Bioengineered surfaces and hydrogels for specific cell capture and release from whole blood

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (leaves 45-47).Microfluidic affinity-based cell capture devices are presently able to isolate specific cell populations from heterogeneous samples, such as whole blood. The impact of this potentially powerful technology, however, is restricted by the fact that there is no reliable method to release the target cells from the capture surface while preserving their integrity. This work presents the development and evaluation of a functional hydrogel coating that supplements microfluidic capture devices to enable both specific capture and release. The hydrogels are formed by ionically crosslinking a microscale pre-functionalized alginate film on top of the capture substrate. After linking the antibody to the exposed functional sites, the gels may be used to capture cells of interest from physiological solutions. The captured cells may be released by applying a gentle chelating buffer which dissolves the gel, eliminating both the specific and the non-specific cell-surface interactions. This system was evaluated for its ability to capture cells from both buffer and blood. Capture efficiency was found to be equivalent to standard affinity-based devices, and the hydrogel system released 90% of the captured cells without affecting their viability. Finally, the system was validated by capturing and releasing rare circulating tumor cells (CTCs) from the whole blood of a prostate cancer patient; specific immunostaining indicated that the released cells were CTCs based on their expression of cytokeratin and prostate specific antigen. This technology has the promise to significantly influence both clinical diagnostics and basic medical research by enabling rapid enumeration and detailed genetic and phenotypic analysis of rare cell populations.by Ajay M. Shah.S.M

Technologies for the isolation of circulating tumor cells

Thesis (Ph. D. in Medical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 125-129).Metastasis, the spread and growth of tumor cells from the primary site to distant organs, is arguably the most devastating and deadly attribute of cancer, and is ultimately responsible for 90% of cancer-related deaths. Circulating tumor cells (CTCs) are exceedingly rare cells found in the whole blood of cancer patients which have the potential to serve as a 'blood biopsy'. The intricate characterization of these cells could result in an entire new class of therapies directly targeting metastasis. Present technologies enable only a susbset of potential analyses to be conducted, principally due to sub-optimal cell isolation sensitivity, purity, throughput, or handling method. Here, we present two novel technologies to address the challenge of CTC isolation. First, we build on affinity-based microfluidic cell capture platforms by developing sacrificial hydrogel coatings to enable the innocuous release of captured cells; we demonstrate that model CTCs captured from whole blood remain viable and proliferative following release and are compatible with downstream immunostaining and FISH analysis. Second, we present a novel cell sorting system that interrogates over 10 million individual events each second, resulting in a high throughput, ultra-efficient rare cell sorter that delivers enriched cells in a vial, readily compatible with virtually any downstream assay. This is the first system combining the high sensitivity and single cell resolution that is characteristic of FACS with the practicality of MACS at a throughput and specificity afforded by inertial focusing, enabling operation in both 'positive selection' and 'negative depletion' modes. We find greater than 90% cell isolation efficiencies with over 2.5 log depletion of contaminating WBCs. Furthermore, the system is applied to clinical patient samples, and proof-of-concept is demonstrated in a cohort of breast, lung and prostate patients. Working in a negative depletion mode to isolate target cells in an unbiased fashion, we used the system to assess single putative CTCs isolated from an endogenous pancreatic mouse model for gene expression of tumor markers. Initial data confirms CTC heterogeneity at the single cell level, and positions us to move forward with single cell transcriptome sequencing, which may reveal a broad array of CTC phenotypes including metastatic precursors.by Ajay Mukesh Shah.Ph.D.in Medical Engineerin

A prospective study of USG guided pigtail catheter drainage in management of liver abscess

