IFITM5-mediated Osteogenesis Imperfecta
Mentors: Tom Gallagher, PhD and Pauline Camacho, MD
Osteoporosis and Paget’s Disease are common bone disorders that arise with advanced age and cause very significant morbidity for millions of patients worldwide. Similar severe bone disorders are the genetic “osteogenesis imperfecta” (OI) syndromes. OI can range from very mildly disfiguring to life threatening cases. OI type V, a rare, life threatening form, has recently been causally linked to a mutation in the “ifitm5” gene. IFITMs are small membrane-associated proteins that impact cell development and cell fate. This proposal aims to understand how an inherited IFITM5 mutation causes OI type V. The proposal will address the hypothesis that the IFITM5 mutation inhibits the development of multinucleated osteoclasts, which are macrophage-like cells that resorb and reshape bones. Completion of the aims of this proposal will shed light on the general functions of IFITMs and will contribute to our understanding of osteoclast activities during bone resorption and remodeling, and will promote novel ways to control dysregulated bone development as occurs during osteoporosis and Paget’s Disease.
Decontamination of Clostridium difficile spores
Collaborators: Adam Driks, Ph.D. and Stuart Johnson, M.D.
Clostridium difficile contamination is widespread in hospitals, significantly increasing the risk of infection for susceptible patients. We seek to reduce this risk by developing chemistries that remove C. difficile from hospital environments which are safe and easy to apply, and do not present additional risks to patients.
The Microbiome and Disease
We have projects working on the urinary microbiome in healthy women and woman with bladder, vaginal or kidney disorders. We have found that most women have Lactobacillus in their urine and that most women’s bladders have a dominant bacterial genera that accounts for >50% of the bacteria that are detected. We have also found that many bacterial genera can be present, such as Staphylococcus, but at a much lower frequency. We are currently studying the relationship between these lower frequency populations and clinical parameters.
We are also studying the ocular microbiome and its role in floppy eyelid disease, Steven’s Johnson Syndrome, graph vs. host disease and aqueous tear deficiency. The eye is another low biomass site, but we have developed robust methods to interrogate the microbiome in this body site. Technology advances are also allowing us to collect and process samples more quickly so we can spend more time on data analysis.
Pearce, M.M., Hilt E.E., Rosenfeld, A.B., Zilliox, M.J., Thomas-White, K., Fok C., Kliethermes, S., Schreckenberger P.C., Brubaker, L., Gai, X. and Wolfe, A.J. 2014. The female urinary microbiome: a comparison of women with and without urgency urinary incontinence. MBio 8:e01283-e01284.
Hilt, E., McKinley, K., Pearce, M.M., Rosenfeld, A.B., Zilliox, M.J., Mueller, E.R., Brubaker, L., Gai, X., Wolfe, A.J. and Schreckenberger, P. 2014. Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J. Clin. Microbiol. 52:871-876.
Pearce, M.M.#, Zilliox, M.J.#, Rosenfeld, A.B., Thomas-White, K.J., Richter, H.E., Nager, C.W., Visco, A.G., Nygaard, I.E., Barber, M.D., Schaffer, J., Moalli, P., Sung, V.W., Smith, A.L., Rogers, R., Nolen, T.L., Wallace, D., Meikle, S.F., Gai, X., Wolfe, A.J., Brubaker, L. Pelvic Floor Disorder Network. 2015. The female urinary microbiome in urgency urinary incontinence. Am. J. Obstet. Gynecol. 213:347.e1-347.e11.
Thomas-White, K.J., Hilt, E.E., Fok, C., Pearce, M.M., Mueller, E.R., Kliethermes, S., Jacobs, K., Zilliox, M.J., Brincat, C., Price, T.K., Kuffel, G., Schreckenberger, P., Gai, X., Brubaker, L., Wolfe, A.J. 2015. Incontinence medication response relates to the female urinary microbiota. Int. Urogynecol. J. [Epub ahead of print].
Malki, K., Shapiro, J.W., Price, T.K., Hilt, E.E., Thomas-White, K., Sircar, T., Rosenfeld, A.B., Kuffel, G., Zilliox, M.J., Wolfe, A.J., Putonti, C. 2016. Genomes of gardnerella strains reveal an abundance of prophages within the bladder microbiome. PLoS One. 11:e0166757.
