Indiana University-Purdue University Indianapolis (IUPUI) / The non-hematopoietic, stationary stromal cells located inside and surrounding skin-draining lymph nodes play a key role in regulating immune responses. We studied distinct populations of lymph node stromal cells from both human subjects and animal models in order to describe their phenotype and function. In the mouse model, we studied two distinct populations: an endothelial cell population expressing Ly51 and MHC-II, and an epithelial cell population expressing the epithelial adhesion molecule EpCAM. Analysis of intra-nodal and extra-nodal lymph node (CD45-) stromal cells through flow cytometry and qPCR provides a general phenotypic profile of the distinct populations. My research focused on the EpCAM+ epithelial cell population located in the fat pad surrounding the skin draining lymph nodes. The EpCAM+ population has been characterized by surface marker phenotype, anatomic location, and gene expression profile. This population demonstrates the ability to inhibit the activation and proliferation of both CD4 and CD8 T cells. This population may play a role in suppressing overactive inflammation and auto-reactive T cells that escaped thymic deletion. The other major arm of my project consisted of identifying a novel endothelial cell population in human lymph nodes. Freshly resected lymph nodes were processed into single cell suspensions and selected for non-hematopoietic CD45- stromal cells. The unique endothelial population expressing CD34 HLA-DR was then characterized and analyzed for anatomic position, surface marker expression, and gene profiles. Overall, these studies emphasize the importance of stationary lymph node stromal cells to our functioning immune systems, and may have clinical relevance to autoimmune diseases, inflammation, and bone marrow transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / It has traditionally been posited that women show a "telescoped" development of alcohol use disorders (Kuhn, 2011). In particular, a number of clinical studies support striking sex differences in the progression from initial use of alcohol to dependence on the compound; with women showing a faster progression through landmark events associated with the development of alcohol addiction (Randall et al., 1999). However, recent studies have challenged this tenet (Keyes et al., 2010). The work presented herein was designed to determine whether females are indeed more vulnerable to the development of behavioral maladaptations following binge drinking and whether sex differences in GABA(A) receptor regulation might underlie this vulnerability. Using a mouse model of binge drinking this dissertation established that, compared to males, females escalate their binge drinking at a faster rate and maintain altered responsivity to the locomotor effects of alcohol after extended abstinence from binge drinking. Female mice also displayed significant increases in ethanol preference and intake in a continuous, two-bottle choice protocol following a shorter history of binge drinking than males. The final goal was to determine if binge drinking results in unique patterns of anxiety- or depressive-like symptoms in males and females and whether these behaviors would be associated with the dimorphic regulation of GABAA receptor subunits across the prefrontal cortex and hippocampus. Male binge drinkers displayed anxiety-like behavior during early withdrawal that dissipated after 2 weeks of abstinence. There were no significant changes in the expression of delta or gamma2 GABAA receptor subunit mRNA at this time point in the regions analyzed. Females also showed temporary anxiety-like behavior during early withdrawal from binge drinking. Additionally, females displayed significant depressive-like behavior after 2 weeks of abstinence from binge drinking. In particular, diestrus-phase females displayed significantly greater immobility in the forced-swim test after ethanol exposure and no longer maintained the reduced swim-time behavior associated with this phase of the cycle at baseline (when compared to the estrus-phase). qPCR analysis of hippocampal tissues from diestrus females supported a significant reduction in expression of gamma2 GABA(A) subunit mRNA after binge drinking. This effect was not noted for RNA isolated from hippocampal tissues taken during the estrus phase of bingers. These final data suggest possible interaction of estrous-cycle and binge drinking history that may result in the unique expression of deficits following binge drinking for females. Taken together, this work supports sex and estrous dependent effects of binge drinking on behavior and gene regulation.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin is the protein product of NF1 and functions as a negative regulator of Ras activity in both hematopoietic and vascular wall cells, which are critical for maintaining blood vessel homeostasis. NF1 patients are predisposed to chronic inflammation and premature cardiovascular disease, including development of large arterial aneurysms, which may result in sudden death secondary to their rupture. However, the molecular pathogenesis of NF1 aneurysm formation is completely unknown. Utilizing a novel model of Nf1 murine aneurysm formation, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/-) results in enhanced aneurysm formation with myeloid cell infiltration and increased reactive oxygen species in the vessel wall. Using cell lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/- aneurysm phenotype in vivo. Additionally, oral administration of simvastatin, a statin with antioxidant and anti-inflammatory effects, significantly reduced aneurysm formation in Nf1+/- mice. Finally, the antioxidant apocynin was administered orally and also resulted in a significant reduction of Nf1+/- aneurysms. These data provide genetic and pharmacologic evidence that neurofibromin-deficient myeloid cells are the central cellular triggers for aneurysm formation in a novel model of NF1 vascular disease, implicated oxidative stress as the key biochemical mechanisms of NF1 aneurysm formation and provide a potential therapeutic target for NF1 vasculopathy.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.

