Development of Portable Diffuse Optical Spectroscopic Systems For Treatment Monitoring

The goal of this dissertation is to demonstrate the utility of portable, small-scale diffuse optical spectroscopic (DOS) systems for the diagnosis and treatment monitoring of various diseases. These systems employ near-infrared light (wavelength range of 650nm to 950nm) to probe human tissue and are sensitive to changes in scattering and absorption properties of tissues. The absorption is mainly influenced by the components of blood, namely oxy- and deoxy-hemoglobin (HbO2 and Hb) and parameters that can be derived from them (e.g. total hemoglobin concentration [THb] and oxygen saturation, StO2). Therefore, I focused on diseases in which these parameters change, which includes vascular diseases such as Peripheral Atrial Disease (PAD) and Infantile Hemangiomas (IH) as well as musculoskeletal autoimmune diseases such as Rheumatoid Arthritis (RA). In each of these specific diseases, current monitoring techniques are limited by their sensitivity to disease progression or simply do not exist as a quantitative metric. As part of this project, I first designed and built a wireless handheld DOS device (WHDD) that can perform DOS measurements at various tissue depths. This device was used in a 15-patient pilot study for infantile hemangiomas (IH) to differentiate diseased skin from normal skin and monitor the vascular changes during intervention. In another study, I compare the ultra-small form- factor WHDD’s ability to monitor synovitis and disease progression during a patient’s treatment of RA against the capabilities of a proven frequency domain optical tomographic (FDOT) system that has shown to differentiate patients with and without RA. Learning from clinical utility of the WHDD from these two studies, I adapted the WHDD technology to develop a compact multi- channel DOS measurement system to monitor perfusion changes in the lower extremities before and after surgical intervention for patients with peripheral artery disease (PAD). Using this multi- channel system, which we called the vascular optical spectroscopic measurement (VOSM) system, our group conducted a 20-subject pilot study to quantify its ability to monitor blood perfusion before and after revascularization of stenotic arteries in the lower extremities. This proof-of- concept study demonstrated how DOS may help vascular surgeons perform revascularization procedures in the operating room and assists in post-operative treatment monitoring of vascular diseases

Macrophage COX-2 As a Target For Imaging And Therapy of Inflammatory Diseases Using Theranostic Nanoemulsions

Personalized medicine can be an approach to address the unsatisfactory treatment outcomes in inflammatory conditions such as cancer, arthritis, and cardiovascular diseases. A common feature of chronic diseases is the infiltration of pro-inflammatory macrophages at the disease loci. Infiltrating macrophages have been previously utilized for disease diagnosis. These features suggest that macrophages can be broadly applicable targets for simultaneous therapy and diagnosis. Cyclooxygenase-2 (COX-2), an enzyme involved in the biosynthesis of a lipid inflammatory mediator, prostaglandin E2 (PGE2), is over expressed in macrophages infiltrating the pathological site. Inhibition of PGE2 leads to reduced inflammation, pain and macrophage infiltration. To utilize macrophages for the purpose of simultaneous therapy and diagnosis, we proposed to integrate therapeutic and imaging capabilities on a single nanomedicine platform, referred as theranostics. A stable 19F MRI visible nanoemulsion platform was developed, incorporating celecoxib for COX-2 inhibition and near-infrared fluorescent dye(s) for fluorescence imaging. We hypothesized that inhibition of COX-2 in macrophages using a theranostic nanoemulsion will reduce the inflammation (and pain), and that this response can be visualized by monitoring changes in macrophage infiltration. In vitro characterization demonstrated that the theranostic displays excellent stability with no toxicity, and significant uptake in macrophages. Furthermore, it delivers celecoxib to macrophages and reduces PGE2 production from these cells. In vivo studies in a murine paw inflammation model showed nanoemulsion presence at the inflamed site, specifically in COX-2 expressing macrophages compared to neutrophils. Supporting our hypothesis, celecoxib delivered through a nanoemulsion demonstrated time-dependent reduction in fluorescence from the inflamed paw, indicative of reduced macrophage infiltration. In a neuropathic pain model, celecoxib delivered to macrophages led to reduced pain concomitant with reduced macrophage infiltration at the inflamed site compared to free drug control (cross reference: Kiran Vasudeva, Dissertation, 2015). In conclusion, inhibition of COX-2 in macrophages using theranostic nanoemulsions proves to be an effective and generalized strategy facilitating simultaneous therapy and diagnosis, which can be applied to many chronic diseases. The diagnostic information during therapy can be used to tailor the treatment and reduce patient variability leading to personalized medicine

