Chronic inflammation is a silent killer, contributing to death in patients with chronic kidney disease (CKD) and other chronic disorders, such as diabetes and obesity. Although the specific molecular pathways linking inflammation contribute to CKD pathogenesis still remain unclear, interleukin-6 (IL-6), the prototypic inflammatory biomarker and a potential mediator, is believed to be able to help us understand the mechanism that leads to CKD. We have been investigating the role of IL-6 family of genes using different animal models, such as the Unilateral Ureteral Obstruction (UUO), to induce interstitial fibrosis. The results from our studies have demonstrated that IL-6 and gp130 are profibrotic molecules. Therefore, our goal was to understand the mechanisms of IL-6 in myofibroblasts, the major contributors of collagen during fibrosis. For these studies, we used wild-type mice, IL6 (+/+), IL6 (-/-) and αSMA-Cre-IL-6(fl/fl), in the UUO model of interstitial fibrosis. We hypothesized that IL-6 regulates the crosslinking of collagen by regulating the lysyl-oxidase gene. Therefore, we assessed the Extracellular matrix expansion and collagen accumulation by measuring the changes in gene expression of Col-1, 3, and 4, IL-6, Lysyl-oxidase, Fli-1 and Hells at different time points during renal fibrogenesis. We consider that the change in collagen crosslinking is an important factor in the turnover rate of collagen and it changes its stability to be degraded/accumulated. The examination of the role of IL-6 could potentially provide a new therapeutic breakthrough for treatment and better detection/assessment of renal fibrosis.

 Craniofacial Microsomia (CFM) is a congenital condition that occurs in approximately 1:5,600 lives births and involves malformations that result in underdevelopment of one or both sides of the face, usually affecting the ear and jaw. It has been previously thought that in patients with unilateral CFM, the other half of the face remains unaffected. Recent studies suggest that the ‘unaffected side’ of the face may be smaller when compared to individuals without craniofacial conditions. This is an expansion of our previous pilot study where we found some support for this claim. We aim to quantify the facial differences clinically observed in the ‘unaffected’ side of the face in a larger sample size of 75 children with CFM compared with 75 children without craniofacial conditions using anthropometric measurements on 3D surface images obtained with the 3dMD® camera system. The outcome variables are coordinates and interlandmark distances using facial landmarks obtained on 3D images from individuals with CFM and controls. We compare distributions (mean and standard deviations) and proportions of the anthropometric measurements between children with CFM and controls using a two-sided Student’s t-test with unequal variance. Ultimately we hope this information will help us to better understand the etiology of this condition and treat children with CFM. We plan to integrate the results of this study into future assessments of patients with CFM to ensure they are receiving the highest quality of care and ensure that interventions are improving patients’ quality of life.

Diffuse Large B-Cell Lymphoma (DLBCL) is one of the most common types of non-Hodgkin's Lymphoma with a 50.4% five-year survival rate.  Previous work has shown that some patients with aggressive DLBCL have increased expression of the transcription factor NF-kß, which can alter normal growth and function of B-lymphocytes.  Interestingly, a protein upstream of NF-kß in the B-Cell Receptor Pathway called PKCß is found to be highly activated in many DLBCL cases, suggesting that PKCß overexpression may be altering the lifecycle of those with DLBCL.  Therefore, PKCß may be a significant new target for therapy.  We designed a transgenic mouse expressing a dominant active form of PKCß expressed only in B-cells (Rosa26 DAPKCß XCD19-Cre).  With this model, we measured differences in B-cell  germinal center development and in antibody responses between WT and DA PKCß-Cre mice immunized with T-dependent antigen NP-CGG.  We hypothesized that DA PKCß-Cre mice may have an increased response compared to WT because of its dominant active form of PKCß, which leads to increased NF-kß and hyper-activation of the responding B-cells.  Thus, we used ELISA and ELISPOT assays to quantify the expressed antibodies and immunohistochemistry to analyze the architecture of the spleen.  Immunohistochemistry has shown that the immunized DA PKCß-Cre mice have slightly larger germinal centers than WT mice, although not statistically significant.  Immunized DA PKCß-Cre mice show no difference in serum antibody levels by ELISA, but have slightly increased numbers of anitbody producing cells by ELISPOT.  Overall, the data suggests that over expression of PKCß in B-cells results in a hyper-proliferative phenotype, but does not strongly alter the antibody response to the T-dependent antigen NP-CGG.  Since we saw a difference in mice immunized with Pertussis, which stimulates the Toll-like Receptor (TLR) Pathway, we will look at different responses of TLR ligands in vivo and in vitro.

