Characterization of MALT1 Inhibitors and Their Effect on Leukemic Cell Growth Properties

Leukemia is the most common childhood cancer, with a combined 40,000 predicted new cases in the United States in 2016 [8]. The two most common subtypes are acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) [9-11]. The commercially available inhibitor of Bruton’s tyrosine kinase (BTK) has shown promising results in clinical trials for CLL because of the importance of BCR signaling in CLL [12-15]. Recent studies suggest that the outgrowth of BTK inhibitor resistant clonal cells in some CLL patients results in a treatment-refractory phenotype [16-18]. MALT1, a protein involved in BCR activation of the NF-κB pathway that functions downstream of BTK, is a promising new target aimed at treating an aggressive BCR signaling-dependent subtype of non-Hodgkin’s lymphoma, known as ABC-DLBCL [1-3, 6]. Targeting this protein for small molecule inhibition may be effective in the treatment of BTKi-refractory CLL patients, as well as other leukemias, such as AML [19]. The effect of MALT1 or BTK inhibition on AML, CLL, and DLBCL cell proliferation was analyzed. Together with examination of protein expression in each cell line and the effect of MALT1 inhibition on cleavage of its downstream target RelB, it is supported that a clinical MALT1 inhibitor would be effective in slowing the growth of CLL, ABC-DLBCL, and AML tumors. Combinatorial experiments supported the hypothesis that combination treatment with MALT and BTK inhibitors could be efficacious at treating patients with relapsed or refractory CLL or ABC-DLBCL

Pathogenesis of light chain-induced dysfunction in cardiac amyloidosis

Although a rare disease, light chain (LC) amyloidosis (AL) is the most common systemic amyloidosis in developed countries. It is caused by an overproduction of immunoglobulin LC proteins in bone marrow plasma cells. In AL amyloidosis, LCs that are prone to misfolding and insolubility will aggregate, form fibrils, and deposit themselves in various tissues, thereby causing organ dysfunction. The most fatal manifestation of AL amyloidosis is associated with cardiac involvement, defined by the presence of extracellular AL amyloid deposits within the heart. Cardiac amyloid infiltration typically leads to diastolic dysfunction followed by heart failure and has a median survival of approximately 6 months from the time of diagnosis if untreated. Clinical observation suggests that a reduction in circulating LCs results in an improvement in heart failure symptoms despite minimal changes in amyloid deposition. This has led to the concept that LCs themselves are cytotoxic to cardiomyocytes. Recent studies indicate that AL LCs induce oxidative stress, cellular dysfunction, and apoptosis (programmed cell death) in cardiomyocytes via a p38α mitogen-activated protein kinase (MAPK) mechanism. They may therefore be a target for amyloidosis therapy. By understanding how LCs cause cardiac dysfunction, we can target this process with therapies and utilize downstream measures of LC activity as diagnostic and prognostic tools. The objective of this study was to determine the role of autophagy in AL amyloidosis. Autophagy is the intracellular process of degrading aging or dysfunctional cellular components. Autophagy can be beneficial by preventing proteotoxicity and providing nutrients, amino acids, and other necessities during times of cellular stress. On the other hand, increased autophagy, like apoptosis, may mediate cellular death depending on the type of stimulus and its duration. Autophagy is induced by a variety of stimuli, including oxidative stress. AL has been demonstrated to increase reactive oxygen species (ROS), and it is unknown if autophagy mediates cardiomyocyte dysfunction in AL cardiac amyloidosis. We thus sought to determine if it is a factor in amyloid cardiotoxicity. We explored the ERK1/2, p38, and JNK MAPK pathways in particular, since MAPK signaling cascades regulate several transcription factors involved in the cell cycle and p38α has been implicated in ROS-induced cardiac AL amyloidosis. Adult rat ventricular myocytes (ARVM) were harvested from healthy adult male rats and exposed to a variety of experimental conditions in vitro. ARVM were treated with vehicle control, human LC obtained from a patient without cardiac involvement, a positive control (aldosterone), and human AL light chains obtained from a patient with AL cardiac amyloidosis in the presence or absence of UO126, SB203580, or SP600125 (specific inhibitors of ERK1/2, p38, and JNK, respectively). The resulting protein expression levels of autophagy indicators LC3II and ATG4B in cardiomyocytes were analyzed by Western blotting. The ratio of phosphorylated to total ERK1/2 protein expression was also explored. We found that AL light chains did not contribute to autophagy via the ERK1/2, p38, or JNK pathways. In contrast to our previous unpublished findings, the protein levels of autophagy indicators in AL-treated ARVM did not differ from vehicle control levels, suggesting that AL did not activate autophagy. However non-cardiomyopathic light chains (LC) did increase LC3II expression in ARVM, despite their human source exhibiting no clinical indications of cardiac involvement. This implies that autophagy induced by non-cardiomyopathic LCs may be beneficial and protect against the development of the cardiotoxicity seen in AL cardiac amyloidosis. Further studies are necessary to understand the effect of autophagy in the heart and its role in cardiac amyloidosis. Continuing to explore the underlying mechanisms of AL light chain toxicity will contribute to the development of diagnostic, prognostic, and treatment strategies for AL amyloidosis

