Angela M. Christiano - US grants

Laminins comprise a family to trimeric extracellular matrix proteins expressed in the basement membranes of many tissues, appearing first during the early stages of embryonic development, and later in a wide variety of tissue-specific isoforms. Laminin 5 is expressed in the basement membrane zone of specialized epithelia with secretory or protective functions, such as the skin. It is observed in the anchoring filaments of the lamina lucida, which connect the hemidosmosomes of the basal keratinocytes to the lamina densa of the dermal-epidermal junction. Laminin 5 is unique within the laminin family, in that it is the only human laminin which has a demonstrated role in a genetic disorder. The junctional forms of EB (JEB) are inherited in an autosomal recessive manner, and are characterized by blisters which form within the dermal- epidermal basement membrane zone, resulting in dysadhesion of the epidermis from the dermis. Junctional EB frequently results in the demise of affected individuals within the first few months of life. Several lines of evidence support the hypothesis that the three genes encoding the anchoring filament protein laminin 5 as candidate genes for harboring the mutations in junctional EB. Mutations in the beta3 chain gene (LAMB3) and gamma2 chain gene (LAMC2) of laminin 5 have recently been shown to underlie the junctional forms of epidermolysis bullosa in some families. In other families, however, both the LAMC2 and LAMB3 genes have been excluded by genetic linkage analysis or by the presence of normal levels of both LAMC2 and LAMB3 mRNAs in cultured keratinocytes. In these cases, the gene encoding the third polypeptide of laminin 5, the alpha3 chain gene (LAMA3) has become an attractive candidate gene for harboring additional mutations underlying JEB. Toward the overall goal of understanding the role of the LAMA3 gene in the junctional forms of epidermolysis bullosa, the principal objectives of this proposal are: Specific Aim 1: To elucidate the intron/exon organization of the LAMA3 gene. This will involve the isolation and characterization of cosmid clones encompassing the sequences encoded by the LAMA3 cDNA. It is anticipated that the gene structure of LAMA3 will be complex, since there is evidence for at least three 5' alternative transcripts and two 3' alternative transcripts. Segments of the LAMA3 cDNA will be used for the design of oligonucleotide primers for direct PCR based sequencing from the genomic DNA clones for the identification of exon borders, flanking intron sequences and the overall gene structure. Specific Aim 2: To design a rapid PCR amplification and mutation detection strategy for the LAMA3 gene. This will involve the design and optimization of PCR primers based on the sequences of the flanking introns. PCR amplimers will be subjected to heteroduplex analysis for the identification of bands with altered mobility in the DNA of unaffected unrelated control individuals. These amplimers will be subcloned and sequenced, and putative neutral polymorphisms will be verified in the normal population. Specific Aim 3: To identify pathogenetic mutations in the LAMA3 gene in junctional EB patients. This will be accomplished using the PCR strategy developed in specific aim 2, and applied to JEB patients in which the candidate gene for mutations is LAMA3. As sequence variants are detected, they will be analyzed to verify that they are indeed pathogenetic and not simple sequence polymorphisms, and that their segregation through JEB families is consistent with the inheritance of the disorder.

The Genotyping and Molecular Diagnostics Core of the Skin Disease Research Center provides expertise in four essential areas of molecular biology central to the investigation of the genetic basis of human disease. These include 1) Genotyping and haplotype analysis 2) Polymerase chain reaction (PCR) technology 3) Heteroduplex analysis and 4) Mutation verification. Core C will assist center investigators in genotype analysis identifying candidate intervals for genes of interest by linkage analysis to be performed in Core A. Pinpointing genes of interest by haplotype analysis in allelotyping studies will also be accomplished in this Core. Design and optimization of a PCR-based mutation screening strategy for candidate genes of interest will be developed in this Core, as well as mutation detection by heteroduplex analysis. Upon identification of putative areas of the gene of interest containing the mutation, PCR products will be prepared for sequence analysis to be performed In Core D. After sequence analysis has identified mutations, the final service provided by this Core will be in assisting the investigator in verifying the mutation and developing a plan for ascertaining its functional consequence, and the way in which it elicits a clinical phenotype. This Core will interact closely with the other three cores, as its success relies on the acquisition of purified DNA from Core B, accurate linkage analysis in Core A, and fidelitous sequencing and oligonucleotide synthesis in Core D. Further, the expertise offered in this Core will be highly utilized and provide a valuable service to several of the Pilot and Feasibility studies, as well as other NIH-funded investigators within the Department.

DESCRIPTION: (Adapted from the applicant's abstract)-This is a resubmission of a grant application proposing to search extensively for mutations that interfere with the normal process of epidermal and in particular palmoplantar keratinization. The project consists of three integrated and interdependent aims. The first one is to search for mutations in eleven candidate genes in a small number of families with a characterized inherited keratoderma. Specifically, the genes are loricrin, involucrin, envoplakin, desmoplakin, plakoglobin, desmogleins 1, 2, 3, desmocollins 1, 2 and 3, and the diseases include Netherton's, Mal-de-Maleda, Vohlwinkel's, Kindler/Weary, erythrokeratoderma, nonepidermolytic PPK, PRP, and eight others for which 1 to 10 families have been enlisted into studies. The second is to identify the mutated genes in large pedigrees with well-characterized, dominant, and fully penetrant keartodermas, one with EB superficialis, the other "Novel Acantholytic Disease." The pedigrees are so large that if the mutations do not map in the region of candidate genes, the first look, random polymorphisms will be used to saturate the map and localize the mutations. The third is to isolate and characterize genes that play a role in epidermal differentiation. Some of these have been cloned at the cDNA level, but the genomic organization, i.e., intron-exon boundaries, is unknown. Others will be obtained from the results of the Specific Aim 2, namely, genes identified as mutated in EB superficialis, and the Novel Acantholytic Disease will be cloned and characterized.

Ectodermal dysplasias (ED) are a group of more than 150 clinically distinct disorders, of which the underlying genes have been positively identified in only two. Many of these syndromes are rare, and can manifest with variable combinations and severity of defects in the development of the four major epidermal appendages: sweat glands, hair, teeth and nails. The conditions have been shown to be attributable to autosomal dominant, autosomal recessive and X-linked dominant and recessive genes. The classical X-linked anhydrotic ectodermal dysplasia (EDA) was linked to Xq12-q13.1, and mutations were identified in a novel transmembrane protein of unknown function, called EDA. More recently, mutations in the plakophilin I gene were found to result in a form of ectodermal dysplasia with skin fragility. In addition to these forms, four other phenotypes have been linked to different chromosomal loci, though no underlying genes or mutations have yet been identified. These include 1) Clouston syndrome in the pericentric region of chromosome 13; 2) EEC (ectrodactyly, ectodermal dysplasia) syndrome to chromosome 7q11.2-21.3; 3) Split hand/split foot malformation to chromosome 7q21.2-q21.3; and most recently 4) (autosomal dominant hypohidrotic ectodermal dysplasia) to chromosome 2q11-q13. We have had an interest in isolated disorders of the hair and teeth, and in this Pilot and Feasibility study, we propose linkage studies and gene discovery in three families with ectodermal dysplasias, with the goal of understanding additional pathways of gene expression in the development of ectodermal structures.