Ultrasound (US) and microbubbles (MBs), as tools for gene transfection, have promising implications for gene therapy. Through permeation, US facilitates the diffusion of a gene or drug from the capillaries into the cell. Binding of the gene or drug to the MBs can further enhance the delivery efficiency, and when exposed to US, the MBs are ultimately cavitated once they have reached their target. Our goal is to create MBs that will enhance US-mediated transfection. Concerning MB production, we have shown that our prepared MBs are more concentrated, stable, and consistent than those of the leading manufacturers, via flow cytometry and Coulter Counter analysis. In in vitro experiments, we have shown that US, by itself, increases transfection efficiency (plasmid concentration in cells) of pGL3 and pGFP in human embryonic kidney 293 cells. 293 cells are transfected very readily and consistently and are therefore useful as a way to accurately quantify gene expression from transfection. Combined with MBs, transfection efficiency is further improved upon. US parameters (intensity, exposure time, frequency) and MB characteristics (concentration, size, distribution, stability) are interdependent, and thus the optimization of each parameter is important. With regards to US parameters, exposure times have been varied from 1 to 5 minutes, at intensities ranging from 0.5 to 2 watts, and the optimal condition (cells transfected minus cells killed) has been found to be 2 watts per square centimeter for 3 minutes. These conditions have been combined to quantify 293 cell gene expression. It has been shown that MBs enhance transfection rates by up to 25 times when compared to those with just US. These results have important implications in gene therapy as a means of non-invasive, localized, and efficient gene delivery.

Many occurrences of glomerulonephritis present recurrent acute inflammation episodes. With time these conditions often lead to progressive fibrosis and chronic renal failure. My project studies the role of Interleukin-6 (IL-6), one of the most proinflammatory cytokines, using the nephrotoxic serum (NTS) nephritis mouse model. The ubiquitous expression of IL-6 makes it a challenge to study this interleukin. We have genetically engineered mice with ablated IL-6 in the F4/80+ macrophages and the Ytgax (CD11c+) dendritic cells. We hypothesize a greater level of glomerular damage and disease in the wild type mice compared to the IL-6 depleted mice. IL-6 +/+ (IL6WT) and -/- mice (IL6KO) were injected with NTS in the tail vein and placed in metabolic cages for seven days. Urine was collected to measure renal failure and progression of the disease. Mice were sacrificed at 7 days after the injection and both kidneys harvested and sectioned. Extraction, quantification and quality analysis (RNA integrity number) from the IL6WT and IL6KO frozen kidney samples were conducted and later cDNA synthesized. Quantitative PCR (qPCR) reactions were carried out to analyze gene expression of IL-6 family genes and pathological analysis of frozen or paraffin embedded tissue was done to assess glomerular and interstitial damage. Preliminary results from histopathology analysis and qPCR suggest a significant role played by IL-6 in glomerular damage. IL6WT diseased mice show upregulated expression of IL-6 and greater degree of disease relative to the IL6KO. We plan to continue gene expression quantifications and perform immunostaining to compile more data and validate our results. These findings will help to establish the efficacy of IL-6 neutralization as a therapeutic option for kidney disease.

