The Pilot and Feasibility Program is a centerpiece of the HSDRC, as it is through this mechanism that collaborations and innovations that fundamentally influence skin disease research occur. Pilot and Feasibility studies have been drawn from multiple sources including young investigators with relatively little experience in skin disease research as well as more established investigators in skin disease research who propose to test a new idea that represents a clear and distinct departure from their ongoing research interests.

The Role of T regulatory cells in Macrophagal Survival During Cutaneous Leishmaniasis
PI : Oleg Akilov, MD, PhD

INTRODUCTION AND SPECIFIC AIMS
The keystone of the host-parasite interaction in cutaneous leishmaniasis (CL) is macrophagal behavior. Macrophages (Mф) serve not only as host cells for the replication of parasites, but they also act as effector cells that are responsible for parasitic killing after Th1 activation. The Mф invasion by Leishmania parasites corresponds to an incubation period, whereas the successful activation of Mф pre-signifies a parasitic eradication. It is known that infected Mф may present Leishmania antigens on their surfaces, which makes them a target for cytotoxic cells. However, the immune destruction of Mф does not affect intracellular parasites. On the contrary, amastigotes are released from the disrupted Mф, which infect new cells, thus invoking a vicious cycle of self-antigen stimulation along with additional damage to surrounding tissues. The hypothesis is that the CD4+CD25+ Foxp3+ regulatory T cells (TR) play a major role in the regulation f the immune attack on infected Mф. Our preliminary studies revealed that mice which received the lysate of apoptotic non-infected Mф prior Leishmania infection demonstrated a significantly lower number of parasites than did the controls. The diminished parasitic burden correlated with a higher number of Mф and TR in the CL lesions of mice immunized with Mф lysate than in the controls. In this pilot and feasibility project, we plan to collect experimental evidence demonstrating that a preservation of Mф integrity can be beneficial for the elimination of intracellular pathogens. The data obtained will have implications not only for the elucidation of pathogenesis of CL, which we chose as a model for our study, but also for the broader spectrum of granulomatous diseases, such as tuberculosis and leprosy.

Specific Aim 1: To evaluate the necessity of TR for regulation of the number of Mф in CL lesions.

Specific Aim 2: To investigate the mechanisms of Foxp3+ cell regulation on the effector immune response.

The role of Msx2 transcription factor in response to UVB light
PI: Marianna Bei, DMD, DMSc

INTRODUCTION and SPECIFIC AIM
Ultraviolet (UV) irradiation from the sun is harmful to human skin. UV-induced skin damage includes sunburn, immuno-suppression, cancer and premature skin aging (photoaging) (Gilchrest and Yaar, 1992). Although skin cancer is relatively common, its prevalence falls far short of that of photoaging, the most common form of skin damage caused by sun exposure.

Photoaging is characterized by loss of the structural integrity of the collagenous extracellular matrix that ultimately leads to the wrinkled appearance of the skin (Gilchrest and Yaar, 1992; Quan et al., 2004; Warren et al., 1991; Fisher et al., 1996). At the molecular level, UV irradiation induces its harmful effects to the skin by altering cellular function via DNA damage and proliferation, generation of ROS, cell cycle de-regulation, impairment of signal transduction pathways and by activating transcription factors including NF-kB, members of the AP-1 complex, such as, c-FOS and c-Jun (Warren R, et al., 1991; Talwar HS, et al., 1995; LoY, et al.,

1995; Whisler R, et al., 1995; Bissett DL, et al. 1996; Bissett DL, et al., 1990). The Msx2 transcription factor is present in normal epidermis and dermis of fetal skin and in the epidermis of adult human and mouse skin, however its role in response to UV light is unknown (Stelnicki et al., 1997; Tureckova et al., 1995). Our preliminary studies indicate that the skin of the Msx2 mutant mice is highly photosensitive and becomes erythematic, hyperplastic and infiltrated when is exposed to acute ultraviolet B (UVB) irradiation. These studies suggest that Msx2 is likely to play an important role in protecting the skin from the adverse effects of UVB exposure. In that context, in this application the following specific aim will be addressed:

Specific Aim: To test the hypothesis that loss of the Msx2 gene function has harmful effects for the skin by increasing its susceptibility to UVB mediated cutaneous photodamage in vivo.