Funding: P20 and R01 from NIDDK
Immune Cell Transcriptomes
With the completion of the human genome project, scientific effort has turned to understanding the transcriptome, epigenome and proteome. Our lab’s interest is in understanding the transcriptome of T cells in antitumor and antiviral responses. Normal gene expression analysis methods are optimized for wet-lab experiments, where researchers have replicates and want to minimize variation. During the immune response, investigators must deal with samples that have increased variation due to cellular activation. To address these issues, our gene expression methods do not look at up- and down-regulated genes, but whether genes are expressed/unexpressed in a given sample. We have recently found that 30% of regulated immune genes are missed due to these problems and we are working to understand their role in protection.
Zilliox, M.J. and Irizarry, R.A. 2007. A gene expression bar code for microarray data. Nature Methods 4:911-913. PMCID: PMC3154617.
McCall, M.N., Uppal, K., Jaffee, H.A., Zilliox, M.J.* and Irizarry, R.A.* 2011. The Gene Expression Barcode: leveraging public data repositories to begin cataloging the human and murine transcriptomes. Nucleic Acids Research 39:D1011-D1015. PMCID:PMC3013751. *Co-corresponding authors.
Wu, G., Yustein, J.T., McCall, M.N., Zilliox, M., Irizarry, R.A., Zeller, K., Dang, C.V. and Ji, H. 2013. ChIP- PED enhances the analysis of ChIP-seq and ChIP-chip data. Bioinformatics 29:1182-1189.
McCall, M.N., Jaffee, H.A., Zelisko, S.J., Sinha, N., Hooiveld, G., Irizarry, R.A. and Zilliox, M.J. 2014. The Gene Expression Barcode 3.0: improved data processing and mining tools. Nucleic Acids Research 42:D938- D943.
Funding: NIH, Loyola University, University of Rochester and Harvard University Institutional Funds
Mechanisms of Clostridium difficile pathogenesis
Collaborators: Adam Driks, PhD and Dale Gerding, MD
Ingestion of C. difficile spores is critical to establishing an infection, yet the fate of the spore in the gut and its role in adhesion to the gut epithelium is unknown. We will identify where within the gut where C. difficile spores reside. We will characterize the association between the spore and the gut mucosa by light and electron microscopy.
Clostridium difficile ecology in the gut
Collaborators: Adam Driks, PhD and Dale Gerding, MD
Ingestion of C. difficile spores is critical to establishing an infection, yet the fate of the spore in the gut and its role in adhesion to the gut epithelium is unknown. It is very likely that C. difficile cells exist within a biofilm in the gut. To begin to address this question, we will search for conditions in vitro that stimulate C. difficile biofilm formation, and characterize any resulting extracellular matricies.
Mechanisms of Clostridium difficile persistence
Collaborators: Adam Driks, PhD and Dale Gerding, MD
Colonization of the gut by C. difficile spores is critical to establishment of disease. Possibly, it is also important in disease recurrence, a growing problem in hospitals. Persistence of C. difficile spores in the gut could explain recurrance. To begin to address this possibility, we will analyze the molecular basis of spore-epithelium interactions.
Human T lymphopoiesis in bone marrow or cord blood transplant
Mentors: Phong Le, PhD and Patrick Stiff, MD
One of the hurdles in immune reconstitution in patients who received radiation or chemotherapy as a treatment of hematologic malignancy is the poor engraftment of T cells. The overall goal of the project is to develop novel strategies to promote the development of T cells from hematopoietic stem cells either from the bone marrow or cord blood. We are currently developing in vitro culture system with human thymic epithelial cells and mouse animal model using thymic organoid to effectively boost the development of T cells from hematopoietic stem cells.