The DNA Sequencing and Synthesis Core will provide instrumentation, general resources and technical expertise in the use of automated DNA sequencing and related molecular biologic techniques for the analysis of gene structure and polymorphisms. Special emphasis will be placed on the use of this information to delineate the genetics of susceptibility to skin diseases. The Core has particular skill in automated sequencing of heterozygous DNA templates to directly define alleles and in computer- assisted sequence analysis. Its two DNA sequencers have been modified to perform automated microsatellite polymorphism analyses as well as conventional automated sequencing. The DNA Sequencing and Synthesis Core will work in concert with the Molecular Diagnostics and Mutation Detection Core on the determination of microsatellite polymorphisms. The DNA Sequencing and Synthesis Core will-also serve as a technical resource center regarding information and training. The Core contains resources and equipment for the synthesis and purification of synthetic oligonucleotides for SDRC investigators and all laboratories interested in the study of skin. Two key features of this core facility are its comprehensive range of services designed to support the needs of both proposed and future SDRC sequencing activities and its objective of training young investigators in the area of its expertise.

[unreadable] DESCRIPTION (provided by applicant): Our work during the first award period was focused on defining the functions of HR in the skin and hair follicle. We have been working to understand what controls hairless expression and have shown that hr is regulated by thyroid hormone as well as VDR in keratinocytes. We localized HR is to the nucleus and identified a new nuclear localization signal in the N-terminal region. We also demonstrated that Hr is tightly associated with the nuclear matrix and forms nuclear speckles in association with the histone deacetylase, HDAC3. We showed that HR and VDR are part of the same complex in keratinocytes. In order place a hairless into a transcriptional regulatory pathway, we performed a microarray analysis of rh/rh versus wild-type mouse epidermis to identify genes that are regulated by hairless. Unexpectedly, we uncovered several genes involved in cell cycle regulation and apoptosis, and in particular, numerous genes that are known downstream targets of NFkB signaling. These novel findings form the basis of our experiments planned for the next five years. We will test the hypothesis that hairless is a key regulator of the balance between cell proliferation and differentiation in the epidermis and hair follicle. We will address this hypothesis in three Specific Aims. Specific Aim 1: Transcriptional Regulation of Hairless by the TNF1/NFkB signaling pathway; Specific Aim 2: Regulation of Downstream Pathways by Hairless; and Specific Aim 3: Hairless functions as an inhibitor of proliferation in epithelial cells in vivo. The crucial role of these signaling pathways in epidermal homeostasis and disease places hairless at the center of many convergent areas of skin research. Advances generated by this work could lead to chemopreventive strategies and innovative treatment options for dermatological diseases. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Our work during the first five years of the grant was focused on defining the functions of the hairless gene in the skin and hair follicle. We have found that hairless is a key regulator of epidermal growth, and that it cross-talks with some key signaling pathways in the skin. In the next five years, we will ask how hairless regulates cell growth and proliferation in the epidermis, both in the normal setting, and in certain skin diseases. These findings may prove useful for gaining understanding a number of different skin disorders where uncontrolled growth is a characteristic of the disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The major emphasis of several laboratories in epidermal biology is on developing gene therapy approaches for skin diseases. As in other fields, gene therapy in the skin has been hampered by the inability to target (or identify) a stem cell, and the lack of sustained gene expression. We instead asked whether we could identify an ectopic source of epithelial cells that could be induced into becoming a skin stem cell. Rather than searching for markers of the epidermal stem cell itself, we asked whether we could reprogram other epithelia into skin under the appropriate inductive (dermal) influences. Much work is currently focused on using the skin as a donor tissue of stem cells for other diseases (neurological, muscular), however, little interest is focused on how to induce other cell types to become skin. We reasoned that if the donor cells were taken from an immunologically-compatible individual or did not elicit an immune response, such cells could overcome the two major obstacles in gene therapy approaches: gene introduction and targeting the stem cell. Donor cells, by definition, would contain an intact gene-of interest, and importantly, others have shown that epidermal stem cells would be sequestered during the induction of the new skin and hair follicle, thus providing a lifelong supply of genetically corrected cells. The success of this project would represent the first demonstration of epithelial reprogramming in human tissues, and more significantly, provide a foundation for the clinical application of reprogrammed skin in the treatment of human genetic skin diseases.

The key features of the Molecular Biology Core of the Skin Disease Research Center are its comprehensive range of services designed to support the needs of both proposed and future SDRC members, and its objective of training young investigators in the area of its expertise. This Core provides assistance in three essential areas of molecular biology central to virtually any studies ranging from the investigation of the genetic basis of human disease to cell biology experiments. These include 1) Disease-gene regulation;2) In Situ labeling of genetic alterations;and 3) Real- Time PCR, automated sequencing and cloning of DNA constructs. The core will assist center investigators in identifying the epigenetic mechanisms of a particular skin disease by promoter and mutagenesis studies and siRNA knockdown models. The Core will assist investigators in automated DNA sequence analysis to identify mutations implicated in skin disease pathogenesis and subsequent histological detection of target cells harboring these genetic mutations. The Core will also assist investigators with the design and optimization of Real-time PCR analysis for candidate disease-genes and developing a plan for ascertaining their functional consequence, and the way in which it elicits a clinical phenotype. The Core will also provide instrumentation, general resources and technical expertise in the use of automated DNA sequencing and DNA cloning. By offering these routinely used services, the Molecular Biology Core will interact closely with the other two cores. Further, the expertise offered in this Core will be almost universally utilized and provide a valuable service to most, if not all, of the Pilot and Feasibility studies, as well as other investigators within the Department.