Background: Treatment of the liver abscess of any etiology has evolved in the recent years. Percutaneous drainage of liver abscess has been an important advancement in the treatment of pyogenic liver abscesses. Aim: to evaluate and assess response, morbidity and complication rates of percutaneous pig tail catheter drainage in treatment of liver abscess.  Methods: During a period of 27 months, 25 patients with liquefied liver abscess ≥5x5 cm underwent percutaneous drainage under sonographic guidance.Results: 18 had solitary abscess, while 7 had multiple abscesses. Pigtail catheters of various sizes (10 F or 12 F) were introduced in these patients using the Seldinger technique. The volume of pus drained ranged from 150 to 400 ml, while the period of catheter drainage ranged from 6 to 17 days. Complications were minor and included catheter blockage in 2 patients and tract pain in 8 patients. There was no mortality associated with this procedure. This study shows a success rate of 96% (successful treatment in 24 out of 25 patients).Conclusion: Percutaneous catheter drainage of liver abscesses is successful with a low morbidity and mortality and should be the first line of management in liquefied moderate to large sized liver abscesses.

The regulation of cardiac intermediary metabolism by NADPH oxidases

NADPH oxidases (NOXs), enzymes whose primary function is to generate reactive oxygen species, are important regulators of the heart’s physiological function and response to pathological insults. The role of NOX-driven redox signalling in pathophysiological myocardial remodelling, including processes such as interstitial fibrosis, contractile dysfunction, cellular hypertrophy, and cell survival, is well recognized. While the NOX2 isoform promotes many detrimental effects, the NOX4 isoform has attracted considerable attention as a driver of adaptive stress responses both during pathology and under physiological states such as exercise. Recent studies have begun to define some of the NOX4-modulated mechanisms that may underlie these adaptive responses. In particular, novel functions of NOX4 in driving cellular metabolic changes have emerged. Alterations in cellular metabolism are a recognized hallmark of the heart’s response to physiological and pathological stresses. In this review, we highlight the emerging roles of NOX enzymes as important modulators of cellular intermediary metabolism in the heart, linking stress responses not only to myocardial energetics but also other functions. The novel interplay of NOX-modulated redox signalling pathways and intermediary metabolism in the heart is unravelling a new aspect of the fascinating biology of these enzymes which will inform a better understanding of how they drive adaptive responses. We also discuss the implications of these new findings for therapeutic approaches that target metabolism in cardiac disease

Synthesis of thyroglobulin in thyroid carcinoma patients after radioiodine therapy

Endogenously radioiodinated thyroglobulin (Tg) and the serum concentration of Tg have been measured in patients with metastatic thyroid carcinoma after therapeutic doses of radioiodine. Serial samples of blood were analyzed for both these parameters over a period of 10 to 22 days. The specific activity of Tg (cpm/ng) was calculated for each sample. Among the six patients studied, three showed constant specific activity. The specific activity of the other three fell, indicating the entering of newly synthesized Tg into the circulation. The respective amounts of Tg entering into the circulation in these three patients were 120, 852, and 20,935 ng/ml serum/day

Triple-Modal Imaging of Magnetically-Targeted Nanocapsules in Solid Tumours In Vivo

Triple-modal imaging magnetic nanocapsules, encapsulating hydrophobic superparamagnetic iron oxide nanoparticles, are formulated and used to magnetically target solid tumours after intravenous administration in tumour-bearing mice. The engineered magnetic polymeric nanocapsules m-NCs are ~200 nm in size with negative Zeta potential and shown to be spherical in shape. The loading efficiency of superparamagnetic iron oxide nanoparticles in the m-NC was ~100%. Up to ~3- and ~2.2-fold increase in tumour uptake at 1 and 24 h was achieved, when a static magnetic field was applied to the tumour for 1 hour. m-NCs, with multiple imaging probes (e.g. indocyanine green, superparamagnetic iron oxide nanoparticles and indium-111), were capable of triple-modal imaging (fluorescence/magnetic resonance/nuclear imaging) in vivo. Using triple-modal imaging is to overcome the intrinsic limitations of single modality imaging and provides complementary information on the spatial distribution of the nanocarrier within the tumour. The significant findings of this study could open up new research perspectives in using novel magnetically-responsive nanomaterials in magnetic-drug targeting combined with multi-modal imaging

Protein Disulfide Isomerase and Host-Pathogen Interaction

Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections