Until recently, B lymphocytes have not been thought to inhabit non-mucosal human skin. They have traditionally been viewed as major elements of the immune system strictly confined to blood and bone marrow. This study reports the novel finding of B cell infiltrates in herpes simplex virus 2 (HSV-2) skin lesion biopsies. Motivated by an interest in the underlying immune mechanisms responsible for the wide spectrum of disease seen in those suffering with reoccurring HSV-2 lesions, I examined the immune microenvironment of tissue biopsies from study participants with active herpes lesions and a history of frequent outbreaks. In order to study the local immune response of an HSV-2 reactivation episode, I analyzed skin sections of herpes lesions using fluorescence in situ hybridization (FISH) to demonstrate the cell marker and immunoglobulin expression profiles of skin-resident immune cells present at the time of herpes outbreaks. Through the use of FISH and advanced deconvolution microscopy, I proved that B cells of the human immune system can home directly to upper layers of the skin to fight HSV-2 reactivation. My work reveals the presence of immune cell populations in HSV-2 lesions showing RNA expression for CD20, CD138, CD4, IgG, IgM, and IgA. This study demonstrates that antibody-producing cells are recruited to sites of HSV-2 reactivation, secrete a variety of antibodies at the site of infection, and appear to interact closely with CD4+ T cells directly in skin. B cells may play a crucial role in the immune response required to effectively control HSV-2 reactivation, which has been underappreciated until now. My work suggests that individuals with immune systems that are less efficient at recruiting B cells to the upper layers of the skin to control HSV-2 infection may explain why some individuals suffer more severe manifestations of the disease than others throughout their lives.

Triple-negative breast cancer (TNBC), the most aggressive form of breast cancer that accounts for 15% of all breast cancers, tests negative for the three common breast cancer oncogene proteins– estrogen receptor, progesterone receptor and HER2. Without specific biomarkers to be targeted for treatment, TNBC treatments are limited to combinations of immunotherapy, chemotherapy, lumpectomy and radiation therapy. Alternative biomarkers such as the PD-L1, PD-L2 and CD-86 are utilized to explore more specific treatment options with minimal side effects. Detecting biomarkers in the tumor tissue typically utilizes immunohistochemistry (IHC), which exhibits limited quantitative precision, sensitivity and multiplex capability. To address the challenge, multiple reaction monitoring mass spectrometry (MRM-MS) has been developed for quantitating multiple tumor biomarkers simultaneously. Accurate detection of targeted biomarkers on the MRM-MS requires samples with high purity. However, the current microbead immunoprecipitation process results in a noticeable level of background impurity. To compensate for this impurity, the assay requires higher peptide yield by using at least 5 mg of tissue (around 100 µg of protein), which may not be available or can only be obtained with more invasive procedures. Additionally, the current biomarker extraction protocol is a 16 manual-step workflow that takes 38 hours. To speed up the biomarker purification process while using less tissue sample, our group has developed a biopsy tissue processing microfluidic device. Instead of magnetic microbeads, the device utilizes smart polymer reagents with higher capture efficiency to improve the peptide purification process and reduce the required sample size down to less than 5 mg of tissue. The device contains pre-loaded reagents, which streamlines the processes of deparaffinization, cell digestion, protein digestion and peptide purification to only 8 hours with 4 manual steps. The new device with the smart polymer reagents can potentially enable comprehensive tumor microenvironment characterization using MRM-MS with needle biopsy tissue.

In the Ratner Lab, our research focuses on engineered biomaterials and surface coatings for improving biocompatibility of implantable medical devices and tissue engineering. Currently, the long-term performance of implantable medical devices is limited by the body’s foreign body reaction (FBR). The body reacts to foreign materials in an inflammatory manner and ultimately encapsulate the device with a dense, avascular scar layer. The Ratner Lab has developed multiple strategies to reduce scarring and improve vascularization, including precision-engineered porous materials. The lab has discovered that materials with uniform 40 μm pores seamlessly heal within the body in a vascularized fashion. Previous research has mostly focused on biostable synthetic materials which remain stable in the body over the duration of implantation. My research will explore the potential of gelatin, a biodegradable, bioderived material, as a precision-engineered porous scaffold to promote healing. IL-4 is a cytokine that directs the inflammatory response towards a healing response. My research will also incorporate IL-4 into the biodegradable porous material to further enhance healing. Our long-term scientific goal is to enable complete regeneration of tissue by first promoting healthy blood vessels growth throughout the porous structure, then allowing the material to completely disappear (biodegrade) to make room for rest of the tissue to heal.