Pathogenesis of Clostridium difficile infection (CDI) (Adaptive Immunity)
Mentors: Katherine Knight, PhD and Dale Gerding, MD
The standard animal model for C. difficile infection (CDI) has been the Syrian Golden Hamster, but more recently a mouse model has been developed that more closely mimics the course of CDI in humans. The availability of additional immunologic reagents in the mouse model also makes this a more attractive model. Previous work in hamsters and data from humans suggest that colonization of the gut by harmless non-toxigenic C. difficile (NTCD) is protective against challenge by toxigenic strains of C. difficile. The mechanism of this protection is unknown. The plan for this project is to evaluate colonization of the mouse by NTCD, dose, effectiveness, duration, and then to evaluate protection against challenge with toxigenic C. difficile. The antibiotic stimulus will be cefoperazone administered in the drinking water for 10d followed by a single dose of clindamycin either orally or by ip injection. Adaptive immune response will be assessed for antibody (surface protein, others) and cellular immunity.
Prevention of CDI in the Mouse Model by Prior Colonization with Non-Toxigenic Clostridium difficile (NTCD)
Mentors: Katherine L. Knight, PhD and Dale Gerding, MD
Colonization of the gut by NTCD has been shown to prevent CDI caused by toxigenic strains in humans and the hamster model, but has not been tested in the mouse model. Demonstration of CDI prevention by NTCD in the mouse is likely to enable uncovering of the mechanism of protection by NTCD which has not been determined to date. This may be immunologic, or related to adherence properties of NTCD that interfere with adherence of toxigenic C. difficile.
Immune response to methicillin resistant Staphylococcus aureus and vaccine design
Mentors: Chris Wiethoff, Ph.D. and Malliswari Challapalli, M.D.
Methicillin resistant S. aureus (MRSA) causes more deaths in the US, than HIV, influenza and viral hepatitis, combined. Skin and soft tissue infection with S. aureus are reaching epidemic proportions in developing countries. Yet no vaccines are currently available to limit this deadly infection. Working with Clinicians, Microbiologists and Immunologists, we will characterize the antibody and T-cell responses to candidate MRSA antigens in patients. Immunodominant epitopes within these antigens will be identified. Immune responses to these epitopes in humans will be correlated with disease severity, disease recurrence and bacterial strain. Using a mouse model for cutaneous MRSA infection, novel T-cell vaccines employing epitopes discovered from these studies will be examined for their capacity to protect against MRSA infections.
Human bladder epithelial culture system for studying host pathogen interactions: Cellular and molecular mechanism for recurrent urinary tract infection
Mentors: Phong T. Le, PhD, Linda Brubaker, MD, MS
The human urinary tract (UT) provides important mechanical barriers to infection; however, UT is one of the most common sites for bacterial infection. UT infections (UTIs) are initiated when uropathogenic Escherichia. coli. (UPEC) invade and colonize the superficial epithelial or bladder umbrella cells (BUC) that line the bladder lumen. Entrance into BUC allows UPEC to proliferate and establish bacterial intracellular communities. In response to the invasion and colonization, BUC respond by mounting innate immune response leading to exfoliation of infected cells. This project will utilize primary human BUC to interrogate host-UPEC interactions and mechanisms by which UPEC escape innate immune response and cause recurrences of UTI.
Mucosal Vaccines for Clostridium difficile infection
Mentors: Dale Gerding, MD and Katherine L. Knight, PhD
Clostridium difficile infection (CDI) rivals MRSA as the most common hospital-acquired infection and results in 15,000 to 20,000 deaths annually. CDI usually occurs after antibiotic treatment and the frequency of CDI is on the rise, in large part because of increased use of antibiotics. Although CDI is generally treatable, it recurs in ~20% of treated patients, and no effective vaccines for prevention of CDI are available.
Because CDI is initiated by an intestinal bacterium, we hypothesize that a mucosal vaccine will provide optimal protection. We propose to develop both DNA and epitope vaccines that will be delivered orally to the gut via a virus-like particle (VLP) that not only delivers the gene or epitope of interest to cells, but also acts as an adjuvant. Similar VLP vaccines, such as the widely used Gardasil (Merck) for human papilloma virus, have been shown to provide mucosal protection.
Immune modulation by Vitamin D
Mentors: Pauline Camacho, MD and Makio Iwashima, Ph.D.
Increasing number of reports show Vitamin D as a potent immunomodulatory factor. Benefit of Vitamin D is recognized to the extent that Time magazine recognized Vitamin D as one of its top 10 medical breakthrough for 2007. Vitamin D deficiency is highly prevalent worldwide and is associated with an increased risk of disabling chronic problems, such as osteoporosis, malignancies, myopathy, hypertension and autoimmune diseases.
