Applications requesting up to $750,000 in total costs are due January 25th, 2018. Background: Diabetic foot ulcers are the most common cause of non-traumatic lower leg amputation in the United States. Despite efforts to prevent and treat foot ulcers, each year about 70,000 Americans with diabetes will lose part of a lower extremity because a foot ulcer becomes infected or does not heal. An obstacle to the development of therapeutics and biomarkers in this area is the paucity of basic fp-information describing the molecular processes and cellular changes underlying diabetic skin and its wound pathobiology. Significant untapped potential for novel wound therapy treatments may lie within the numerous molecular, cellular, and structural aspects of the skin that have been implicated in impaired diabetic wound healing. However, to-date, most research studies have tended to focus on a limited number of proteins or pathways that are altered within specific cell types in diabetic wounds. Thus, developing a detailed, comprehensive knowledge base of the molecular features associated with foot ulcer development in diabetes patients will address a significant gap in our understanding of how and why diabetic foot ulcers fail to heal. Purpose:The purpose of this Pilot Program is to adapt and evaluate emerging techniques focused on defining cell types and subtypes and their three-dimensional context to the study of diabetic foot ulcers. This will be accomplished by funding pilot studies to conduct key establishing experiments that will be necessary to fp-inform any future construction of an unbiased atlas of diabetic skin and foot ulcers. Pilot projects will be composed of multidisciplinary teams that consist of partnerships between: 1) clinical scientists with experience in diabetic foot ulcer biology and access to fresh human biopsies/surgical material for analysis, and 2) investigators with expertise in applying a diverse set of agnostic, medium- to high-throughput technologies with single cell-resolution to the systematic exploration of the cellular diversity of human tissues (e.g., CLARITY combined with highly multiplexed profiling of proteins and/or RNAs, imaging mass cytometry, etc.) or human tissue-derived cells (e.g., RNAseq, Dropseq, ATACseq, etc.). Objectives: The objectives of the Pilot Program are to 1) establish protocols for the collection, preservation, storage, and processing of diabetic skin and foot ulcer tissue, 2) adapt and utilize agnostic, single cell-resolution technologies of tissues and tissue-derived cells to assess the cellular complexity of intact and wounded tissues in diabetic patients, and 3) critically evaluate limitations of current methodologies for their application to the context of diabetic foot ulcers and explore potential solutions to begin to bridge these gaps. Clinical Sample Collection: Pilot projects should collect samples from routine clinical debridement that includes tissue at various locations in the wound and adjacent tissue. Samples for future analysis such as blood and microbiome/biofilms should also be collected and stored. It is expected that applicants will obtain appropriate consent to broadly share samples, de-identified patient fp-information, and experimental data. Consent should also permit following the course of wound healing after sample collection in order to include that fp-information in metadata associated with samples and their analysis. Essential Elements of Proposals Applications should include the following elements as subsections of the Research Plan (see below in Application & Eligibility for overall application instructions):