[unreadable] DESCRIPTION (provided by applicant): Alopecia areata (AA) affects approximately 4.6 million individuals in the United States alone, including males and females of all ages and ethnic groups. Despite its high incidence, its pathomechanism is largely unknown. Although the presence of genetic components conferring susceptibility for developing AA is now widely accepted, this has not translated into studies investigating the genetic basis of AA. Up to now, genetic studies have been limited to association analyses, which suggest that a permissive HLA status may potentiate the development of the AA phenotype. A systematic screen for identifying the primary genetic mechanisms underlying this disorder has never before been undertaken. Some authors have openly advocated in favor of conducting genome-wide linkage analyses in AA, irrespective of the challenges inherent to complex diseases:"... the extent to which this disorder causes morbidity and its incidence warrant investigation for the genes underlying alopecia areata (Green and Sinclair 2000). It is now generally accepted that AA fits the paradigm of a complex or multifactorial trait, in which a combination of genetic and environmental factors combine to result in the final phenotype. In this application, we outline further evidence supporting a polygenic model of inheritance in AA, such as clustering in families, no clear pattern of Mendelian inheritance, a Gaussian distribution of phenotypes, high population prevalence, concordance in twin studies, and an increased risk to first-degree relatives. Importantly, the genomics and computational technology for the analysis of such diseases has vastly improved in recent years. There are currently a number of examples of complex diseases of the skin, such as atopic dermatitis and psoriasis, in which genetic studies have been successfully undertaken that substantiate the timeliness of this approach. We have initiated a comprehensive genetic analysis of AA, as outlined in this proposal. Importantly, we provide evidence for suggestive linkage at four independent loci in the human genome. These include loci on chromosomes 6, 10, 16 and 18. In the context of this proposal, we will perform fine-mapping and gene identification in AA, and go on to demonstrate the association of pathogenic variants in candidate genes with the inheritance of disease. We anticipate these studies will provide a foundation for understanding the interactions of these genes with each other and with other variables such as the immune system. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this Project is to identify the genetic variation contributing to the risk of developing Alopecia Areata (AA). AA is one of the most common human autoimmune diseases, with a lifetime risk of approximately 2%. It affects approximately 4.6 million individuals in the United States alone, including males and females of all ages and ethnic groups. AA fits the paradigm of a complex or multifactorial trait, in which combinations of genetic and environmental factors combine to give rise to the final phenotype. Our lab has focused on using unbiased genome-wide approaches to identify susceptibility loci for AA. We recently pioneered the use of genome-wide family-based linkage as applied to AA for the first time, and identified four susceptibility loci in a small cohort of large, multiplex pedigrees. This study was designed to identify rare alleles with relatively large effects. In this Project, we now seek to do the converse. Here, we propose to carry out a genome-wide association study (GWAS) to identify common alleles with small effect that contribute to risk of AA, using 1000 cases and 3000 controls. All of these cases and a proportion of the controls were collected from the Alopecia Areata Registry and will be genotyped with the Illumina 550K SNP array. We will utilize a strategy that has proved successful with other GWAS and obtain the majority of our control samples from a database of shared controls. We will perform replication studies in an additional 3 independent samples of cases and controls. Each of these four studies will be analyzed independently and jointly, greatly increasing our power to detect association of disease alleles with moderate genetic effects. Once susceptibility alleles for AA have been identified, candidate genes containing the SNPs of interest will be prioritized using mRNA and protein expression patterns in the hair follicle. The positional information will be cross-referenced with information derived from expression studies in mouse models for AA. Finally, should some of the variants identified be coding-sequence SNPs, we will then analyze these candidate genes in depth, to look at mRNA and protein expression, and to formulate mechanistic links to the human disease. We expect that identification of SNPs that confer susceptibility to AA will uncover the network of pathways of disease pathogenesis and lead to new approaches for treating this disorder. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE. The long-term goal of this Application is to identify the genetic variation contributing to the risk of developing Alopecia Areata (AA). AA is a form of hair loss in which the body attacks the growing hair follicle, and can result in hair loss ranging from patches on the scalp to complete hair loss over the body. AA is one of the most common human autoimmune diseases, with a lifetime risk of approximately 2%. AA affects approximately 4.6 million individuals in the United States alone, including males and females of all ages and ethnic groups. While AA is a non-lethal skin disease, its impact as measured in the Burden of Skin Disease Report is profound as it relates to quality-of-life measures. Ultimately, it is anticipated that discovery and modulation of the genes for AA will provide novel therapeutic targets, and eventually eliminate this psychologically devastating dermatologic disorder. The studies outlined in this Application aim to systematically pinpoint common susceptibility alleles for human AA for the first time. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human Genetics has become a central focus of basic and biomedical research over the past decades, and is truly an integrative field. The study of the molecular basis of human disease promises to yield significant insights into our basic understanding of disease pathogenesis, and holds enormous promise in terms of therapeutic and clinical applications. This application requests funds to establish a Medical Genetics Training Program at Columbia University Medical Center to support three postdoctoral trainees per year, in a University-wide training program on the topic of Human Genetics. Training focuses on the clinical, molecular and genetic aspects of both Mendelian and complex diseases in children and adults. The major strength of the Medical Genetics Training Program is the outstanding quality and diversity of the training faculty, which provides trainees with the opportunity to perform postdoctoral research in laboratories and clinical settings that are internationally recognized. Twenty three faculty members representing many different Departments are included in this training program and have extensive experience and outstanding track records as trainers and mentors. Our mission is to advance the science underlying human genetics, and incorporate these advances into clinical practice through translational research. The goals of this Training Grant are directly aligned with NIGMS-supported postdoctoral training programs in medical genetics, which are designed to foster research careers in both basic and clinical aspects of human genetics. The clinical, didactic and research components of our Training Program are intended to prepare physician- scientists for a career in academic medicine. The Medical Genetics Training Program will provide an outstanding well-rounded training experience for postdoctoral trainees that will allow them to pursue a future academic career, and create a forum for the advancement of human genetics from many different programs at CUMC.

DESCRIPTION (provided by applicant): Inherited genetic risk is a major component of Type I diabetes (T1D). Major international research efforts have identified a large number of T1D susceptibility genes through genome wide association studies (GWAS). Despite these advances, in nearly all cases GWAS "hits" have not yielded insight into protein coding alterations but rather are presumed to reflect regulatory SNPs or haplotypes that have refractory to detection. To address the intractable problem of missing hereditability in the post-GWAS era we have assembled a uniquely qualified interdisciplinary team, both to extract maximum information from GWAS and to pursue "post-GWAS" pathogenic epigenetic events. Our innovative approach to this question focuses on both aberrations of T cells and end- organ vulnerability. We have assembled both T1D immunologists and beta cell biologists to team with molecular geneticists/epigeneticists and an experienced bioinformatician in a systems approach to identify therapeutically targetable genome wide epigenetic events in the relevant cytotoxic T cells and their targets. Using mouse models and human subjects recruited through the diabetes clinical centers at Columbia/Yale, T1D specific epigenetic hits will be identified with clinical predictive utility and mechanistic importance. Therapeutic opportunities for reversal will be pursued using novel antisense approaches. Our approach has already identified key epigenetically regulated immunological targets modulating tolerance (CD3, PD-1), and has developed pioneering T1D monitoring tools to define the prediabetic window of insulitis (hypomethylated insulin). Novel epigenetic-based therapeutic approaches are proposed and tested that represent an innovative strategy for inducing endogenous anti-inflammatory isoforms, including endogenous soluble cytokine receptors and ligand independent inhibitory costimulatory molecules. In addition, we will use allele-specific mapping of DNA methylation and gene expression to help extract maximum information from GWAS and re-sequencing data for T1D susceptibility loci. In sum this project will identify genetic and epigenetic events responsible for T1D susceptibility and disease progression, and pursue therapeutic approaches directed at known and novel genetic and epigenetic targets. PUBLIC HEALTH RELEVANCE: Heritable risk in Type 1 diabetes has been extensively studied but remains poorly defined. We will use epigenetic tools to bolster the genetic data, and to find genetic variants that are responsible for aberrant activity of killer T cells and the vulnerability of the end organ islet cells. Treatment opportunities to correct genetically and epigenetically dysregulated genes will be pursued with antisense approaches.