X-linked severe combined immunodeficiency (X-SCID) is caused by a mutation in the interleukin 2 receptor gamma (IL2Rγ) gene, which is required for lymphocyte development. Affected patients lack functional lymphocytes and die unless cured through bone marrow transplant or gene therapy. However, gene therapy treatment was stopped due to risk of leukemia resulting from insertional mutagenesis, and this prompted the pursuit of alternate treatments, such as gene repair. Gene repair requires the use of two essential components, a homing endonuclease and a repair template, that harness homology directed repair mechanism, which repairs double stranded DNA breaks in cells. Homing endonucleases are enzymes used to target and cut DNA near a mutation in the genome, while a repair template is the DNA sequence used to correct the mutation. My project involved performing experiments to optimize specific components of gene repair using the Sce-SCID mouse model that contained a recognition sequence for the homing endonuclease, I-SceI proximal to the IL2Rγ mutation. I used adeno-associated viral vectors (AAV) to deliver I-SceI and a repair template to hematopoietic stem cells (HSC) isolated from Sce-SCID mice. I tested the hypothesis that delivery of AAV encoding I-SceI homing endonuclease to Sce-SCID HSC would result in recognition and cutting of the Sce locus. Cutting of the Sce locus is essential to gene repair because it initiates the repair of DNA double stranded breaks through homology directed repair with the repair template. Previous experiments in the Rawlings lab demonstrated cutting of the Sce locus with stable I-SceI expression, but my experiments involved transient AAV delivery. Transient delivery is critical for the safety profile of gene repair. Once I-SceI cutting was established with transient delivery, I performed experiments that invovled both I-SceI and repair template in an attempt to correct the SCID phenotype of the Sce-SCID mouse.

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Interleukin-6 (IL-6) is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine. From our preliminary studies we have demonstrated that IL-6 and its receptor gp130 are pro-fibrotic in renal fibrogenesis. Here we measure mast cell function in unilateral urethral obstruction, a well-characterized model of renal fibrosis, using mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter bred to IL-6 flox/flox mice. Mast cells from the C57BL/6 Cpa3-IL-6 (fl/fl) mice do not express IL-6. We have groups of 10 for each of the following strains: control1; Cpa3-Cre, control2; IL-6fl/fl and experimental mice; Cpa3-IL-6. Mice under general anesthesia had the ureter ligated at 2 sites distally from the kidney and sacrificed after 14d. We measured the level of fibrosis by measuring hydroxyproline colorimetrically and by measuring area of Picro Sirius red in stained kidney sections, and quantified by pixel analysis. Our preliminary results indicate that the absence of IL-6 in mast cells is important in renal fibrosis. When IL-6 was ablated from mast cells the level of fibrosis measured by collagen and the expansion of extracellular matrix was reduced. These preliminary results suggest that mast cell-derived IL-6 promotes renal fibrosis after unilateral urethral obstruction.

The inferior olive is heavily involved in implicit timing, the unconscious encoding of timing of sensory or motor events. Performance of these tasks relies on synchronous output from ensembles of neurons in the inferior olive, which are electrotonically coupled to one another via gap junctions. Previous attempts to understand coupling properties in the inferior olive have relied on technically challenging dual whole-cell recordings. Here, I demonstrate an optogenetic approach which provides rapid evaluation of electrotonic coupling in the inferior olive using femtosecond pulsed infrared stimulation of olivary cells expressing the light-activated cation channel channelrhodopsin-2 (ChR2 H134R). Targeted expression of ChR2 in olivary neurons was achieved through the Ai32 mouse line engineered at the Allen Institute for Brain Science, which were crossed with mice expressing Cre-recombinase targeted to the pancreatic transcription factor 1a (Ptf1a) locus. Parasagittal brainstem slices were prepared from young (P20-P40) Ptf1a-Ai32 positive mice. Electrodes were guided under IR-DIC for whole-cell recording of a single olivary neuron. Neighboring cells were stimulated using 920 nm light (<1 mW/µm² average power), in which a single infrared beam was split into 64 beamlets and distributed evenly over the neighboring cell's cross sectional area (100-400 µm²). Coupling properties determined by optogenetic stimulation of neighboring cells replicated previous results produced by dual whole-cell recordings (CC=1-10%). Furthermore, optogenetic evaluation of coupling following pharmacological manipulation of olivary neurons were consistent with previous findings. This optogenetic approach will provide a rapid and efficient method for future exploration of coupling properties in the inferior olive, which may accelerate the study of their involvement in pathologies such alcoholism, autism and sudden infant death syndrome.