• Clinical Sample Rationale: A detailed rationale for and description of clinical samples to be collected (e.g., total number of samples, number of samples per patient, location in/around wound bed, cross-sectional versus longitudinal design, etc.) and their overall importance to the goal of exploring the diabetic foot ulcer niche must be presented. This should include estimates of the amount of tissues to be collected and the needed quantities in experiments. Key phenotypic characteristics of the patients and what patient fp-information will be associated with samples should be described. Only samples from people with diabetes (type 1 or type 2) are allowed for this program.
• Overall Research Design: A description of the overall research design and technologies that will be undertaken to adapt and evaluate emerging techniques focused on defining cell types and subtypes and their three-dimensional context to the study of diabetic foot ulcers. It expected that applications will utilize skin and wound samples that include both the epidermis and dermis and will discuss how cells from various relevant sub-tissue types (e.g., fibroblasts, nervous tissue, vasculature, keratinocytes, eccrine glands, macrophages, etc.) will be sub-classified.
• Technology Development and Validation: A detailed plan to evaluate how collection, preservation, storage, and processing of cells and tissues affects the down-stream readouts of the proposed technologies. This plan should address how relevant metadata (e.g., patient phenotype, location, and orientation on the body of collected tissues, handling steps, high-resolution images of the wound at the time of collections and during the healing phase that follows, etc.) will be associated with samples and resulting data.
• Analysis and Statistical Approaches: A description of the analytical and statistical approaches that will be used to compare cell/tissue composition at the different sample sites within individuals (e.g., unhealing tissue, leading edge, distal tissue, etc.) and how these approaches could be used to frame questions and generate hypotheses about the differences between healing versus non-healing wounds. Applicants should indicate how data will be stored, shared, and visualized between sites and with NIDDK staff.
• Prior Experience of the Multidisciplinary Team: The clinical component of the team should demonstrate its ability to collect samples from clinically well-phenotyped individuals with diabetic foot ulcers and to rapidly transport fresh biopsy/surgical samples to the experimental laboratory. The technology-driven component should demonstrate its ability to apply state-of-the-art single cell-resolution approaches to explore the cellular diversity of complex tissues as well as the needed expertise to analyze, define cells by subtypes, and visualize the resulting data.
• Commitment to Work as a Consortium: The team should indicate its commitment to work with other awardees in the Pilot Program, DiaComp, and other related NIDDK research efforts to communicate regularly to discuss common concerns, develop standards and quality control metrics, and share pre-publication data to assure the inter-operability of approaches. An in person kick off meeting will be held at the start of awards to harmonize tissue collection protocols to the maximum extent possible and develop a set of common analysis techniques across pilot projects. Applicants should indicate their willingness to share clinical samples with other Pilot awardees and to deposit excess samples in the NIDDK Central Repository (https://www.niddkrepository.org/home/) at the end of the Pilot award.
• Protection of Human Subjects: When applicable, proposals must include a protection of human subjects section that addresses 1) risks to human subjects, 2) human subjects involvement and characteristics, 3) recruitment and fp-informed consent, 4) inclusion of woman and minorities, and 5) inclusion of children. Additional sub-items that should be included in this section can be found under "Application & Eligibility". If the investigative team does not obtain private identifiable fp-information, the proposed research may not be considered human subjects research. An example would be the use of discarded tissue from a wound debridement in which the individuals involved in the research do not have private identifiable fp-information on the patient. Please see https://humansubjects.nih.gov/human-specimens-cell-lines-data for more fp-information. If applicants determine proposed studies are not considered human subjects research, they should provide a rationale for this. For questions, please contact Dr. Pawlyk.

• Specific Aims,
• Research Design and Methods,
  1. Clinical Sample Rationale,
  2. Overall Research Design,
  3. Technology Development and Validation,
  4. Analysis and Statistical Approaches,
• Prior Experience of the Multidisciplinary Team,
• Protection of Human Subjects (If applicable - For studies that are not considered human subjects research, applicants should instead provide a rationale for this.)
  1. Risks to Subjects
  2. Human Subjects Involvement and Characteristics
     1. Sources of materials
     2. Potential risks
     3. Adequacy of Protection against Risks
  3. Recruitment and fp-informed consent
     1. Protections against Risks
     2. Potential benefits of the proposed research to the subjects and others
     3. Importance of the knowledge to be gained
  4. Inclusion of Women and Minorities (including PHS Inclusion Enrollment Report)
  5. Inclusion of Children
• Commitment to Work as a Consortium,
• Literature Cited,
• Sharing Plan

Applications requesting up to $100,000 in total costs for one year are due March 10, 2017.

Diabetic foot ulcers are the most common cause of non-traumatic lower leg amputation in the United States. Despite efforts to prevent and treat foot ulcers, each year about 70,000 Americans with diabetes will lose part of their lower extremity because a foot ulcer becomes infected or does not heal. An obstacle to the development of therapies is the paucity of validated biomarkers that assess healing, infection, and recurrence risk, and validated outcomes that measure patient satisfaction and quality of life. In addition, systematic reviews of clinical studies on the prevention and management of diabetic foot ulcers consistently point to the urgent need for clinical trials with standardized definitions, outcomes, biomarkers, and protocols that will enable a thorough evaluation of treatments and comparison of results across trials.