DESCRIPTION (provided by applicant): The goal of this application is to define a translational platform of candidate biomarkers for Alopecia Areata (AA). AA is one of the most common autoimmune diseases, with a lifetime risk of approximately 2%, affecting 5.3 million individuals in the United States alone, including males and females of all ages and ethnic groups. Autoimmunity is acquired against the hair follicles in the skin, which causes hair loss associated with an accumulation of immune-response cells around the affected hair follicles. The prognosis of AA is unpredictable and currently there is no definitive treatment. The chronic nature of this disease profoundly impacts patients. A dearth of information about the underlying pathology has been the major obstacle in identifying effective treatments. In 2000, NIH/NIAMS funded the National Alopecia Areata for the registration and data collection of individuals with a confirmed diagnosis of AA and their family members. For the purpose of this Ancillary Studies application, we require both fresh tissue biopsies as well as freshly-collected blood samples from a subset of 60 subjects. Therefore, it is essential that this grant be carried out in parallel with the NAAR contract. We will develop a translational platform for AA by characterizing the genetic networks of two interacting tissues in the context of an autoimmune disease: cells of the immune system as well as the skin. Translational advances in AA will likely have broad impact in autoimmunity. Our recent GWAS study in AA unexpectedly revealed a number of risk loci shared by rheumatoid arthritis (RA), type 1 diabetes (T1D), celiac disease (CeD), SLE, MS and psoriasis (PS). The commonality with RA, T1D, and CeD is especially noteworthy in light of the shared pathogenic expression of NK ligands in the target organ of each of these three autoimmune diseases. Thus, pursuit of biomarker development in AA, by integrating signatures in the blood and the target organ, could accelerate candidate biomarker discovery for other related autoimmune states in which the target organ is not as accessible. Importantly, these studies will provide the basis for a translational platform in AA;to identify molecular drivers of AA subtypes enabling drug development to be appropriately targeted and monitored in this disease. PUBLIC HEALTH RELEVANCE: In this proposal, we will apply high-throughput, genomic characterization methods to identify AA transcriptional anomalies across two interacting tissues, the scalp skin and T cells, for the first time. This project will generate genome-wide gene expression and miRNA profiles of T-cells in circulation as well as the accompanying gene expression profile of the skin itself. We will then integrate this analysis with our GWAS findings, together with serum cytokine measurements and semi-quantitative methods such as qPCR and IHC. These putative biomarkers may identify molecular subtypes of AA and shed light on novel therapeutic pathways for intervention in AA. Importantly, they provide the basis for a translational platform in AA that is a crucial element for development and monitoring of biologically-targeted clinical intervention trials.

DESCRIPTION (provided by applicant): One of the goals of the Regulatory Science program within the Common Fund is to develop innovative in vitro models of complex tissues to facilitate evaluation of new drugs in a rapid manner. In this proposal, we will develop a 3D model of skin using iPSCs that have been differentiated to form keratinocytes and fibroblasts, for the first time Our lab has led the field in the development of differentiation protocols to form keratinocytes from human iPSC, and we are currently doing the same for dermal fibroblasts. In addition, we will develop a contextual model of a representative complex human skin diseases, psoriasis, by generating patient-specific iPSC from psoriatic lesions as the basis for the constructs. These constructs will also incorporate different populations of immune cells, such as Th17 cells, with the goal of modeling the inflammatory component of the disease as well as the skin barrier component. Lastly, in order to model both topical and systemic drug delivery for skin diseases, we will incorporate perfusable microvascular networks within the skin equivalents, which can be infused with selected therapeutic agents. Currently, there are no reliable in vitro models by which the efficacy and safety of psoriasis drug candidates can be tested, and this represents a major unmet pharmaceutical need. In this proposal, we will use microbiofabrication techniques to produce skin equivalents that better mimic systemic drug delivery to the skin, by incorporating perfusable microvascular networks within the skin equivalents. Our iPSC-derived constructs will represent a significant advance over currently available models. Current skin models comprise of fibroblasts seeded either in a collagen gel, or into a scaffold with keratinocytes seeded on top There have been several adaptations of these models in recent years, for example, pigmented models have been developed by incorporation of melanocytes, which can be used to assess product efficacy for solar protection. Immunocompetent skin models have been established containing Langerhans and dendritic cells with which pharmaceutical companies can evaluate allergenic or sensitizing agents. In all of these models, the cells used to comprise the equivalent are obtained directly from donor skin, and as such, these constructs require large amounts of input cells, and are often limited by the size of the original biopsy. Within this proposal we aim o establish a skin construct composed entirely of fibroblasts and keratinocytes derived from induced pluripotent stem cells (iPSCs), to produce an unlimited supply of disease- specific donor cells for use in skin constructs, which can be differentiated into multiple cell lineages. More broadly, this platform can be expanded to generate models other complex skin disorders for which new therapeutic options are in great demand.