Our recent work showed that a correlative evidence showing that vitamin D deficient lung transplant patients have a lower success rate than vitamin D sufficient patients. We hypothesize that vitamin D deficiency in these patients cause reduction in immunoregulatory cell functions. To test this hypothesis, we will determine if peripheral blood cells from vitamin D deficient patients have impairment in induction of immunoregulatory lymphocytes.
The results of this study will contribute significantly to our understanding of the vitamin D role in regulation of immune responses and may provide a background study for future studies that can improve tolerance among organ transplant recipients and treatment of autoimmune disorders through vitamin D
Cellular and molecular basis of fetal tolerance
Mentors: Makio Iwashima, PhD and Paula White, MD
Fetal and neonatal immune system is known to be immature and antigen stimulation can cause tolerance rather than effector responses. Monocytes in peripheral blood differentiate to macrophages and dendritic cells (DC) which drive adaptive immunity, and they also likely provide cytokine support for T-independent B cell responses. We have recently identified a population of monocytes in cord blood that drive development of immunosuppressive regulatory T (Treg) cells. We hypothesize that the immunosuppressive state of infants is due in part to the predominance of these immunosuppressive monocytes which promote expansion of Treg cells. In this project, we will determine the genes expressed selectively by these immunomodulatory monocytes to elucidate the mechanism by which these monocytes induce Tregs. Results from these studies are expected to provide a basis for understanding fetal/neonatal tolerance and will help developing safer and long lasting immunesuppression. The outcomes of this study will also help overcoming the immunocompromised state of infants by a combination of more effective vaccine adjuvants that will abrogate the immunosuppressive state of infants as well as enhance their immunity.
Development of anti-tumor CD4 T cells
Mentors: Makio Iwashima, PhD and Christopher Wigfield, MD
One of major obstacles in promoting anti-tumor immunity is the regulatory wing of the immune system that prevents attack on self-tissues. To promote effective immune responses, it is critical to overcome the dominance by these regulatory mechanisms. One such mechanism is apoptotic cell death of antigen-activated T cells whereby continuous stimulation by antigen (such as tumor specific antigens) leads to apoptosis of reactive T cells. We demonstrated that , under the culture conditions that include TGF-beta, naive CD4 T cells become resistant to apoptosis and undergo robust expansion instead of apoptosis. A substantial fraction of expanded T cells expressed IL-17, which are highly pro-inflammatory and can be effective in destruction of tumors. In this project, we will generate tumor-specific Th17 cells from human peripheral blood using these culture conditions and test if they can promote ant-tumor immunity.
Development of a Clostridium difficile Oral Vaccine using Non-Toxigenic C. difficile as a Vector
Mentors: Katherine L. Knight, PhD, Dale Gerding, MD and Lorinda Wright, MD
Non-toxigenic C. difficile (NTCD) lacks the genes for production of toxins A, B, and binary toxin and does not produce these toxins, but is able to colonize the gut of animals and humans following disruption of the microbiota by prior antibiotics. These animals and patients do not have any symptoms of C. difficile infection (CDI) but colonization by these strains protects against challenge with a toxigenic strain of C. difficile. One NTCD strain is currently undergoing clinical testing in patients to determine if it will colonize and prevent CDI in these patients. Once colonization ends in animals and humans, it is assumed that the normal microbiota has recovered, but if additional antibiotics are given, the subject will once again be at risk for infection by a toxigenic strain.
The goal of this project is to use NTCD as an oral vaccine vector to induce a mucosal immune response to binding elements of C. difficile toxins A and B. To do so requires the genetic introduction of the binding regions of the toxins plus promoters, and expression of these antigenic binding regions either on the surface or in the supernatent in the colon of the recipient. Genetic introduction using both plasmids and introduction into the chromosome using Clostron are being employed. The project is currently still in the stages of introducing the genetic changes in the NTCD organisms. When this is achieved testing for in vitro expression of the binding regions will begin followed by testing for immune response in the hamster and mouse models of CDI, measurement of serum and mucosal antibodies and testing of prevention of CDI when challenged with toxigenic strains.