Indiana University-Purdue University Indianapolis (IUPUI) / To cope with the high metabolic demands of the body during pregnancy, the maternal liver adapts by increasing its mass and size. This increase is proportional to the increase in total body weight during the course of gestation. The pregnancy-induced maternal liver growth is a result of both hepatocyte hypertrophy and hyperplasia. Microarray analysis of pregnant maternal livers shows markedly different gene expression profiles when compared to a non-pregnant state. Most interesting was the 2,500-fold up-regulation in the mRNA expression of Ascl1, a transcription factor responsible for the differentiation of neural progenitor cells into various neuronal types, during the second half of pregnancy. Our investigation aimed at (1) characterizing the identity of maternal hepatic Ascl1-expressing cells and (2) tracing the fate of Ascl1-expressing cells in the maternal liver during pregnancy. Timed pregnancies were generated and non-pregnant (NP) and pregnant maternal livers were harvested and analysed. To identify the maternal hepatic Ascl1-expressing cells we used the Ascl1GFP/+ reporter mouse line. NP and gestation day 15 (D15) maternal livers were immunostained for green fluorescent protein (GFP). The result shows that GFP-positive, Ascl1-expressing cells are hepatocyte-like cells, which are present in D15 maternal livers, but absent in NP livers. The Rosa26floxstopLacZ/ floxstopLacZ;Ascl1CreERT2/+ mouse line was used to trace the fate of Ascl1-expressing cells during pregnancy. LacZ staining of gestation day 13 (D13) and 18 (D18) maternal livers demonstrates that D13 hepatic Ascl1-expressing cells (labeled with LacZ) undergo hyperplasia to repopulate a large portion of D18 maternal livers. Furthermore, LacZ and HNF4α co-staining of D13 and D18 maternal livers shows the presence of two populations of LacZ-expressing cells: HNF4α+ population and HNF4α- population. HNF4α+ LacZ-expressing cells represent hepatocyte lineage cells that are derived from Ascl1-expressing cells. We observe that, towards the end of pregnancy, a considerable portion of the maternal liver is comprised of hepatocytes derived from Ascl1-expressing cells. Taken together, our preliminary study suggests that pregnancy induces maternal liver turnover via Ascl1-expressing cells.

A recently emerging engineering design approach entails studying the brilliant design solutions found in nature with an aim to develop design strategies that mimic the remarkable efficiency found in biological systems. This novel engineering approach is referred to as bio-inspired design. In this context, the present study quantifies the structure-property relations in bighorn sheep (Ovis canadensis) horn keratin, qualitatively characterizes the effects of a tapered spiral geometry (the same form as in a ram’s horn) on pressure wave and impulse mitigation, describes the stress attenuation capabilities and features of a ram’s head, and compares the structures and mechanical properties of some energy absorbent natural materials. The results and ideas presented herein can be used in the development of lightweight, energy absorbent, bio-inspired material designs. Among the most notable conclusions garnered from this research include: Horn keratin behaves in an anisotropic manner similar to a long fiber composite. Moisture content dominates the material behavior of horn keratin more than anisotropy, age, and stress-state. This makes moisture content the most influential parameter on the mechanical behavior of horn keratin. Tapered geometries mitigate the impulse generated by a stress wave due to the convergent boundary and a continually decreasing cross sectional area such that greater uniaxial stresses and subsequent axial deformation arises. Furthermore, the tapered geometry introduces small shear stresses that further decrease the impulse. Spiral geometries attenuate the impulse generated by a stress wave by the introduction of shear stresses along the length of the spiral. These shear stresses introduce transverse displacements that function to lessen the impulse. When both a taper and spiral geometry are used in a design, their synergistic effects multiplicatively reduce the impulse Tough natural materials have a high porosity, which makes them light-weight, while increasing their compressive energy absorption ability. Biomaterials whose functions include protection and energy absorption feature a multiscale, hierarchical, composite structure. The constituent materials are arranged in such ways to achieve a synergistic effect, where the properties of the composite exceed the properties of its constituents. Biological materials are therefore not confined to the law of mixtures.