The primary cause of morbidity and mortality in cystic fibrosis is progressive pulmonary disease, an important component of which is a hyperactive inflammatory response, the mechanisms of which are not completely understood. Previous studies have highlighted alterations in polyunsaturated fatty acid and eicosanoid metabolism in cystic fibrosis airway epithelial cells leading to increased arachidonic acid and its metabolites, the pro-inflammatory leukotrienes and prostaglandins. This work tests the hypothesis that these underlying metabolic abnormalities play an important role in the excessive inflammatory response. Using airway epithelial cell culture models of cystic fibrosis, we demonstrate that there is increased release of AA in response to stimulation in CF cells. Additionally, we show that enzymes of fatty acid and eicosanoid metabolism, including Δ6-desaturase, cytosolic phospholipase A2, cyclooxygenase-2, microsomal PGE2 synthase, and 5-lipoxygenase, are upregulated in cystic fibrosis cells compared to wild-type counterparts. These increases are exaggerated when the cells are stimulated with products of Pseudomonas aeruginosa culture, leading to increased production of PGE2 and LTB4 and the pro-inflammatory cytokines IL-6 and IL-8. Inhibition of the LTB4 synthesis pathway at any of multiple points results in blunting of both cyclooxygenase-2 expression and IL-6 and IL-8 production, while inhibition of cyclooxygenase-2 itself results in only minor suppression of cytokine production. Similar results are seen with docosahexaenoic acid, an anti-inflammatory n-3 fatty acid. Taken together, these results indicate that fatty acid and eicosanoid metabolic abnormalities that are intrinsic to epithelial cells in cystic fibrosis, particularly the LTB4 synthesis pathway, may play an important role in the hyperactive inflammatory response characteristic of this disease.

Atherosclerosis is the driving force behind cardiovascular disease, the principal cause of death in the world today. To complement currently available interventions, such as statins which reduce risk of a major cardiac event by only approximately 30%, a search for atheroprotective genes was initiated. Our study uncovered SPRR3 as the first identified atheroprotective protein expressed specifically in atheroma VSMCs. Loss of SPRR3 led to increased atherosclerosis progression independently of bone-marrow-derived cell SPRR3 expression. However, the observed increase in atherosclerosis was associated with a reduction in maintenance of atheroma VSMC content. This reduced VSMC content occurred, at least in part, due to an Akt-dependent reduction in VSMC survival in SPRR3-deficient atheroprone animals. Loss of SPRR3 expression also contributed to features of atheroma instability, including increased lipid-rich core size, reduced fibrous cap collagen content, and reduced fibrous cap thickness in brachiocephalic artery atheromas. In coronary arteries, SPRR3 loss contributed to a significant increase in the burden of complex atheromas as compared to fatty streaks and intermediate stage atheromas. In vitro, we observed a reduction in VSMC collagen type I mRNA and protein synthesis in SPRR3-deficient VSMCs. Our data indicate that SPRR3 promotes an atheroprotective response in atheroma VSMCs by enhancing VSMC survival and increasing collagen type I synthesis. The atheroma-specific expression pattern of SPRR3, moreover, uniquely positions this protein as a potential source of interventions for delaying progression and promoting stability of atheromas.

Sepsis is a systemic inflammatory response to infection. It is often accompanied by the pathologic production of thrombin, the key enzyme in blood plasma responsible for coagulation, and by contact activation, which is a pro-inflammatory pathway in plasma. The project described in this dissertation focused on factor XI (FXI), the zymogen of FXIa, a protease that contributes to thrombin generation during coagulation. FXIa is formed by proteolytic cleavage of FXI by thrombin or by factor XIIa, a protease that is generated during contact activation. Building upon earlier work that indicated an influence of FXI on the pathology of murine sepsis, we investigated the importance of FXI to mortality and to the cytokine and coagulation responses after cecal ligation and puncture (CLP). Compared to wild type (WT) littermates, FXI-deficient (FXI-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in WT mice 24 hours post-CLP, but not in FXI-/- mice, supporting the impression of a reduced inflammatory response in the absence of FXI. There was no evidence of a severe coagulopathy after CLP, with a notable lack of enhanced thrombin generation in any of the mice. Plasma levels of the contact proteins factor XII (FXII) and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation, while levels of these proteins were not reduced in FXI-/- animals. Additional in vivo studies confirmed that FXI deficiency reduces contact activation, with in vitro analysis revealing that FXIa activates FXII in a reaction that is enhanced by polyanions such as polyphosphate and nucleic acids (which may be released during infection). Our data indicate that FXI deficiency confers a survival advantage after CLP by altering the cytokine response to infection and blunting activation of the contact system. The findings support a novel hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.

