison of the pre- and post-treatment metastasis region median SUVmax levels in patients with metastatic involvement showed a decrease from 2.1 to 0 in the surviving patients but only from 4.8 to 3.2 in the deceased patients (P < 0.01). Survival results indicated that 28.6% of the patients receiving classical treatment only died while all the patients receiving additional sorafenib and anti-VEGF survived. Conclusions: 18F-PET may be a useful technique before and during the follow-up of neoadjuvant treatment in pediatric metastatic bone tumor patients. The addition of sorafenib and anti-VEGF to classical treatment has a favorable contribution to the response and therefore the survival duration.