The purpose of this program is to support planning activities for the development of a multi-center study for clinical biomarkers and outcome measures for diabetic foot ulcers. It is also part of an effort by the NIDDK to support clinical studies that involve two or more institutions to build a collaborative framework for ongoing research on diabetic wound healing.

Applications should propose a future clinical study based on the criteria listed below. The application should include a rationale for the future clinical study, documenting significance and need, and describe the potential impact of the clinical study on biomarkers and research infrastructure. Preliminary results and background to support the study and an overview of the study design should be provided. The latter should include an estimate of the number of subjects, entry criteria, and outcome measures. In addition, the application should describe the planning activities proposed to be conducted during the single year of this program. These may include meetings to develop the protocol, focus groups for the patient-centered outcomes, clinical studies to obtain preliminary data, quality control testing of patient sample analysis, biostatistical analysis to develop sample size calculations, or analysis of databases to fp-inform recruitment strategies. The goal of the planning activities is to develop a complete study protocol for the validation of a biomarker or outcome measure for diabetic foot ulcers.

The future, proposed clinical study should meet the following criteria:

• The proposed study should address a significant gap in ulcer and patient assessment that obstructs progress in clinical research or patient care in this area. A biomarker that would fill a critical need for clinical research, but due to its complexity or expense, may not be practical for widespread adoption, would still be responsive to this program.
• The future clinical study should be supported by preliminary data or published results that justify a multi-site clinical study. Discovery research, such as an "-omics" approach to wound samples, is not appropriate for this program. Obtaining additional preliminary data that would support the clinical study, can be proposed as a planning activity.
• The scope of the project can range from a biomarker of a specific process of wound healing or infection to a broad model with a multi-factorial prediction of healing.
• The proposed clinical study must involve recruitment of new subjects at multiple institutions. The use of stored samples or databases could be used to augment findings in the main study. A goal of this program is to foster collaboration in the standardization of protocols. The application for planning activities can come from a single institution, but should discuss the organization of multi-site clinical study and plans to recruit additional sites.
• The research needs to focus exclusively on subjects with diabetes. The biomarker may be applicable to wound healing in non-diabetic subjects, but the proposed clinical study should only include subjects with diabetes. The study may propose to exam a subgroup of patients with diabetes, such as patients with end-stage renal disease, if it is justified scientifically.

It is anticipated that the NIDDK will publish a future FOA for a Cooperative Agreement for a Data Coordinating Center (DCC) to coordinate multi-site, clinical studies on diabetic foot ulcers. In conjunction with the NIDDK, the DCC will request the completed study proposals in Spring, 2018 and convene an External Evaluation Committee to make recommendations on the priority of the studies for implementation by the investigators and the DCC. Study proposals will not be limited to awardees from this DiaComp program. 

Topics for a clinical study include, but are not limited to:

• Molecular or histologic detection of infection and biofilms in diabetic wounds.
• Surrogate end-points that when applied early in treatment can predict eventual healing.
• Assessment of the healed skin that can be used as an outcome measure and predictive biomarker for recurrence.
• Cellular markers of inflammation and regeneration that assess the effect of surgical and medical therapies.
• Risk factor models that combine wound and patient characteristics to predict healing or recurrence.
• Patient questionnaires and clinical scales to assess patient satisfaction, quality of life, and function with treatment and outcomes.

The application budget needs to reflect the actual needs of the proposed project and cannot exceed $100,000 in total costs (direct + F&A costs). The project period is one year. The program anticipates funding up to five awards, which is dependent upon the number, quality, and cost of the applications received. Awards will be made as subcontracts from the DiaComp Coordinating and Bioinformatics Unit (CBU) at the Augusta University and not directly by the NIH.