Microbiome of the urinary tract
Mentors: Alan Wolfe, PhD and Paul Schreckenberger, PhD, Linda Brubaker, MD, Elizabeth Mueller, MD, or Cynthia Brincat, MD
Urinary tract infections (UTI) are common in women, with an estimated lifetime risk of 60%. In 2000, the estimated cost of UTI treatment in the USA was about $2.47 billion. There is a critical, unmet clinical need for diagnostic & treatment advances for women with symptoms typical of UTI, but who have culture-negative urine samples. Given the dogma that ‘normal’ urine is sterile, UTI-like symptoms in culture-negative women have been enigmatic. Using the latest DNA sequencing technologies, we have obtained information that may advance our understanding of the problem: women have a bladder microbiome; i.e, bacteria colonize the bladders of women (Wolfe et al., 2012; Hilt et al., 2014; Pearce et al., 2014; Nienhouse et al., 2014; Brubaker et al., 2014; Pearce et al., 2015; Thomas-White et al., 2015). Now that we know that a female bladder microbiome exists, our translational research team composed of clinicians from Urogynecology, basic scientists within the Department of Microbiology & Immunology & Clinical Microbiology Lab, in collaboration with the Loyola Genomics Facility & Bioinformatics Center want to know how that microbiome affects women’s health. Our primary hypothesis is that bacterial biodiversity exists in the bladders of normal women & that perturbations in this normal bacterial biodiversity may contribute to symptoms in culture-negative symptomatic populations. In simpler terms, we wish to learn the answers to the following questions: Which bacteria are present in the female bladder? How does the composition of the bladder microbiome compare to the microbiomes of the surrounding organs or surfaces, e.g. the vagina and the perineum? Which bacteria are protective? Which ones are pathogenic? We anticipate that this study will facilitate a paradigm change in our understanding of the female urinary tract in health and disease.
Developing novel methods to decontaminate bacterial spores
Mentors: Adam Driks, PhD, and Dale Gerding, MD
There is a need for novel treatments that destroy infectious agents under conditions that are mild and, therefore, can be used in situations where a more reactive or harsh treatment would be inappropriate. This goal is especially important in the case of bacterial spores, which are highly resistant to most decontamination treatments. In this project, we will study the effects of novel decontamination formulations including both mild chemicals as well as degradative enzymes, on bacterial spores. We will study the decontamination of the biological weapon Bacillus anthracis as well as the hospital acquired pathogen Clostridium difficile.
Adenoviral Immunopathogenesis: Reduced Innate Immune Modulation by Highly Pathogenic Adenovirus 14 Clinical Isolates
Mentors:Jay R. Radke,PhD, and James L. Cook, MD
Adenoviruses modulate host immune responses through a variety of mechanisms. This might explain the relatively mild nature of most Ad infections in immunocompetent people. We recently discovered a novel Ad mechanism that represses pro-inflammatory responses by mimicking the immunomodulatory effects of apoptotic cells. We mapped this function to the Ad E1B 19kD gene. Over the last few years there have been outbreaks at military bases and in civilian populations of emerging Ad14 strains that can induce strong, pro-inflammatory reactions in infected, but otherwise healthy people. In some cases, the lung inflammatory responses to these Ad14 infections have been so intense that they have resulted in Acute Respiratory Distress Syndrome (ARDS), resulting in death from respiratory failure. Based upon our previous studies, we postulated that one reason for the increased inflammatory reactions in these patients could be viral mutations that reduce the E1B 19kD immunomodulatory activity of these emerging Ad14 strains. To date, our studies have revealed that cells infected with these highly pathogenic Ad14 strains fail to repress pro-inflammatory responses as effectively as cells infected with wild type Ad14. In addition, cells infected with the emerging Ad14 strains display characteristics consistent with defects in either E1B 19kD expression or function. Sequencing of the E1B 19kD gene showed a single silent point mutation, which suggested that any alterations in E1B 19kD activity are more likely explained by changes in its expression level than its function. That hypothesis has been supported by our observation that expression of E1B 19kD mRNA is markedly reduced in cells infected with the highly pathogenic Ad14 strains, when compared with E1B 19 kD mRNA expression in cells infected with wild type Ad14. Further sequencing analysis has revealed mutations scattered throughout the E1B 19kD promoter region, suggesting that promoter dysfunction could be one possible mechanism of reduced E1B 19kD mRNA expression. In addition, sequencing of the E1A gene has revealed a mutation in the region of E1A that is associated with transcriptional activation of E1B 19kD and other adenoviral genes. The objectives of this project include the study of these two possible mechanisms of reduced E1B 19kD expression in cells infected with the highly pathogenic strains of Ad14 and pursuit of related studies of the immunobiology of disease caused by this emerging viral pathogen.