The role of PAD4 in periodontal disease, autoimmunity and inflammation

Periodontal disease (PD) and rheumatoid arthritis (RA) are multifactorial chronic inflammatory diseases with high prevalence among the global population. There is evidence of a bidirectional relationship between PD and RA, although the underlying mechanisms remain undefined. Both PD and RA are associated with a dysregulated immune response and citrullination, a post-translational modification of proteins catalysed by peptidylarginine deiminases (PADs). PADs, in particular PAD4, are involved in formation of neutrophil extracellular traps (NETs) and may play a role both in generating potential auto-antigens and in host defence against bacterial infections. RA onset is preceded by a breach of self-tolerance and presence of anti-citrullinated protein antibodies (ACPAs). These ACPA have also been found in PD patients. Porphyromonas gingivalis is a key pathogen in PD and uniquely among prokaryotes expresses a PAD enzyme (PPAD), which is also potential source of citrullinated self-antigens. One hypothesis linking PD and RA suggests that the combination of PPAD and PAD4 activity in an inflamed environment may predispose to autoimmunity to citrullinated proteins and generation of ACPAs. This project aimed to determine the effect of PAD4 activity in PD and RA disease progression. Using PAD4 deficient animals or wild type controls, PAD4 was confirmed to be essential for NETs formation as bone marrow derived neutrophils from PADi4 knockout (KO) mice were unable to generate NETs in vitro. Experimental PD was initiated by oral infection with P. gingivalis and animals demonstrated a robust antibody response to P. gingivalis. However, there was limited evidence of bone loss in the animals, possibly due to inherent resistance in the strain. The immune response to P. gingivalis appeared unaffected by absence of PAD4, implying that NETS do not play a substantial role in the response to oral infection in this system. In experimental arthritis (EA) models, inflammation in EA was greater in absence of PAD4. Further investigation of the underlying mechanisms of PAD4 modulation of inflammation showed no direct impact in the innate response mediated by neutrophils, but confirmed a sexually dimorphic behaviour in PAD4 regulation of T-cell mediated inflammation. Pharmacological inhibition of PAD4 has been proposed and trialled as an RA therapy. These data suggest that PAD4 may impart subtle modulations on inflammation, which may impact on the outcome of such intervention

In vitro assessment of the primary stability of the acetabular component in hip arthroplasty

In Europa, più di 700'000 interventi di artroplastica d’anca vengono effettuati annualmente. Il tasso di fallimento della chirurgia è del 2-8 % (a 10 anni). Di questo, più del 50% è dovuto alla mobilizzazione asettica della componente acetabolare (più che ad un fallimento legato alla componente femorale). Lo scopo centrale di questo progetto di tesi è quello di creare un pilot-test per la valutazione in vitro della stabilità primaria di una componente acetabolare commerciale, impiantata in una emipelvi sintetica (senza cemento, attraverso la procedura chirurgica press-fit). La valutazione dei micromovimenti prevede un approccio multiplo, costituito dall’utilizzo della Digital Image Correlation (DIC) e di sensori lineari di spostamento. Per adeguare e migliorare le prestazioni dei due strumenti di misura, lo studio prevede: (1.a) l’ottimizzazione delle misure ottenute dalla correlazione di immagini, (1.b) creare ed effettuare la procedura di calibrazione interna dei sensori di spostamento e l’ottimizzazione delle misure ottenute dai sensori stessi come monitor dell’intero pilot-test. La seconda parte del lavoro si prone di implementare una metodologia affidabile per il calcolo delle roto-traslazioni relative tra coppa e osso. La creazione di un algoritmo dedicato, prevede, quindi, di valutare: (2.a) la migrazione permanente e (2.b) i micromovimenti inducibili dai picchi di carico.L’utilizzo della correlazione di immagini è risultato un gran punto di forza dello studio. Grazie al potere della DIC nell’elaborare spostamenti e deformazioni a tutto campo, senza contatto e in stereofotogrammetria, per la prima volta è stato possibile ottenere informazioni 3D del vettore migrazione della coppa. Inoltre, creando una procedura ottimizzata dell’allineamento del provino sotto la macchina, si sono potute riferire tutte le misure ottenute dal pilot-test, all’Aneterior Pelvic Plane (sistema di riferimento di rilevanza clinica)