Microneedles are an effective method for transdermal delivery of a variety of pharmaceutically active agents primarily because of their ability to puncture the stratum corneum. Tissue puncture using microneedles also has potential to improve drug delivery at mucosal sites such as the buccal mucosa, where topical dosing is limited by a thick epithelial layer and continuous salivary flow. However, the low tissue stiffness and wide variance in epithelial thickness present in the oral mucosa preclude direct translation of currently available transdermal microneedle application methods. Further studies of microneedle drug delivery in the oral mucosa require methods for complete and reproducible microneedle application. This project aims to address this need with a device that can apply microneedles to the buccal mucosa with reproducible penetration depth and force, metrics which are correlated with delivery efficiency. The device is designed to be tunable to accommodate microneedle arrays with various dimensions and mechanical properties. Physical parameters of the device are optimized in silico via finite element analysis simulation of tissue puncture with a microneedle array. A prototype of the device is then evaluated using a tissue phantom model to assess penetration depth and force. Performance of the lead candidate device is then validated in tissue explants. This project provides insights for future improvements to microneedle application in the oral mucosa.

Natural Killer cells (NK) are rare, naturally-occurring white blood cells that attack tumor cells, virally-infected cells and cells that do not display a ‘self’ signal. Cancer cures may be possible if patient NK cells can be removed, then expanded and activated in ex vivo culture and given back in sufficient numbers to kill cancer cells. For this purpose, NK cells can be selected from blood for expansion or un-selected cells can be cultured in conditions that favor NK expansion over the other cells. Patient NK cells in bone marrow transplant recipients are responsible for donor graft failure , even though they have been irradiated as part of the patients conditioning regimen. This raises the possibility that NK cells are relatively radioresistant compared to T cells or NKT cells which also have NK function but retain T cell markers. The data presented here explores NK, T-cells and NKT responses to irradiation in an attempt to define these three distinct cell types and differentially kill them. Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation, then irradiated at 1, 2, 5, 5.625, 6.25, 7.5 or 10 Gray (Gy) with a 137Cs source delivering 5.7 Gy/minute. Both irradiated and non-irradiated control cells were labeled with a stable vital dye CFSE and placed into NK expansion cultures. After a week the number of NK, NKT and T-cells was determined by flow cytometry using CD3 and CD94 antibodies to define the cell types. ( CD3+ T cells, CD 94+ NK cells, and CD3/94+ NKT cells. Intensity of CFSE staining indicated the number of cell divisions that occurred since the level of dye/cell is reduced by ½ every cell division. T cell proliferation was the most affected by the irradiation. Dose responses to proliferation were similar for NK and NKT. However after 6.25 Gy both the NKT and T-cells ceased proliferating, whereas NK proliferation was still evident. In conclusion the CD3/CD94 + NKT cells behaved more like the innate immune system NK cells than the adaptive immune system T-cells up to 6.25 Gy. At higher doses the NK and NKT could be distinguished from each other. To eliminate T cells while retaining NK cells one could use between 5.625 and 6.25 Gy of irradiation at a dose rate of 5.7 Gy/min. Future studies will home in on the optimal dose of irradiation and possibly investigate lower dose-rates.