How stress and the cholinergic axis influence antimicrobial peptide (AMP)
Mentors: Kathrine Radke, PhD
How stress and the cholinergic axis influence antimicrobial peptide (AMP) and Toll-like receptor signaling in models of tissue injury and infection in relation to the urinary tract. This is a new research focus in the Radke Lab.
Urinary Research Interests
Mentors: Kathrine Radke, PhD and Paul Schreckenberger, PhD
Research in the Radek laboratory focuses on how stress and the cholinergic axis influence antimicrobial peptide (AMP) and Toll-like receptor signaling in the urinary tract. Antimicrobial peptide (AMP) production is critical for epithelial homeostasis and the response to injury and infection. Urothelial cells possess a non-neuronal cholinergic system comprised of acetylcholine (ACh) and acetylcholine nicotinic (nAChR) receptors. We previously determined that systemic cholinergic activation via psychological stress in mice diminished epithelial AMP expression, which was restored by topical application of nAChR antagonists. This association is relevant to the bladder, as several urinary tract disorders (e.g. overactive bladder, painful bladder syndrome) are aggravated by psychological stressors, in part, due to altered nAChR activation. Toll-like Receptors (TLRs) are innate immune receptors that can sense bacterial components to promote AMP induction at the site injury or infection. We recently identified that nAChR activation in urothelial cells dampens TLR2 and TLR4-mediated AMP induction. Thus, we are investigating the mechanisms by which stress and the cholinergic axis may promote AMP dysregulation in urothelial cells in the context of urinary tract infection. Impairment of this pathway is likely a novel cause for recurrent UTI development that contributes to secondary clinical manifestations (i.e. pyelonephritis; urosepsis).
Skin Research Interests
Mentors: Katherine, PhD and Paul O’Keefe, MD
Dermatology research in the Radek laboratory focuses on how stress and the cholinergic axis influence cutaneous antimicrobial peptide (AMP) and Toll-like receptor signaling in models of tissue injury and infection in the skin. Antimicrobial peptide (AMP) production is critical for cutaneous homeostasis and the response to injury and infection. Epithelial cells possess a non-neuronal cholinergic system comprised of acetylcholine (ACh) and acetylcholine nicotinic (nAChR) receptors. We previously determined that systemic cholinergic activation via psychological stress in mice diminished epithelial AMP expression, which was restored by topical application of nAChR antagonists. In parallel, we observed an increased susceptibility to Staphylococcal and Streptococcal infection, supporting clinical observations that stress promotes or exacerbates many skin disorders associated with AMP dysregulation. We are investigating how stress and the cholinergic axis may promote AMP dysregulation and subsequent epithelial repair processes in keratinocytes in the context of impaired wound healing and inflammation. Disrupting this tight balance via changes in AMP regulation can shift the balance to restrict the ability of the skin to combat infection or initiate detrimental inflammatory processes.
Does the urinary microbiome influence urinary incontinence (UI)?
Mentors: Alan Wolfe, PhD and Linda Brubaker, MD, Elizabeth Mueller, MD, or Cynthia Brincat, MD
UI affects as many as 30-60% middle-aged and older women in the general population. Treatment is often not effective, suggesting that UI may have multiple and sometime unknown contributing etiologies. One distinct possibility is the existence of previously unidentified urinary bacteria that cause symptoms similar to UI (Pearce et al., 2014; Pearce et al., 2015). An InDIRI MS student would test this hypothesis using the latest in DNA sequencing technologies and bioinformatic analyses. Our aim is to improve our understanding of the disease etiology and to devise individualized treatment accordingly.
Does the urinary microbiome influence treatment of overactive bladder (OAB)?