<p>Glioblastoma is the most common and aggressive malignant brain tumor among all primary brain and central nervous system (CNS) tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Medulloblastoma is another major malignant brain tumor that most frequently occurs in children. Although recent advances in surgery, radiotherapy, and chemotherapy have led to an increase in 5-year survival rates of medulloblastoma patients, treatment-related toxicity often has a major impact on long-term quality of survival. </p><p>As a result, there is an urgent need to develop more efficient and novel therapeutic approaches that specifically target tumor cells while preserving the surrounding normal CNS to improve the poor survival and quality of life of patients with malignant brain tumors. To address this need, we have developed two novel targeted immunotoxins (ITs), D2C7-(scdsFv)-PE38KDEL (D2C7-IT) and NZ-1-(scdsFv)-PE38KDEL (NZ-1-IT). D2C7-IT was developed by fusing the single-chain variable fragment (scFv) of the D2C7 monoclonal antibody (mAb) with domains II and III of Pseudomonas exotoxin A (PE38KDEL), and NZ-1-IT was developed by fusing the scFv of the NZ-1 mAb with PE38KDEL. D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and the EGFR variant III (EGFRvIII), two overexpressed proteins in glioblastomas. NZ-1-IT reacts with podoplanin (PDPN), a protein that has a high expression in glioblastomas and medulloblastomas. </p><p>In vitro cytotoxicity data shows that both ITs effectively inhibited protein synthesis in a variety of epitope-expressing glioblastoma and medulloblastoma xenograft cells and human tumor cell lines. Furthermore, the direct anti-tumor efficacy of D2C7-IT was examined in orthotopic glioma models in immunocompromised mice, while the direct anti-tumor efficacy of NZ-1-IT was observed in medulloblastoma xenograft-bearing immunocompromised mice. Both immunotoxins showed a robust anti-tumor efficacy in the preclinical brain tumor models. D2C7-IT was first investigated in the subsequent studies to accelerate its translation to the clinic. The preclinical toxicity of intracerebral D2C7-IT infusion was subsequently determined in normal Sprague-Dawley (SD) rats. The maximum tolerated dose (MTD) of D2C7-IT was determined to be between a total dose of 0.10 and 0.35 μg, and the no-observed-adverse-effect level (NOAEL) of D2C7-IT was a total dose of 0.05 μg in SD rats. Both the MTD and NOAEL were utilized as references for the D2C7-IT clinical trial design.</p><p>In addition to direct tumor cell killing, immunotoxin monotherapy has been shown to induce a secondary anti-tumor immune response through the engagement of T cells. Therefore, the D2C7-IT-induced secondary anti-tumor immune response was investigated using syngeneic mouse glioma models in immunocompetent mice. Moreover, previous studies have demonstrated that immune checkpoint inhibitors have a robust anti-tumor efficacy by augmenting the T cell response to the tumor cells. Thus, immune checkpoint inhibitors were combined with D2C7-IT in order to enhance the immunotoxin-induced anti-tumor immune response to eliminate residual tumor cells and prevent tumor recurrence in the long term. Meanwhile, studies with NZ-1-IT remain preliminary; thus, this IT will not be as robustly discussed as D2C7-IT throughout this text.</p> / Dissertation