Congenital heart defects are the most common form of birth defect, affecting about one out of every 100 newborns in the US. Ten percent of these afflicted newborns will experience a narrowing of their aorta, formally called coarctation of the aorta. Coarctation can result in extreme hypoxia in tissues that do not receive sufficient blood supply, and can eventually lead to congestive heart failure; surgical repair is required for the infant to regain normal circulation. The etiology of coarctation has been linked in many cases to migration of cells from the ductus arteriosus into the aorta. The ductus arteriosus is a fetal vessel that must close at birth, and does so by constriction of its smooth muscle cells until its lumen is occluded. If these contractile cells migrate into the aorta, they can constrict, narrowing the aortic lumen. Preliminary data from experiments on the region where the ductus inserts into the aorta show a differential expression of chemorepulsive signaling proteins from the Slit and Robo families in ductal versus aortic tissue, suggesting that a distinct tissue boundary forms in this region. This is supported by previous studies in lineage mapping, which demonstrated a distinct somite-neural crest smooth muscle lineage boundary in the aortic arch. In order to elucidate the role that Slit and Robo proteins play in determining the ductus-aorta boundary, I use reverse transcription PCR analysis to examine the expression of Slit-Robo ligand and receptor proteins in perinatal ductal and aortic tissue. By comparing expression in normal samples with samples from patients with coarctation, more insight into the influence of Slit-Robo chemorepulsive signaling on boundary formation will be gained. Characterization of the ductus-aorta boundary by identification of differential ligand-receptor expression is critical to understanding the role that abnormal boundary formation plays in coarctation of the aorta.

Ulnar-Mammary Syndrome (UMS) is a congenital syndrome that is characterized by ulnar abnormalities and breast hypoplasia. The Bamshad Lab discovered that UMS is caused by mutations in the gene, TBX3. The goal of our project is to find novel TBX3 mutations that cause UMS, and expand the knowledge of how different mutations correlate to the severity of the disease. We collected DNA samples from three UMS families, and sequenced the exons of TBX3 via Sanger sequencing. Sequence analysis software allowed us to search the sequence for mutations and their locations within the gene. In the family K31928, previous screening showed that the proband and her sister, have a mutation called 1294_1301dup8. We screened the parents for mutations in TBX3, and we found that the father had the insertion mutation as well. The father’s clinical findings were very mild, but this screening confirmed that he passed the mutation causing UMS to his daughters. No mutations were found in members of the other two UMS families. The next step of this project is to investigate, in the families without TBX3 mutations, other candidate genes, as well as regulatory regions of TBX3, for mutations that could cause UMS.

Autosomal DNA, the non-recombining Y chromosome (NRY), and mitochondrial DNA (mtDNA) are all efficient means of predicting an individual's genetic ancestry. Commercially available autosomal DNA ancestry estimates are obtained using loci that show large frequency differences between continental groups known as ancestry informative markers (AIMs), and NRY and mtDNA ancestry estimates rely on haplogroup information to trace an individual's genetic lineage. Since autosomal DNA is bi-parentally inherited and the NRY and mtDNA are uni-parentally inherited, autosomal DNA potentially contains more information regarding an individual's genetic heritage than the NRY or mtDNA. Our goal is to determine the accuracy of NRY and mtDNA ancestry estimates for predicting autosomal DNA ancestry. Ancestry estimates have been obtained for 965 individuals from a global panel of human populatins from the Human Genome Diversity Project (HGDP-CEPH). Ancestry estimates were compared based on one's biogeographical ancestry or the geographic location of one's ancestors. Autosomal DNA ancestry was determined by the greatest proportion of ancestry based on seven biogeographic groups: African, European, Middle Eastern, Central/South Asian, East Asian, Oceanian, and American. Since each haplogroup is associated with a biogeographic group, the accuracy of NRY and mtDNA haplogroups in predicting autosomal DNA ancestry was determined based on a statistically signifcant number of individuals classified with their correct autosomally determined biogeographical ancestry. This information is useful to assess the quality of different ancestry estimations for use in population-based studies and direct-to-consumer ancestry tests. 