Mentors: Alan Wolfe, PhD and Linda Brubaker, MD, Elizabeth Mueller, MD or Cynthia Brincat, MD
OAB affects at least 15% of adult women, with higher incidence with aging. Miragebron is an oral medication marketed by the pharmaceutical company Astellas for OAB. While Mirabegron is effective for many patients, others do not experience these preferred results. We hypothesize that the urinary microbiome may contribute to urinary symptoms and there is a difference in microbiota between responders and non-responders to Mirabegron. A similar study asking the same questions of an OAB therapy from a different class of medications was recently published (Thomas-White et al., 2015). An InDIRI MS student would test this hypothesis using the latest in DNA sequencing technologies and bioinformatic analyses. Again, our aim is to improve our understanding of etiology and to devise individualized treatment.
Is there a protective urinary microbiome?
Mentors: Alan Wolfe, PhD, and Linda Brubaker, MD, Elizabeth Mueller, MD or Cynthia Brincat, MD
Several new lines of evidence suggest the existence of a community of bacteria that protect individuals against UTI. For example, we used quantitative PCR to seek urinary bacteria in UUI patients undergoing treatment (Brubaker et al., 2014). Surprisingly, evidence of urinary bacteria was negatively associated with the risk of UTI, a disease caused by bacteria. Similar results were observed by 16S rRNA sequencing (Pearce et al., 2015). On the basis of these unexpected but fascinating observations, we hypothesize that certain urinary bacteria help protect UUI patients from UTI caused by other bacteria. An InDIRI MS student would sequence and analyze bacterial DNA obtained from the urine of these UUI patients.
Phenotype bacteria isolated from urine by the clinical microbiology laboratory
Mentors: Alan Wolfe, PhD and Paul Schreckenberger, PhD
From the urine samples that we have sequenced, the clinical microbiology laboratory has isolated a variety of bacteria (Hilt et al., 2014; Pearce et al., 2014). To understand how these bacteria influence women’s health and disease, we must characterize them. An InDIRI MS student would phenotype these bacteria, monitoring relevant behaviors, e.g. resistance to anti-microbial peptides and hydrogen peroxide, ability to produce hydrogen peroxide and acid, tolerance to pH and lactic acid, ability to grow or persist in urine, ability to be motile, the capacity to build a biofilm or to attach, enter and/or grow in bladder epithelial cells.
Culture and characterize bacteria identified by sequencing but not yet cultured
Mentors: Alan Wolfe, PhD and Paul Schreckenberger, Ph.D
Many of the bacteria detected by sequencing have not been cultured, although protocols for culturing do exist for some. Once successfully cultured, the bacterium would be identified and characterized.
Mechanism of neutralization resistance in an HIV-1 strain circulating among IV drug users
Mentors: Edward Campbell, PhD and Paul O'Keefe, MD
Recently, a circulating recombinant strain of HIV-1 has been shown to contain a small mutation encoding a small deletion in the viral gag protein. Although this mutation attenuates viral replication, its continued transmission among IV drug users suggests that this virus maintains a selective advantage in its ability to be transmitted via contaminated needles. Preliminary evidence suggests that this virus may maintain viability ex vivo through increased incorporation of the HIV-1 envelope during viral production, allowing it to remain infectious for longer periods of time while in contaminated syringes. We will directly test this hypothesis by measuring envelope incorporation into HIV-1 particles with this mutation, and measuring the ability of these particles to remain infectious following their release from producer cells. Completion of this project will shed light on the mechanisms by which viruses are selected to enhance their ability to be transmitted between individuals.
Genetic manipulation of potential uropathogens
Mentors: Karen Visick, PhD and TBD
Until recently, urine was believed to be sterile. Now, work from Loyola, in particular from the Wolfe lab, has demonstrated the existence of numerous microbes in urine. Some of these microbes may be “normal flora” bacteria, while others have been associated with the disease state. In collaboration with the Wolfe lab, our long term goals seek to understand the roles of specific urinary isolates in disease. To achieve these goals, we propose first to develop tools for the genetic manipulation of the urinary isolates. Then, we will mutate specific genes with the potential to be involved in virulence. Finally, through collaborations, we will develop assays of virulence to determine if the mutant bacteria exhibit reduced or loss of virulence.