Extracellular matrix (ECM) functions as the insoluble scaffold of tissues that controls cellular behaviors and regulates environmental cues that are essential for many aspect of biology. Basement membranes (BMs) are specialized forms of ECM comprised of a collection of proteins, including collagen IV and laminin, which establish a sheet-like supramolecular arrangement. BMs create barriers between cell populations and modulate cellular crosstalk by underling polarized cells of vascular, ductal, and glandular tissues and encasing essential cells of the reproductive, nervous, muscular systems and fat tissues. Recent discoveries suggest that BMs contribute to physiology and disease through dynamic regulation of BM composition and organization within tissues. Moreover, extracellular components peroxidasin and Goodpasture-antigen binding protein (GPBP) are emerging as members of the BM that have important roles in developmental and pathological process involving dynamic transitions of tissues. The works described in this dissertation addresses the dynamic interplay of BM components in pregnancy. We sought to test the hypothesis that BM structure and components are dynamically regulated during uterine, embryonic, and extraembryonic tissue development for pregnancy. Electron microscopy, polymerase chain reaction, and immunofluoresce revealed the novel finding that BM components are have distinct and integrated patterns of localization in uterine tissues during decidualization. Findings from In uteruo and in vitro culture studies of early embryonic development suggested that specific BM components were utilized for early epithelial genesis. In extraembryonic tissues, BM components changed localization patterns throughout development. These findings advance fundamental knowledge about BM dynamics in pregnancy that translates to other cellular and molecular physiological and pathological processes, such as tissue morphogenesis and cancer.

Diagnostic laboratory tests are critical to patient care as they help dictate the most appropriate treatments and procedures. To that end, hospitals and laboratories must ensure their diagnostic assays are optimized so as to afford patients their best chance for recovery. The pathology laboratory at Boston Medical Center, like all testing labs, must validate their IHC protocols yearly according to ASCO/CAP guidelines. Furthermore, BMC has a diverse patient population similar to the Atlanta population-based study published by Lund, et al. in 2010. Based on the results of the yearly ASCO/CAP testing and compared with the results of the Atlanta study, it was found that BMC’s HER2 testing was most likely not capturing all positive cases consistently. This prompted an optimization procedure for HER2 to be implemented. In addition to HER2 testing, BMC is looking at ways to optimize and implement PD-L1 IHC protocols as a way to identify those patients who might benefit from more targeted therapy. METHODS: HER2 IHC was performed with an altered protocol to attempt a higher concordance rate with PhenoPath, a reference lab in Seattle, and the Atlanta study data. ER and PR validation IHC protocols were also performed to ensure adequate concordance with required yearly testing. RESULTS: ER and PR protocols were found to be >95% accurate as compared to reference lab results. The new HER2 protocol yielded more vibrant staining results when compared to known positive reference data and previous BMC testing of the same sample. DISCUSSION: Optimizing lab assays is a critical step in ensuring proper clinical therapies are being utilized. Regular testing of a lab’s IHC output must be continuously verified, and continued data collection of the improved HER2 protocol is needed to make sure appropriate standards are being met. Further testing of PD-L1 IHC protocols will be warranted to maintain maximum efficiency.