Obesity and atherosclerosis (AS) are major contributors to poor health in the developing world. Hepatic lipase (HL), an enzyme produced in the liver that plays a central role in plasma lipid metabolism, may affect both obesity and AS. HL converts large, cholesterol rich high density lipoproteins (HDL) into cholesterol poor HDL that will circulate through the body and collect cholesterol, possibly reducing AS development. However, HL also converts intermediate density lipoproteins (IDL) into low density lipoproteins (LDL), which is a major risk for AS development. We hypothesized that lipoprotein hydrolysis by HL releases free fatty acids that may modulate satiety and therefore play a role in obesity. It was recently discovered that HL promotes weight gain and food consumption (in mice with HL) whereas the absence of HL protected against weight gain (in mice without HL), supporting our hypothesis. To apply this discovery to human disease, we created a transgenic mouse model expressing human HL. We created a catalytically inactive HL (ciHL) mouse model to study the contribution of non-hydrolytic properties of HL to obesity. Our current study utilizes these mouse models to assess the role of catalytic and non-catalytic effects of HL in promoting weight gain by assessing daily food intake and weight gain and by determining energy expenditure using indirect calorimetry. Because HL lipoprotein hydrolysis might have both pro-and anti- atherogenic effects, we are also examining the contribution of active and catalytically inactive HL to AS in mouse models that have been crossed with LDL receptor-deficient mice, a validated atherosclerosis model with high plasma cholesterol. In this model we will test the hypothesis that catalytically active HL promotes atherosclerosis. If our hypotheses that HL contributes to obesity and atherosclerosis are supported in the humanized mouse models,  a pharmaceutical agent could be developed to inhibit HL in humans.

Sepsis is a complex, incompletely understood and often fatal disorder that is one of the most common causes of death in children worldwide. The overlapping pro- and anti-inflammatory responses, and resulting complications contribute to the complexity of sepsis and have made it difficult to find biomarkers and therapeutics targets for this disorder. Mast cells and basophils are well-known for being key players in allergic responses. However, it is less known whether these cells can influence the outcome of bacterial infections. We hypothesize that mast cells and/or basophils can limit the severity of the inflammatory response against bacterial infections to preven sepsis. We found that mast cell and basophil deficient mice (Cpa3-Cre; Mcl-1^flox/flox mice) exhibit signs of morbidity (reduced body temperature), kidney dysfunction (high blood urea nitrogen levels), and are unable to govern the inflammatory response induced by cecal ligation and puncture (CLP), a well-established model of sepsis. The inability to regulate the inflammatory response induced by CLP is shown by the increased number of neutrophils in the peritoneal cavity of the mast cell and basophil deficient mice. Our initial results indicate that Cpa3-Cre; Mcl-1^flox/flox mice that have been previously engrafted with mast cells show similar signs of morbidity, kidney dysfunction, and neutrophil numbers in the peritoneal cavity than non-engrafted mice. These results could indicate that basophils are more responsible than mast cells for the down-regulation of inflammation and reduced morbidity after CLP. Considering these new results, the next step will be to obtain basophil deficient mice to see the response after CLP and what effect basophils might have on the anti-inflammatory response during sepsis.