DESCRIPTION (provided by applicant): The goal of this application is to develop a new outcome instrument, the Alopecia Areata Disease Activity Index (ALADIN) that has the potential for use in the determination of clinically relevant endpoints for Alopecia Areata (AA). AA is one of the most common autoimmune diseases, with a lifetime risk of approximately 2%, affecting 5.3 million individuals in the United States alone, including males and females of all ages and ethnic groups. AA represents the second most common form of human hair loss, and causes significant disfigurement and psychological distress to affected individuals. AA carries one of the highest burdens among any skin diseases, particularly among children and adolescents whose self-image is so closely linked to their appearance. Integration of patient reported outcomes (PROs) as disease endpoints is needed in the determination of drug efficacy and in the comparison of outcomes across drug trials for AA. Because of the high psychological burden associated with AA, it is essential that the patient's perspective be considered as a core outcome in the development of new treatments for AA. For this reason, PROs need to be developed that can capture the emotional and psychological impact of the disease. To our knowledge, the emotional and psychological impacts of AA on patients' health related quality of life have not yet been included in any completed RCT studies. In order to incorporate an AA specific PRO into the assessment of disease progression/status, there needs to exist a reliable and valid instrument that measures a uniform set of outcomes in clinical trials for AA. In the context of our ongoing clinical trials in AA at Columbia University Medical Center, we have the unique opportunity to develop and deploy the Alopecia Areata Disease Activity Index (ALADIN) into clinical trials for AA, where we will be able to assess its psychometric properties in an AA population. ALADIN will be constructed by integrating a patient reported health-related quality of life instrument; physician reported hair loss assessments, clinical measures, a genotype risk score, and gene expression analysis and serum biomarkers. ALADIN will be modeled after the well-accepted and extensively validated Crohn's disease activity index (CDAI) which similarly contains patient reported measures; physician reported measures, and biological data. We will apply innovative statistical approaches in developing ALADIN, to create a classifier that quantitatively distinguishes disease state, prognosis and therapeutic response. The final instrument will be a single index that can be used to monitor disease activity in AA, and will be easily adaptable to multiple clinical trial sites and allow comparison across trials.

DESCRIPTION (provided by applicant): Antitumor antibody therapeutics have significantly impacted the treatment of cancer over the past two decades, yet challenges and opportunities remain. For advanced stage solid tumor treatment, complete clinical remissions are infrequent and transient, even with concomitant chemotherapy. A better understanding of the common therapeutic and resistance mechanisms is urgently needed to improve clinical outcomes of this emerging class of monoclonal antibody (mAb) therapeutics. The first clinical successes, Rituximab (anti-CD20), Trastuzumab (anti- HER2) and Cetuximab (anti-EGFR) were initially thought to work exclusively through interruption of their respective downstream signaling pathways. Unexpectedly, however, our own studies demonstrated that Fc-FcR interactions were required for efficacy of these drugs in the mouse, defining an essential role for effector immunity. Consistent with our observations in mice, several clinical studies have since correlated favorable clinical outcomes in cancer patients harboring higher IgG affinity FcR alleles. The FcR-mediated immunological mechanisms have been collectively described as 'antibody- dependent cellular cytotoxicity' (ADCC), a concept that stems from in vitro observations of cytotoxic FcR-bearing innate effectors (macrophages, neutrophils and NK cells) that rapidly kill IgG-coated tumor cells, within hours. Yet in vivo, with the exception of Rituximab-mediated clearance of normal and malignant B cells in the blood (e.g., CLL), there is little evidence for an 'ADCC-like' acute inflammatory response for solid tumor treatment. Instead, the slower kinetics of tumor responses are more consistent with an induced adaptive immune response, elicited as a vaccinal response to combination chemotherapy and antitumor antibodies. In this model, antibody opsonization of apoptotic/necrotic material provides a therapeutic window to modulate the immunological outcome of tumor antigen uptake by antigen presenting cells. Our recent studies demonstrated clinical relevance of this vaccinal pathway in breast cancer patients treated with chemotherapy + trastuzumab. Patients treated with this regimen developed antitumor HER-2 specific immunity, which we found were enriched in patients with favorable clinical outcomes, indicating that passive mAb therapy induces active tumor immunity that may contribute to efficacy. In this proposal, we will further define the importance of the vaccinal pathway mechanistically (Aim 1) and pursue efforts to enhance these effects using two innovative approaches. We will first use antitumor antibodies engineered to selectively engage activating human Fc receptors on dendritic cells (Aim 2), and secondly, we will use pharmacologic strategies to block inhibitory pathways that limit T cell activation (Aim 3). Our Aims are designed to test the hypothesis that removal of negative regulatory pathways could promote this burst of antibody-enhanced antigen delivery, leading to effective anti-tumor cell T cell generation and conversion of a suboptimal antitumor treatment response into meaningful clinical benefit.

? DESCRIPTION (provided by applicant): We request partial support for the 9th World Congress of Hair Research (WCHR), to be held on November 18-21, 2015 at the InterContinental Hotel in downtown Miami, Florida. The WCHR co-Chairs are Angela M. Christiano, Ph.D., Wilma Bergfeld, M.D. and Maria Hordinsky, M.D. continuing the tradition set by the international hair research societies of Japan, Korea, Europe and North America, this biannual Congress will be a comprehensive, international hair research meeting for the advancement of knowledge in hair growth, hair and scalp disease, and clinical care. Hair research has undergone a transformative period of growth over the past ten years, and now represents among the most rapidly growing categories of research in the NIAMS portfolio. The Congress will bring together hair biologists, dermatologists, cosmetic scientists and hair transplantation surgeons for a unique three and a half day comprehensive hair research meeting that will establish new directions for the research community both in the United States and internationally. The Congress will include general sessions, scientific posters, pre-Congress workshops, networking opportunities, a full exhibits program, company-sponsored satellite symposia, and other educational activities. International colleagues will present new research, share experiences, and discuss new directions for the advancement of knowledge in hair growth, hair and scalp disease, and clinical care.

? DESCRIPTION (provided by applicant): Alopecia Areata (AA) is one of the most common autoimmune diseases in the US, with a lifetime risk of 1.7%, it affects approximately 5.3 million individuals across all ethnic groups. We recently carried out a genome-wide association study (GWAS) to identify common alleles that contribute to risk of AA, and identified several genomic regions harboring potential susceptibility genes. In our Preliminary Studies, we have generated a significant and unique resource of genomic and genetic datasets in alopecia areata patient samples using several approaches, including GWAS genotyping on >1000 individuals, ImmunoChip on >400 individuals, Linkage analysis on >50 families, Gene expression profiling on >100 individuals, Targeted resequencing in GWAS regions in 124 individuals, and Whole exome sequencing in >10 alopecia areata probands selected from linkage families. These robust and highly integrated datasets provide a rich foundation from which to interrogate the functional significance of variants in AA candidate genes in the context of this innovative proposal. As a first step towards understanding the biological significance of these results, we must now perform deep sequencing analysis in these regions to identify causal variants that are driving the association of tagSNPs. The emerging picture of the genetic architecture of common diseases contains niches for both common and rare variants acting independently or in concert to influence phenotypes. Understanding the impact of these variants in disease pathogenesis is the first key step in moving toward novel therapies for AA. These studies are extremely timely, and will allow us to place AA into the context of other autoimmune diseases in which GWAS and deep sequencing is already underway. We postulate that causal variants in candidate genes underlie the susceptibility to develop AA. This grant is focused on carrying out deep sequencing studies to identify new variants in candidate genes, followed by functional genomics of variants in several in vitro contexts, which will allow us to determine: 1) the nature of the specific variats contributing to AA susceptibility; and 2) the mechanism(s) by which they contribute to disease pathogenesis. In this proposal, we will carry out Exome-plus sequencing with enrichment in our previous GWAS regions to identify causal variants. We will then interrogate the functional consequences of variants within candidate genes expressed in both the hair follicle and immune cells, with an emphasis on the NKG2D pathway.