Infectious diseases caused by antibiotic resistant bacteria are difficult to treat using traditional antibiotics and have emerged as a global public health problem (Bartlett, Gilbert, & Spellberg, 2013). An alternative strategy to bypass antibiotic resistance in bacteria is to enhance the host immune system (Hancock, & Sahl, 2006), especially innate immunity (Ajesh, & Sreejith, 2009), in order to combat bacterial infections. To verify the augmentation of innate immunity under certain stimuli, a quantifiable measurement of bacterial killing ability is needed. A new method of using heparinized human peripheral whole blood and mild heated blood samples from the same individuals as comparisons has been developed. This assay was used to quantify the bacterial killing ability of phagocytic cells by measuring the bacterial load after co-culturing with the Gram negative bacteria Pseudomonas aeruginosa. The assay shows that peripheral whole blood of healthy humans is capable of being activated ex vivo upon bacterial challenge and suppressing the bacterial load, while blood gently heated at 48oC for one hour failed to do so. The morphology of the phagocytic cells was examined microscopically. Phagocytosis of bacteria in neutrophils is observed in intact blood, and no such changes were found in heated blood. The level of inflammatory cytokine interleukin 8 (IL-8) was elevated in intact blood after bacteria inoculation, while in the heated blood the cytokine level stayed at baseline, indicating a persistence of leukocyte viability in whole blood and damaged leukocyte activity in gently heated blood. The data show that the human peripheral whole blood assay is suitable as a method for ex vivo bacterial killing quantification with the mildly heated blood sample from the same individual as a comparison. When kept at 37oC ex vivo, human whole blood can be activated upon bacterial challenge, produce inflammatory cytokines, reduce bacterial load, and phagocytes in the whole blood can successfully maintain their cell viability and capacity to phagocytose bacteria.

The effects of tissue preservation techniques are known to have meaningful impact on diagnostic and prognostic variables. This study aims to evaluate and improve the quality of biospecimens utilized in cancer research. Protein Death Ligand 1 (PD-L1) is a cell surface molecule with an important role for immune suppression. Its utility lies within providing a basis for limiting the immune response. We aimed to evaluate the expression levels of PD-L1 in tissues that had varying lengths of delay to fixation. 21 placenta, renal, and colon surgical specimens were collected and divided into delay to fixation cohorts. Samples were then evaluated through Hematoxylin and Eosin (H and E), PD-L1 Immunohistochemistry (IHC), and for the colon samples Microsatellite Instability (MSI) Status. A total of 75% of slides were positive for PD-L1 expression. In relation to Placenta all were positive for PD-L1 expression. While colon samples were 41.25% positive for PD-L1 expression. Finally 49.5% of renal samples were positive for PD-L1 expression. Of the three colon samples one was MSI Low status while the remaining were Microsatellite Stable (MSS). PD-L1’s status as a utile biomarker for prognostic and diagnostic reasons remains uncertain. The stability of PD-L1 expression across tissue type and sample delay shows the stability of the biomarker. Therefore our work shows that a delay while significant in affecting other biomarkers does not significantly alter PD-L1 expression.

The skin contains a highly regionalized stem and progenitor cell niche system, where a particular epithelial progenitor population primarily displays unipotent or bipotent behavior by contributing to their local niche under homeostatic conditions. Some of these populations, however, can also contribute to lineages beyond their local niche in response to wounding or other traumas. The goal of this thesis was to identify additional epithelial progenitor niches in the skin and characterize them. I focused on identifying and investigating the role of epithelial progenitors residing in two discrete compartments, the upper isthmus (UI) of the hair follicle and the touch dome (TD) in the epidermis, during skin development and homeostasis. The UI niche was identified by immunofluorescence studies based on the expression profile of a6 integrin low, Sca-1 negative, CD34 negative cells. The progenitor characteristics of UI keratinocytes were confirmed using skin reconstitution and in vitro colony formation assays. The transcriptional profiling of UI cells led to the identification of Tbc1d10c, one of the uniquely upregulated genes in UI cells. The comprehensive characterization of the UI niche by immunofluorescence led to the identification of a neural complex called palisade nerve endings (PN) present outside of the UI. Further investigation of PN using immunofluorescence and animal models revealed the presence of a glutamate transport to the HF via sensory nerve fibers. I elucidated that the presence of glutamate was critical for the organization of Schwann cells in the developing HF. Although the relationship between UI keratinocytes and PN still needs to be investigated, the presence of PN in the UI niche implicates that there is more function ascribed to UI cells in addition to serving as epithelial progenitors. The next epithelial progenitor niche I identified and characterized was the touch dome (TD) in the epidermis of hairy skin. The TD is a specialized niche, in which both epidermal Merkel cells and TD keratinocytes reside. The unexpected and exclusive presence of both Tbc1d10c and CD200 proteins in TD keratinocytes eventually led us to find that they were epidermal Merkel cell progenitors. Their progenitor characteristics were validated using skin reconstitution assay and in vivo lineage tracing by EdU incorporation. Our results collectively suggested that TD keratinocytes were bipotent progenitors that gave rise to both squamous and neuroendocrine epidermal lineages. Lastly, I generated a UI-specific Cre transgenic mouse line driven by the Tbc1d10c promoter, which will be utilized for further characterization of UI keratinocytes, including in vivo lineage tracing. I also generated Tbc1d10c complete knockout mice, which I will investigate to determine whether Tbc1d10c plays a role during skin development and homeostasis.

