Magnetic microbeads are a common tool used in biological assay purification, however, they don’t diffuse quickly nor do they have a high surface area, which results in low binding efficiency. Magnetic nanoparticles have more total surface area and can diffuse quickly, but cannot be separated magnetically as individual particles. The research I do uses magnetic nanoparticles (MNPs) coupled with pNIPAAm (p(N-isopropylacrylamide)) as a temperature-responsive reagent system to purify solutions for diagnostic tests in order to increase the sensitivity and specificity. This works by combining MNPs coated with pNIPAAm and free pNIPAAM-antibody conjugates. The antigen binds to the antibody and when heated to the cloud point temperature (around 37°C for pNIPAAm), the polymers aggregate, allowing the MNPs and the antibody conjugates to be magnetically separated together. The conjugates are synthesized using NHS chemistry and purified through HPLC. The separation efficiency of these conjugates is determined though ELISA and can reach near 100% and the total aggregation and capture time for this system is less than 4 minutes. This is useful because it can rapidly concentrate and purify samples to reduce false positives and allow for a lower limit of detection. This can be incorporated into a multitude of point-of-care tests to deliver earlier, faster, and more accurate results.

Thirty-five million people worldwide currently live with HIV. Despite the availability of highly-active antiretroviral therapy (HAART) which may prolong the lifespan of HIV patients, HAART does not target the latent viral reservoir in infected cells, enabling viral rebound in the absence of treatment. Introducing genetically-engineered DNA cleavage enzymes (endonucleases) into HIV-infected cells may disrupt the integrated provirus. The HIV-directed site-specific DNA cutting activity of these endonucleases operating in conjunction with cellular error-prone DNA repair mechanisms can introduce targeted mutations in the integrated HIV genome that prevent viral reactivation. We are investigating three classes of engineered endonucleases: homing endonucleases (HEs), zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENS). While the DNA binding domains of these endonucleases allow for customization to a desired target sequence, delivering these enzymes to HIV-infected cells poses a challenge. TALENS in particular require a DNA coding sequence beyond the capacity of many viral vectors. Thus, a method of co-transduction must be pursued, whereby two halves of the endonuclease coding sequence are delivered by separate viral vectors and coordinately expressed in the target cell. My project aims to determine the optimal capsid serotype, promoter, and multiplicity of infection (MOI) for co-transducing HIV-infected cells using adeno-associated virus (AAV) vectors. I first determined AAV-1 to be the optimal serotype of AAV for infecting SupT1 cells, a cell line used as a model for HIV-infected T-cells. I then constructed two sets of AAV-1 viral vectors to express either Green Fluorescent Protein (GFP) or a red fluorescent protein (mCherry) as surrogates for endonuclease genes and infected SupT1 cells with both sets of vectors at variable MOIs, using flow cytometry to determine the rate of co-transduction. Through this method, I have successfully co-transduced cells with two AAV vectors. These results will have applications in future HIV research and clinical therapy.

The high antigenic diversity HIV-1 presents an enormous challenge for vaccine design as effective vaccines must be broad and potent in order to recognize and effectively counteract diverse circulating HIV strains. The identification of broadly neutralizing antibodies to recognize and counteract these diverse strains provides optimism for the ability to elicit broad and potent responses against HIV via vaccination. PGT128 is one such antibody and has been shown, in combination with other broadly neutralizing antibodies, to improve neutralization coverage of both viruses transmitted heterosexually and vertically, i.e. from mother to infant. Thus it is crucial that we understand the molecular determinants of the virus that leads to its sensitivity to broadly neutralizing antibodies. We studied viruses from longitudinal time points, one at 21 days post infection and one from 560 days post infection, of one infected individual who displayed increasing sensitivity to PGT128. Using nested PCR to create chimeras between the two viruses, we found that the loss of a single potential N-linked glycosylation site in the V1 region of HIV envelope conferred sensitivity to PGT128. This emphasizes the importance of N-linked glycosylation sites in conferring PGT128 recognition.

The current approach for FDA drug approval requires an exorbitant amount of time and money, often spent in pre-clinical animal studies, and has a high late-stage failure rate.  This is partly due to the fact that pharmaceutical companies develop drugs to treat the average patient with any given disease.  A personalized medicine approach would increase the effectiveness of a new drug for each patient and decrease the need for animal testing.  Induced pluripotent stem cells (iPSCs) are becoming the next-generation tool to study disease and discover new drugs.  Patient-specific somatic cells are reprogrammed to a stem-like state, and then differentiated in culture into the cell type most likely to manifest features of a particular disease.  High-throughput drug screens can be designed to find new compounds that can correct the disease defect in the culture dish.  Using this technology, we extracted somatic cells from the urine of patients with autistic syndrome disorder (ASD).  In addition to their cognitive deficiencies, these young patients suffer from chronic and debilitating constipation, which profoundly impacts their quality of life.  This lack of gut motility is suspected to be caused by a defect in enteric neuron function.  Our hypothesis is that the same synaptic malfunction causing cognitive deficits in ASD children also causes the enteric neuron defect.  Preliminary data also suggests that neural crest cells - an intermediate cell type in the enteric neuron lineage - can be generated using a combination of growth factors.  Therefore, I am conducting growth factor dose-response experiments on iPSCs to optimize neural crest cell differentiation.  I will phenotype the resultant cells via immunocytochemistry and qPCR analysis.  If successful, this project will lead to the discovery of new compounds able to relieve ASD patients of their constipation and further validate iPSCs as a tool for disease modeling and drug discovery.