ABSTRACT The Research Project serves as the principal research directive of the AACORT. The overall goal of the AACORT is the comprehensive study of the pathology of Alopecia Areata, which the Research Project formalizes into four key goals: 1) the study of the microbiome, 2) proteomic research into AA autoantigens, 3) mouse model studies of AA pathology and drug discovery, 4) and studies of human AA patients for molecular pathology and drug response. These four goals will result in the full characterization of the microbiome, genomic, transcriptomic, and proteomic loads that contribute to AA pathology. This, in turn, will provide the foundation for the development of biomarkers to track AA progression and response to treatment (both genomic and molecular), and the identification of drug targets and drug candidates. To drive the pursuit of these objectives, the Research Project will serve as a key mediator between the TRAC Core, which will be responsible for designing and acquiring human patient biosamples for analysis, and the DATA Core, which will oversee the annotation, storage, and analysis of all data generated by the TRAC. It will be one of the Research Project?s roles to identify targetable biological objectives to these two cores, which will then acquire the data and analyze it for the key molecular drivers that the Research Project can then validate as a complement to their ongoing research. Experiments pursued by the Research Project, in human or mouse models, will be continuously cross-checked with the bioinformatic analyses of human cohorts to develop an unprecedented systemic model of AA pathology. In this manner, all ongoing research in the AACORT will be mediated by the central biological questions posed by the Research Project, thereby providing uniform objectives for which the AACORT can leverage its multiple analytic pipelines.

ABSTRACT The role of the Skin Immunity, Integrity, and Disease (SIND) Core is to facilitate skin-focused research by providing a range of in vivo and ex vivo models that aim to mimic human skin conditions resulting from immune and inflammatory diseases and cutaneous photodamage. The SIND will provide guidance and training in the manipulation of these models, and will incorporate state-of-the-art techniques at molecular, cellular, tissue, and intact animal levels. The SIND will comprise four clusters, each with a specific focus. Cluster A: Immune and Inflammation models component will provide a wide array of highly specialized tools for studying T cell biology in both healthy and pathologic skin. The SIND will assist core investigators with the isolation of high quality skin- resident T cells using a 3-D matrix, with flow cytometric immunophenotyping of T cell populations, and with cytokine profiling using a panel of the SIND?s validated antibodies. The SIND is equipped with special technical expertise and knowledge to provide in vitro screening for T cell antigen reactivity (e.g., lipid antigens) and T cell receptor (TCR) sequencing. Cluster B: UV radiation models component will provide guidance for in vivo assessment of photodamage. The SIND will provide both standard and unique murine models of UV, and will assist with an array of molecular, biochemical, and cellular assays with endpoints that reflect tissue integrity and the biological function of the skin. The SIND will assist with the quantitation of pre-mutagenic DNA lesions, exome-sequencing?with analysis targeted to molecular signatures of UV damage?and assessment of cellular processes relevant to epidermal homeostasis. Cluster C: Alopecia Areata (AA) models component is equipped with state-of-the-art methods for AA modeling in ex vivo and in vivo platforms, and will provide assistance with a battery of experimental techniques and approaches relevant to the study of AA pathogenesis. Cluster D: High- throughput screening component will provide assistance with genetic (via cas9 mediated gene disruption) and chemical (via reporter cell lines) screening techniques to facilitate the development of screening assays applicable to skin cells. The SIND will provide a 3-D human skin culture system, and will provide assistance with 96-well high-throughput imaging analysis. Finally, the SIND will provide proof-reading and suggestions for Materials & Methods sections for publications and applications for extramural research funding utilizing SIND services, and assist with the preparation of IACUC protocols. Combined with expertise of the SIND investigators, the scientific services provided by the SIND core have the potential to make a unique contribution to advancing research in skin biology and disease.

Administrative Core Abstract The Administrative Core provides the essential organizational framework for the Alopecia Areata Center for Research Translation (AACORT) and in a very real sense functions as its nerve center. It serves to coordinate and integrate all Center functions; provides rigorous and regular fiscal oversight of the Project, Core facilities and the Pilot and Feasibility studies; offers advice to the Director and Associate Director regarding all aspects of the Center?s activities; and assists in assuring that the Enrichment Programs function effectively. The Administrative Core provides the major infrastructure of the AACORT and is designed to ensure the most efficient utilization of the Center?s resources toward advancing translational research in AA. The members of the Executive Committee and the Advisory Group are talented and experienced research scientists capable of providing competent oversight and direction to the Center. An additional lay member is responsible for assuring that the interests and concerns of the AA patients. The AACORT will strive to develop collaborative and cooperative interactions among the clinicians and scientists comprising the Cores and members of the AACORT at Columbia and focus attention on the opportunities available for research in AA by coordinating and facilitating these activities on the health sciences campus. The Administrative Core will function as a clearinghouse to leverage resources by partnering with other existing Cores for the benefit of AACORT investigators. The Administrative Core will provide administrative and fiscal support for the Cores and the P&F studies proposed under the aegis of the AACORT and will ensure integration across the Project, Cores, the P&F programs of the AACORT will foster novel ideas generated in AA research that can be nurtured and expanded into funded programs utilizing the P&F studies. The Enrichment Program will serve as a vehicle to promote institutional and community awareness of the importance of AA translational research and the tremendous opportunities that exist for the creation of new knowledge in this vitally important area of biomedical science.

Overall AACORT Abstract The Alopecia Areata Center of Research Translation (AACORT) at Columbia University Medical Center (CUMC) is focused on the singular goal of developing new treatments for alopecia areata (AA). The mission of the AACORT is to leverage our recent discoveries beginning to unravel the genetic basis of AA and the fact that its pathogenesis is linked to dysregulation of both innate and acquired immunity, and to synergize these findings into translational studies that will lead to novel therapies for this disease, including the repurposing of existing drugs. In the past few years, the AACORT team has made transformative progress in understanding the genetic basis of AA, as well as initiating a series of clinical trials employing mechanism-driven targeted JAK inhibitors aimed at reversing the disease. While this class of drugs is having a profound impact on guiding clinical development efforts in AA, there is much more work to be done, since clearly many patients do not respond to these drugs. The goal of this AACORT is to build upon our established translational pipeline to leverage our basic and preclinical discoveries into the clinical setting. This AACORT is centered around a central Research Project that focuses on the Translational Science of Alopecia Areata, and deals with innovative translational aspects of the disease, including initiating factors such as the microbiome; preclinical studies focused on antigen discovery and testing new therapeutic approaches in vitro and in vivo; and clinical correlative studies designed to rigorously assess the efficacy and safety of new drugs coming available for patients with AA. The Research Project is supported by the Translational and Clinical Resource Core (TRAC), and the Data Analysis and Translational Applications (DATA) Core, which bring together an outstanding cadre of talented, committed and scientifically diverse faculty from across the institution. The AACORT is supported by a highly capable and experienced Administrative Core that provides its vitally important organizational infrastructure. The goal of the AACORT is to surmount barriers to translational application of basic discoveries that have the potential to revolutionize the management of this heretofore intractable and debilitating disorder.