Hexavalent chromium Cr (VI) is one of the environmental carcinogens, which is considered as highly toxic metal to human health. According to epidemiologic studies, long term Cr (VI) exposure is highly associated with increased rate of lung cancer risks. Underlying mechanisms of Cr (VI) induced carcinogenicity and its induced malignant cellular transformation need to be explored. Transcription factor Nrf2 is a redox sensitive and it is involved in regulating heavy metal induced oxidative stress balancing under cellular environment. Interestingly, Nrf2 plays in two different roles in metal induced carcinogenesis. At initiation process of metal induced cellular transformation Nrf2 protects cells from oxidative stress and acts as a tumor suppressor. However, at later stage of malignant cell transformation Nrf2 is constitutively activated and acts as an oncogenic by preventing malignant cells from excessive oxidative stress. Therefore, there is a need to investigate the underlying reason of Nrf2 activation in hexavalent chromium induced cellular transformation. Nrf2 signaling pathway is regulated by many different cellular signaling pathways including autophagy dysregulation, ER stress dysregulation and epigenetic reprogramming. Among them, epigenetic alterations such as DNA methylation, histone modifications and microRNA dysregulation have been growing area to understand Cr (VI) carcinogenesis as well as abnormal activity of Nrf2 in metal induced malignancy. The purpose of this study was to determine the underlying mechanisms of Nrf2 upregulation and microRNA dysregulation in Cr-T cells. Our microRNA differential expression studies among parental B2B and Cr-T cells revealed that oxidative stress sensitive miR-27a/b expression is downregulated in Cr-T cells compared with B2B. Additionally, miR-27a/b contains the seed sequences which could potentially target redox sensitive transcription factor Nrf2. Here, we aimed to determine underlying mechanisms of Nrf2 upregulation and loss of activity of miR-27a/b in Cr-T cells, and miR-27a/b functional role in Cr-T cells. By exposing immortalized and nontumorigenic human lung epithelial BEAS-2B (B2B) cells to Cr (VI) at lower dose for longer time, we created in vitro model BEAS-Cr (Cr-T). This cell culture model of malignant chromium transformation allowed us to do research on Cr (VI) induced carcinogenicity. Throughout our studies, Cr (VI) free medium treated passaged control B2B and Cr-T cells were used. Our results collectively demonstrate one of the possible mechanisms for constitutive activation of Nrf2 is through loss of miR-27a/b in Cr-T cells. We also highlighted the Cr (VI) induced oxidative stress and ROS generation could be one reason of dysregulation of Nrf2 and miR-27a/b by showing the restoration result of Nrf2 and miR-27a/b expression in ROS inhibitor treated Cr-T cells. Potentially, Nrf2 3’UTR is a direct target of both miR-27a and miR-27b. Additionally, miR-27a/b acts as a tumor suppressor in Cr-T cells by inhibiting cellular proliferation, migration, colony formation, angiogenesis and tumor growth rate of Cr-T cells. Those microRNAs may reflect its tumor suppressor function by targeting 3’UTR of Nrf2. Such findings enhance our current understanding of Cr (VI) induced carcinogenicity: this knowledge related with Cr (VI) induced molecular mechanism may increase the potential prevention of Cr (VI) carcinogenicity and further tumor development.