Acute Lymphoblastic Leukemia (ALL) is a particularly aggressive hematologic malignancy. While highly curable in children and adolescents, treatment outcomes for adults are poor and novel therapies are needed. Increased evidence suggests that antibody-based therapeutics may be effective and relatively well tolerated in patients that failed to respond to conventional chemotherapeutics or experience disease relapse. Promising early clinical trials were obtained with inotuzumab ozogamicin (IO), an immunoconjugate consisting of an anti-CD22 antibody and a toxic calicheamicin moiety. IO specifically targets the CD22 receptor, a cell surface protein that is expressed by the lymphoid cells responsible for ALL. Nevertheless, despite the presence of these receptors on the majority of malignant cells, IO is not effective in many ALL patients. It is thought that IO exerts its anti-ALL effect through calicheamicin-induced DNA damage and subsequent apoptosis, but the exact molecular mechanisms remain poorly understood. I performed a series of phenotyping assays (n=3) using flow cytometry and chemical labelling to quantify the amount of CD22 receptors expressed on a panel of 8 well-characterized human ALL cell lines. I then executed IO and calicheamicin cytotoxicity assays (2 cycles of n=3) on these cell lines to identify which of these appeared to be IO- and/or calicheamicin-resistant. Future studies will be aimed at using these cell lines - and at a later stage - primary ALL specimens, to better understand the mechanism of action of IO and identify clinically relevant mechanisms of resistance. The longer-term goal is to develop strategies to improve the anti-ALL activity of IO to optimize treatment outcomes for ALL patients receiving IO-based therapies.

Colorectal cancer (CRC) is the third most prevalent (136,830 new cases estimated for 2014) and fatal cancers (50,310 deaths estimated) in the world, according to the American Cancer Society. Despite an expanding knowledge of its molecular pathogenesis during the past two decades, robust biomarkers to enable surveillance, screening, and primary prevention of CRC are still lacking. In the present study, we propose a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolic profiling approach for highly sensitive and specific colorectal cancer (CRC) detection using human serum samples. 158 metabolites from 25 metabolic pathways of potential significance were monitored. 234 serum samples from three groups of patients (66 CRC patients, 76 polyp patients, and 92 healthy controls) were analyzed. We detected 113 metabolites out of the 158 monitored, with 42 of them showing statistical significance between CRC cancer and healthy controls, 48 showing statistical difference between CRC cancer and polyp patients, and 8 between healthy controls and polyp patients. Partial least squares-discriminate analysis (PLS-DA) models established using 13 or 14 metabolites proved to be powerful for distinguishing CRC patients from either healthy controls or polyp patients, respectively. Receiver operator characteristic (ROC) curves generated based on these PLS-DA models gave high sensitivities, good specificities, low false discovery rates, and excellent areas under the curve (0.93 and 0.95 respectively, for differentiating CRC patients from healthy controls or polyp patients). Monte Carlo cross validation (MCCV) was also applied, demonstrating the robust diagnostic power of this metabolic profiling approach. To the best of our knowledge, this is the first time that an LC-MS/MS targeted serum metabolic profiling approach has been applied for comparing CRC patients to healthy controls and polyp patients, and our results demonstrate that a panel of serum metabolites enhanced by clinical factors (age, gender, smoking and alcohol status), can potentially serve as novel disease biomarkers for CRC diagnosis.

It is well known that tumors can be “seen” by the immune system, but immune responses differ depending on the nature and context of the target antigen: tumor antigens that are self-proteins are generally weakly immunogenic due to pre-existing self-tolerance, whereas tumor antigens that are truly tumor-specific (viral and mutated proteins) are potentially highly immunogenic because the immune system has not been previously exposed to these antigens. Therefore, tumor-specific T cells that recognize these antigens as non-self should be able to eliminate cancer cells expressing such tumor-specific neo-antigens. We developed a spontaneous Tamoxifen-inducible cancer mouse model to investigate the fate of naïve tumor-specific T cells encountering a tumor-specific neo-antigen during the pre-malignant phase of tumor development. We found, contrary to our expectation, that tumor-specific T cells were rendered tolerant and unresponsive to the cancer cell as early as 8 days post-tumor initiation and displayed very similar phenotypic and functional characteristics compared to self-tolerant T cells. Interestingly, this unresponsiveness was not due to global tumor-induced immune suppression in the microenvironment but instead the result of continuous antigen encounter, as tumor-infiltrating control T cells specific for an antigen not expressed by the cancer cell remained functional in the tumor site. Transcriptional profiling uncovered the molecular program underlying this early cancer antigen-induced T cell dysfunction. Thus, an antigenic stimulus in a non-inflammatory context, whether by a self-antigen or tumor-specific antigen, induces a distinct state of “tolerance-specific” functional unresponsiveness. While many immunotherapies aim to reverse the immune suppression mediated by the tumor microenvironment, we demonstrate that there is another level of antigen-specific, cell-intrinsic dysfunction that must also be overcome.