Project Summary Lichen planopilaris (LPP) is a subtype of primary cicatricial (scarring) alopecia caused by chronic lymphocytic inflammation centered around the hair follicle (HF) bulge stem cell region. The origin of LPP, like the other primary cicatricial alopecias (PCAs), remains poorly understood, however, they share the common finding of a targeted folliculocentric attack, which leads to irreversible HF destruction and permanent hair loss. LPP is a true dermatologic emergency, since delayed intervention will lead to irreversible, lifelong hair loss and permanent disfigurement of the scalp. Currently, few effective treatments are available for LPP, due to the lack of understanding of disease etiology and the difficulty of diagnosis among the subtypes of PCAs. Recently, one study of human LPP scalp skin implicated the collapse of immune privilege (IP) around HF epithelial stem cell niche during LPP pathogenesis, which was accompanied by the infiltration of cytotoxic CD8+ T lymphocytes that produce IFNgamma. This presentation is remarkably similar to another type of non-scarring inflammatory hair loss, alopecia areata (AA), in which the collapse of hair follicle IP results in the infiltration of inflammatory lymphocytes around the lower, bulb region of HFs, where it does not cause permanent scarring since the bulge stem cells are spared. Due to the striking similarities in both the collapse of HF immune privilege and the nature of the CD8+ T cell immune infiltrate in LPP and AA, we recently postulated that tofacitinib might also have efficacy in LPP, as we have shown in AA. Indeed, we recently treated 8 patients with LPP with tofacitinib, and observed a clinical response in about half of those treated. These exploratory studies have led to the initiation of a new Open-Label Pilot Study of Tofacitinib in Patients with LPP at Columbia University Medical Center, which will open in July 2017 and serves as the Parent Study for this application. In this Ancillary Study, we have the opportunity conduct a careful Immunophenotyping of LPP for the first time, directly from patient scalp and Tcells. We will identify pathogenic T cell populations, gene expression profiling from the skin and the blood, serum cytokine analysis, immunohistochemical definition of the Tcell infiltrate and high dimensional TCR sequencing. In line with the Parent Study, we will conduct subsequent immunomonitoring of LPP patient responses to tofacitinib during an open label clinical study. This Ancillary Studies project affords us the unique opportunity to conduct these critical immunophenotyping studies in LPP, which may have broader clinical relevance to other inflammatory and autoimmune diseases of the skin and hair follicle.

Project Summary Psoriasis is an inflammatory skin disorder with abnormal epidermal hyperproliferation, which affects 2-3 % of the US population. There are no reliable in vitro models with which the efficacy and safety of psoriasis drug candidates can be tested. To meet this major unmet pharmaceutical need, we will combine iPSC reprograming and 3D bioprinting technologies to construct a 3D skin model to screen for drugs to treat this disease. iPSC methodology will be used, because iPSCs have unlimited proliferation potential and the capability to differentiate into different cell lineages, which allows us to circumvent the limitation imposed by the limited availability of primary cells that can be procured from patients. We successfully constructed 3D skin using exclusively iPSC-derived cells (fibroblasts and keratinocytes), demonstrating the feasibility of this approach. 3D bioprinting can allow us to reproducibly construct complex structures to recapitulate human tissues/organs. In this project, we will first construct immunocompetent 3D skin with primary keratinocytes, fibroblasts and T cells. Then, iPSCs will be reprogramed from fibroblasts or blood cells obtained from normal and psoriatic individuals. Keratinocytes and fibroblasts will be differentiated from iPSCs for construction of 3D skin models, whereas the abundant availability of T cells allows us to use cells directly from primary sources. Bioprinted psoriasis-specific iPSC derived skin constructs will be used for disease modeling and pharmacological validation. CRISPR technology will be used to integrate fluorescent reporters in iPSC-derived keratinocytes to monitor disease induction of psoriasis and effect of drug treatment. Completion of this project will provide us with a reliable 3D bioprinted model to develop drugs to treat psoriasis. Moreover, our 3D skin model can be easily adapted and modified to screen for drugs to treat other skin diseases, using a platform approach.

Project Summary Alopecia areata (AA) is a common autoimmune form of hair loss that affects approximately 5.3 million people in the US, including males and females across all ethnic groups. Autoimmune diseases collectively carry a tremendous public health burden, imparting a severe economic and social impact globally. The prevalence of this class of disorders is currently estimated to be 7.6-9.8%, and results in annual direct health care costs of at least $100 billion in the U.S, as estimated by the NIH. AA typically presents as loss of distinct patches of hair that can hair loss can spread to the entire scalp or the entire body. We recently conducted regulatory modeling of alopecia areata from gene expression analyses. Reverse-engineered regulatory networks have recently demonstrated great promise in the analysis of other complex diseases, such as cancer and Alzheimer?s disease, and have been used for drug prediction. The DeMAND algorithm was designed to leverage high-throughput drug screens conducted at Columbia University, where panels of FDA-approved drugs were used to treat several cancer cell lines. We utilized the DeMAND algorithm interrogated with our regulatory networks from alopecia areata patient samples, and identified a repurposable drug candidate, Vorinostat, an HDAC inhibitor, which we will evaluate for pre-clinical efficacy in alopecia areata in this proposal. There is a strong use-case for Vorinostat in alopecia areata, particularly because this predicted repurposing crosses indications. This therapeutic/indication pair represents an example of a drug developed for cancer that is being proposed for the treatment of autoimmunity. The goal of this proposal is the characterization and validation of the potential of Vorinostat to treat alopecia areata, as a therapeutic/indication pair as well as to design a clinical study to evaluate the efficacy of Vorinostat in human patients.