Automated analysis of 16-color polychromatic flow cytometry data maps immune cell populations and reveals a distinct inhibitory receptor signature in systemic sclerosis

Background. The phenotypic profiles of both peripheral blood and tissue-resident immune cells have been linked to the health status of individuals with infectious and autoimmune diseases, as well as cancer. In light of the promising clinical trial results of agents that block the Inhibitory Receptor (IR) Programmed Death 1 (PD-1) axis, novel flow cytometric panels that simultaneously measure multiple IRs on several immune cell subsets could provide the distinct IR signatures to target in combinational therapies for many disease states. Also, due to the paucity of human samples, larger (14+ color) ‘1-tube’ panels for immune cell characterization ex vivo are of a high value in translational studies. Development of fluorescent-based panels offer several advantages as compared with analogous mass cytometric methods, including the ability to sort multiple populations of interest from the sample for further study. However, automated platforms of multi-dimensional single cell analysis that allow objective and comprehensive population characterization are severely underutilized on data generated from large polychromatic panels. Methods. A 16-color flow cytometry (FCM) panel was developed and optimized for the simultaneous characterization and purification of multiple human immune cell populations on a 4- laser BD FACSARIA II cell sorter. FCM data of samples obtained from healthy subjects and individuals with systemic sclerosis (SSc) were loaded into Cytobank cloud, then compensated and analyzed with SPADE clustering algorithm. The viSNE algorithm was also employed to compress the data into a 2D map of phenotypic space that was subsequently clustered using SPADE. For comparison, the FCM data were also analyzed manually using FlowJo software. Results. Our novel 16-color panel recognizes CD3, CD4, CD8, CD45RO, CD25, CD127, CD16, CD56, γδTCR, vα24, PD-1, LAG-3, CTLA-4, and TIM-3; it also contains a CD1d-tetramer and a live-dead dye (with CD19 and CD14 included as a combined dump channel). This panel allows combinational IR signatures to be determined from CD4+ T, CD8+ T, Natural Killer (NK), invariant Natural Killer (iNKT), and gamma delta (γδ) immune cell subsets within one sample. We have successfully identified all subsets of interest using automatic SPADE and viSNE algorithms integrated into Cytobank services, and demonstrated a distinctive phenotype of IR distribution on healthy versus systemic sclerosis subject groups. Conclusions. Methods of automatic analysis that were originally developed for processing multi-dimensional mass cytometry can be applied to polychromatic FCM datasets and provide robust results, including subset identification and distinct IR signatures in healthy compared to diseased subject groups

Captives of the Dark and Bloody Ground: Identity, Race, and Power in the Contested American South