New Research Focus in the Chouldhry Lab
Mashkoor Chouldhry, PhD
• Gut mucosal immunity and epithelial barrier in response to traumatic injury and alcohol
• Gut microbiota and its role in maintenance of gut immunity and epithelial barrier
• Alcohol role in inflammatory responses associated with inflammatory bowel disease
• Neutrophil role in tissue damage and in maintenance of mucosal defense in setting of alcohol and trauma
• Autophagy in the maintenance of mucosal defense
• microRNA in the maintenance of mucosal defense
The effect of scavenger receptor ligand on blood cells
Makio Iwashima, PhD
We have recently identified a subset of blood cells that prevent inflammatory and auto-immune responses. Those cells express a surface protein called scavenger receptor. Known ligands for this receptor includes low density lipoprotein (LDL) and beta-amyloid. These molecules are associated with atherosclerosis and Alzheimer’s disease, respectively. We are currently studying the effect of scavenger receptor ligand on these blood cells.
Research Projects in Iwashima's Lab
Makio Iwashima, PhD
Immunoregulatory monocytes and Alzheimer’s disease
Effect of LDL on Immunoregulatory monocytes - with Erin Lowery, MD
T cells effect on fibrosis and tumor metastasis - with Erin Lowery, MD
Cytotoxic CD4 T cells and anti-tumor immunity - with Mamdouh Bakhos, MD
Mechanisms underlying commensal bacterial metabolic products regulating host immune function
Liang Qiao, Ph.D
Human gastrointestinal (GI) harbors a complex microbial community composed of trillions of commensal bacteria. The profound influence of these microbial communities is very important in many aspects of health and also plays important roles in food digestion and synthesis of nutrients as well as regulation of immune function. We will determine how gut commensal bacteria regulate host innate immune cell functions.
Development of mucosal HIV-1 vaccines
Liang Qiao, Ph.D
Although anti-HIV-1 (human immunodeficiency virus type 1) drugs are effective in controlling development of AIDS, HIV-1 infection still remains an enormous problem worldwide. Thus, an effective HIV-1 vaccine is urgently needed. We are developing novel mucosal HIV-1 vaccines that can provide effective protection.
Investigating mechanisms of macrophage activation during viral infection
Mentors: Susan Baker, PhD, Francis Alonzo, PhD and Jennifer Layden, MD, PhD
Macrophages are critical immune mediators and key players in the clearance of viral infections. This project will investigate how the model coronavirus, mouse hepatitis virus, affects the phagocytic activity of macrophages. Students will learn how to: isolate and propagate bone marrow-derived macrophages; to analyze transcriptional activation; to monitor phagocytic activity and to determine the effect of viral replication on macrophage activation.
Operational Mosquito Control Research
Mentors: Justin Harbison, PhD
In response to the Zika epidemic that occurred in during 2015-2016, the CDC funded proposals from four groups to create “Regional Centers of Excellence for Vector-Borne Disease” to address emerging and exotic vector-borne diseases in the United States. One of these groups, that includes faculty from University of Wisconsin – Madison, Michigan State University, Iowa State University, University of Illinois at Urbana-Champaign, and Loyola University Chicago has created the “Upper Midwestern Center of Excellence in Vector Borne-Diseases” headquartered in Madison, Wisconsin. As part of this collaborative research and education initiative, Dr. Harbison and his students focus on 1) enhancing surveillance of the invasive mosquito species and potential Zika vector, Aedes albopictus, and 2) validation of existing mosquito control methods (ie. type and timing of pesticide use).
Mechanisms of Biofilm Formation
Mentors: Karen Visick, PhD and TBD
Bacteria adhere to each other and to surfaces in communities that are termed biofilms. Bacteria within biofilms are highly resistant to antibiotics and to host defenses, making biofilms problematic in clinical settings. Thus, understanding how biofilms form and how bacteria are released or dispersed from biofilm communities is a critical area of research. We probe these aspects of bacterial biology using the microbe Vibrio fischeri as a simple model for animal-relevant biofilm formation. Various specific projects are available, including investigating the signal transduction machinery that leads to biofilm formation, environmental signals that promote biofilm development, and cues and factors that lead to dispersal.