Project Summary Psoriasis affects 2-3% of the US population. Currently, there are no high throughput screening methodologies to develop new psoriasis drugs. To meet this significant unmet pharmaceutical need, we will screen for drugs to treat this disease by using a 3D skin system established with 3D bioprinting, induced pluripotent stem cell (iPSC) reprograming and CRISPR genome-editing technologies. We successfully constructed 3D skin using exclusively iPSC-derived fibroblasts iFBs) and keratinocytes (iKCs), demonstrating the feasibility of this approach. 3D bioprinting can allow us to reproducibly construct complex structures to recapitulate human tissues/organs, and miniaturize the 3D fabricated skin for high throughput screening. Psoriasis is an inflammatory skin disorder with abnormal epidermal hyperproliferation, compromised barrier function, upregulated keratin 16 (KRT16), downregulated filaggrin (FLG) and excessive secretion of cytokines such as IL-8, which can be monitored with transepithelial/transendothelial electrical resistance (TEER), fluorescence reporters and IL-8 ELISA as readouts. We postulate that compounds will manifest their anti-psoriatic effects through the reduction in disease phenotypes that can be detected by relevant readouts. Psoriasis can be recapitulated and assessed using cytokine treatment on skin constructs derived from primary fibroblasts (FBs) and keratinocytes (KCs), however it is labor intensive and inefficient to produce large quantity of T cells and reporter-containing iPSC-derived keratinocytes (iKCs), Therefore, in this project, we will use cytokine treatment and skin constructs fabricated with primary cells for initial screening and then use iPSC-derived 3D skin containing T cells and reporters for subsequent compound validation, confirmation and the mechanism of actions (MOA) studies. Specifically, we will use FBs and KCs to bioprint 3D skin in a 96 well format, and treat with a cytokine cocktail containing TNF-a, IL-1a, IL6 and IL17A, for a pilot screen of 50 inflammation-related compounds to optimize the high throughput procedure that then will be applied to screen a library of 1000 compounds using TEER and IL-8 ELISA. Subsequently, we will validate the lead compounds using lactate dehydrogenase (LDH) toxicity assays and iPSC-derived skin constructs that are generated from healthy donor cells and containing GFP reporters for KRT16 and mCherry for FLG. Finally, bioprinted psoriasis-specific iPSC derived skin constructs containing reporters and Th17/Th1 cells will be used to confirm the lead hits and delineate the MOA of compounds using RNA-seq. Completion of this project will provide us with compounds that may be developed into drugs to treat psoriasis. Moreover, our 3D skin model-based high throughput system can be easily adapted to screen for drugs to treat other inflammatory skin diseases.

ABSTRACT Alopecia areata (AA) affects as many as 6.8 million people in the U.S. and 147 million worldwide, with a lifetime risk of 2.1%, making it one of the most common autoimmune diseases. AA causes significant disfigurement and psychological distress to affected individuals and carries one of the highest emotional burdens amongst all skin diseases, particularly among children and adolescents whose self-image is so closely linked to their appearance. At present, the prognosis is unpredictable and there is no FDA approved treatment for AA. AA shows several genetic and immunopathogenic similarities to other autoimmune conditions, suggesting that similar environmental triggers as well as the inflammatory responses leading to damage in the end-organ, the hair follicle (HF), may have common mechanisms. While our group and others have made significant advances in understanding the genetic architecture of AA, the environmental triggers of systemic autoimmune response in AA have not been identified. Therefore, we recently turned our attention to determining potential environmental triggers, in particular, the microbiome. In our Preliminary Studies we analyzed the gut microbiome composition of a cohort of 26 AA patients and 10 healthy subjects, and found striking dissimilarities between the composition of the gut microbiome of AA patients and those of healthy controls. We observed an overrepresentation of firmicutes and an underrepresentation of bacteroides in AA patients, findings which are also seen in other autoimmune diseases. Our additional Preliminary Studies on the well-established C3H/HeJ mouse model of AA demonstrated that pre-treatment with a wide-spectrum antibiotic cocktail results in protection against AA after skin grafting, suggesting the gut microbiome is required for the onset of AA. In addition, three cases have been reported in the literature of patients with chronic alopecia areata/universalis who were treated with a fecal microbiota transplant (FMT) for unrelated conditions, who subsequently experienced significant regrowth of hair. Taken together, these findings suggest an unexpected role of the gut microbiome in alopecia areata, and provided the rationale for the Parent Study of this Ancillary Studies Grant, entitled ?Fecal Microbiota Transplantation as a Potential Treatment for Patients with Alopecia Areata?. The purpose of this Ancillary Studies grant is to conduct microbiome analysis and immunological studies in AA patients undergoing FMT, in order to understand the pathogenic role of environmental factors, such as changes in the composition of the gut microbiome, in the reversal of AA by FMT.

OVERALL: PROJECT SUMMARY Defining the molecular and cellular heterogeneity underlying senescent cell states is a critical knowledge gap in the field. The Columbia University Senescence Tissue Mapping (CUSTMAP) Center is uniquely poised to address this gap by creating a multi-scale atlas of senescence in diverse tissue types across the adult human lifespan. CUSTMAP is a highly collaborative effort that builds upon long-standing and established collaborations between Columbia University Irving Medical Center (CUIMC), The University of Edinburgh, New York University (NYU), and the New York Genome Center (NYGC). Using state-of-the-art spatial genomics technologies and leveraging our established experimental workflows and analytical pipelines, CUSTMAP will generate three- dimensional maps of senescent cells in tissues with vulnerability to age-related degenerative processes: the central nervous system (brain and spinal cord) and the skin. We will perform spatially resolved transcriptomics (ST), single-nucleus RNA-sequencing, and multiplexed proteomics using iterative indirect immunofluorescence imaging (4i) experiments in central nervous system (CNS) and skin tissues across the human lifespan, allowing for unprecedented genome-wide molecular characterization at single-cell resolution in space. CUSTMAP is structured around three scientific Cores, as well as an Administrative Core to support these integrated efforts. Human tissue samples characterized and collected through the Biospecimen Core (BIO) will be analyzed using the tools and techniques developed through the Biological Analysis Core (BAC) and Data Analysis Core (DAC), leading to detailed maps of senescent cells and their effects in human tissues at single-cell resolution. The success of CUSTMAP is predicated on access to a continuous supply of human tissues from healthy individuals across the lifespan. CUSTMAP investigators have access to post-mortem samples and prospective tissue collection from local resources at CUIMC as well as through established collaborations. Our unique geographic location in Upper Manhattan enables tissue acquisition from a local population of patients that is rich in racial and ethnic diversity. Thus, CUSTMAP is uniquely poised to obtain high-quality tissue from these diverse populations and apply cutting-edge multiomic approaches to build integrated 3D molecular atlases of senescent cells in CNS and skin tissues. Our approach provides a unique platform for driving transformative discoveries of novel molecular, cellular and regional correlates of age-related changes in cellular senescence in human tissues. The CUSTMAP workflow and computational tools are readily generalizable to other tissue types and can be efficiently shared with and deployed across the SenNet Consortium.