In this dissertation, I use the lens of captivity to explore how Native Southerners defined themselves and the other. Before they encountered one another in the colonial era, the peoples of Africa, Europe, and North America considered enslavement a legitimate fate for captured enemy peoples, though their attitudes about the status and roles of captives differed. In the South during the colonial and early national periods, violent conflict often erupted as Indian nations labored to maintain their territorial integrity and political autonomy, Euro-Americans desired to control Indian land and African labor, and Africans sought freedom. During such episodes, Native groups took enemies-white, black, and Indian-as captives. Victors then subjected their captives to a variety of fates: they ritually killed some to satisfy the demands of clan vengeance; they adopted others to replace deceased family members; they made chattel slaves out of the remainder. Throughout the colonial period, Native Southerners largely determined a captive's fate based on his or her sex and age. By the late eighteenth century, however, race became a captive's most salient characteristic, and African-American captives were overwhelmingly targeted in warfare and then sold or held in transgenerational bondage. This study, in part, explores why that shift toward racialization occurred, and how it reflected Native Southerners' changing sense of identity. More broadly, "Captives of the Dark and Bloody Ground" addresses the construction of race and racism in America and contributes to a growing body of scholarship on the diversity of enslavement in North America. This dissertation traces the dynamic institution of captivity from the precolonial past, when Native chiefdoms competed for regional power, through the conclusion of the Second Seminole War in 1842, which marks the final captive-taking episode in the contested American South. It draws upon a wide variety of English- and Spanish-language sources including legal documents, personal and official correspondence, journals, ethnographies, and the archaeological record

Beyond the Volcanoes: A Community Partnership for Health in Rural Nicaragua

Background: Health inequities related to gender, ethnicity, socioeconomic status, and geography exist in rural Nicaragua. The purpose of this ongoing project is to improve health equity in rural Nicaragua through social transformation using community-based participatory action research. Bronfenbrenner\u27s ecological model of human development, school health, and primary health care theories provided the framework for this research. Methods: Community-based participatory action research involves six phases: partnership, assessment, planning, implementation, evaluation, and dissemination. In the evaluation phase, the goal was to use the data obtained during the assessment, planning, and implementation phases to evaluate the cookstove intervention in its ability to reach the community\u27s health-related goals. Pre- and post-test surveys were used to assess indoor air pollution including: kitchen layout, stove type, fuel usage, and women and children\u27s health. Results: Forty-eight community members participated in the cookstove evaluation. Pre-test surveys indicated that the community members used open fire stoves in closed kitchen spaces with wood being the primary fuel source. Women reported suffering from headaches, eye irritation, and chronic coughing. One year following the implementation phase, post-test surveys indicated a sustainable, significant improvement in women\u27s health (p=.05) but no significant change in the amount of wood used for cooking. Conclusion: Results from the cookstove evaluation were used by community members to guide the re-engineering of the cookstoves\u27 firebox to decrease wood consumption and improve deforestation. Partnership in community health research provides a mechanism to engage community members in social justice through working toward a common goal – sustainable health for all

Phyllodes Tumor vs Fibroadenoma: Diagnosis and Management

Case: The patient is a 71 year-old woman who presented with enlarging painful breast mass. She had history of previous excision of a fibroadenoma in her left breast in 1993. She underwent menopause at 52 and does not take estrogen. Diagnostic imaging revealed 4.7cm breast mass, which had increased from prior measurement of 2.8cm to 4.7cm. Core biopsy demonstrated a fibroepithelial neoplasm areas of hypercellular stroma and occasional stromal mitotic figures most consistent with phyllodes tumor. Lumpectomy was performed. Final pathology showed a 4.8cm well-demarcated tumor with mildly pleomorphic spindled cells in the stroma and up to 1 per 10 mitoses per high powered field, consistent with benign phyllodes. The patient was followed every 6 months with imaging for 2 years without recurrence. Conclusions: Phyllodes tumors are rare fibroepithelial tumors of varying metastatic potential that can be mistaken for benign fibroadenomas. Phyllodes tumors should be surgically excised with wide margins, needing radiation or chemotherapy only if recurrent or large (>10cm), whereas fibroadenomas can be managed expectantly if asymptomatic (Gnerlich, 2014). Phyllodes tumors are often diagnosed in women ages 35-55. The patient in this case was diagnosed at a more advanced age with benign disease, although older age is more often associated with increased histologic grade (Mishra, 2013) (Karim, 2009). Borderline and malignant tumors are more likely to recur within two years of resection; there is less data on recurrence rates of benign tumors. Clinical Significance: Phyllodes tumors should be suspected with rapid growth of a known fibroadenoma. Core biopsy should be performed rather than fine needle aspiration for accurate diagnosis. Although phyllodes tumors comprise less than 1% of all breast neoplasms, it is crucial that uncommon pathologies are diagnosed correctly so that patients receive appropriate treatment