Liver disease is a significant worldwide health burden, causing over two million deaths worldwide each year. Currently, the only curative treatment for end-stage liver disease is transplant, but a critical organ shortage leaves many patients to die on a waiting list. Regenerative medicine is an important branch of translational research that aims to restore normal function to damaged tissue. To this end, our group and other researchers have developed micropatterned tissue constructs that combine engineered liver cells, bioactive molecules, and biomaterials that expand and restore tissue function after implantation. Despite the therapeutic promise of these tissue-mimetic implants, there remains a significant knowledge gap regarding the engineered tissue’s morphology and its comparability to human liver tissue architecture, severely limiting its clinical translation. Clinicians must know, ideally via quantitative methods, that they are implanting healthy engineered liver tissue, rather than something that inadvertently remodels in the body to recapitulate diseased liver tissue. To address this, I have developed a user-friendly, quantitative tissue morphology analysis software package to aid the informed design of next-generation engineered human liver tissue. This software combines deep learning, morphological transformations, and statistical quantitative image analysis to generate high-throughput, end-to-end histology image analysis pipelines. This software allows researchers to train a deep convolutional neural network that classifies and clusters single cells in both 2D images, as well as 3D volumes. To show the efficacy of this software, analysis pipelines were generated to characterize cellular distribution in mouse embryos and engineered liver tissue constructs. Importantly, this work will provide researchers across many disciplines with a tool to understand the microstructural differences between engineered tissue and native/natural tissue, enabling the development of more physiologically relevant and biomimetic tissue-based therapies and disease models.

The CRISPR-Cas system is an adaptive immune system that provides protection to bacteria against bacteriophages. This system offers a clear advantage for bacteria as they need to defend themselves against the threat of bacteriophages. However, bacteria that have CRISPR systems can experience autoimmune damage, if they take up a spacer targeting a lysogen incorporated into their genome. This threat gives bacteria an incentive to encode CRISPR inhibitors. Through a metagenomics screen to identify novel inhibitors of the CRISPR-Cas9 system, it was found that a bacterial nickel binding chaperone protein, HypA, inhibits Cas9. This unexpected discovery may offer insight into how bacteria regulate their CRISPR systems and prevent autoimmunity. Through my work, I have determined that a diverse set of homologs can inhibit Cas9. I have also determined that HypA may act to destabilize Cas9’s structure. In order to investigate whether HypA can prevent autoimmune damage, I co-expressed HypA and a self-targeting Cas9, and found that HypA can protect the bacterial genome from Cas9. This work may provide insight into the evolution and regulation of the CRISPR-Cas system in bacteria, by helping explain how bacteria navigate the challenges caused by having an immune system. This work may also illuminate why some bacteria retain and others lose their CRISPR systems when they encode self-targeting CRISPR spacers. 

Antiretroviral therapy (ART) cannot eliminate latently infected human immunodeficiency virus (HIV) reservoirs, the barrier to HIV cure. A “shock and kill” strategy has been proposed to cure HIV by using latency-reversing agents (LRAs) to reactive latent proviruses and allow for reservoir elimination. Due to the low potency and high toxicity of LRAs, none have yet been effective in reducing reservoir size in vivo. Here, we hypothesize that delivery of LRAs using nanocarriers (NCs) will improve drug solubility and safety, provide sustained drug release, and simultaneously deliver multiple drugs to reservoir tissues and cells. We developed hybrid nanocarriers to incorporate physicochemically diverse LRAs and target reservoirs in lymphatic CD4+ T cells. LRAs were formulated by physical encapsulation or covalent conjugation to the biodegradable polymer (PLGA) core. Drug combinations were evaluated in vitro using a J-Lat reporter cells and validated in CD4+ T cells from virologically suppressed patients. CD4+ T cell targeting specificity was tested ex vivo in non-human primate (NHP) peripheral blood mononuclear cells (PBMCs). Targeting and toxicity were also evaluated in vivo in mice following size optimization for increased passive drainage to lymph nodes. Optimized nanocarriers were used for identification of an LRA combination displaying synergistic latency reversal and low toxicity in vitro in model and patient cells. Long-term and specific activation of CD4+ T cells in NHP PMBCs ex vivo and in mouse lymph nodes in vivo was observed, with significant reduction in toxicity compared to free LRA delivery. This nanocarrier platform targets CD4+ T cells, successfully inducing latency reactivation in HIV reservoirs. The platform additionally enables new solutions for HIV cure with the potential to deliver anti-HIV agents, vaccines, immunomodulating agents, and gene-modifying oligonucleotide drugs.