Exploring Infantile Hemangioma Regression by High-Throughput Screening of Small Molecule and SiRNA libraries
PI: Shosanna Greenberger, MD, PhD

INTRODUCTION and SPECIFIC AIM
Infantile hemangioma (IH) is the most common tumor of infancy, affecting about 10% of white babies. The lesions, which are usually solitary and cutaneous-only, appear early in post-natal life, grow rapidly during infancy, and involute spontaneously in early childhood. IH usually follows a benign course. However, in some cases, hemangiomas can become disfiguring and even vision- or life-threatening.  Systemic or intra-lesional corticosteroids are often effective treatments for hemangiomas. However, severe side effects limit their usefulness. In life-threatening cases that have not responded to corticosteroids, interferon- has been used as an alternative. Nevertheless, its use has been limited by reports of severe and irreversible neurotoxicity in as many as 20% of patients treated. Thus, the search for new drugs, which are both effective and safe in the treatment of IH, is a crucial need.

In the present study, a high-throughput screening (HTS) will be employed. By using robotics, liquid handling devices, sensitive detectors and data processing and control software, HTS allows a researcher to investigate numerous active compounds, antibodies or genes which modulate a particular biomolecular pathway ⁸. In order to discover new drugs for the treatment of IH we will screen small molecule compounds for their effect on HemSCs proliferation. As a result of such a screen, we expect to identify a number of active compounds that will halt HemSC growth. Subsequently, we will focus our efforts on further testing of these compounds for their ability to prevent the formation or accelerate the involution of IH using our established in vitro and in vivo models.

Specific Aim 1: To perform high-throughput, microscopy based screening of a small molecule library consisting of known bioactive molecules and natural compounds for the ability to arrest the proliferation of HemSCs.

Specific Aim 2: To test the activity of the most potent compounds identified in Aim 1 in vitro and in vivo models of IH

Evaluation of Ly6G as a novel mediator of neutrophil recruitment
PI: Peter Nigrovic, MD

INTRODUCTION and SPECIFIC AIM
The neutrophil is the paradigmatic innate immune effector cell, participating in the defense against a variety of microorganisms. In autoimmune and inflammatory diseases affecting the skin, neutrophil recruitment can represent a key pathophysiologic step in the development of tissue injury.  Dermatoses featuring neutrophilic infiltration include severe and/or pustular psoriasis, Behcet’s disease, Sweet’s syndrome, and rashes associated with autoinflammatory syndromes, among others. A pathologic role for these cells can be identified experimentally. As an example, depletion of neutrophils reduces epidermal thickness by >50% in the psoriasiform lesions of flaky skin (fsn/fsn) mice.

These and similar experiments utilizing the canonical anti-neutrophil antibody (anti-Gr-1; clone RB6-8C5; antigen Ly6G/C) have been interpreted as reflecting depletion of the circulating neutrophil pool. However, in the course of work on experimental murine arthritis, we have observed that suppression of this neutrophil-dependent process occurs at antibody doses that cause no detectable neutropenia of blood or marrow. In contrast, preliminary findings indicate that antibody ligation of Ly6G blocks neutrophil recruitment to peritoneal inflammation. The mechanism of this block to cell migration is completely uncharacterized and may represent a novel target in tissue specific recruitment pathways. We propose to explore this phenomenon, in collaboration with the Lymphocyte Migration Core and other facilities of the HSDRC, and to develop preliminary data that could serve as the foundation for a broader investigation and future applications for funding. Our ultimate goal is to identify a novel mechanism of neutrophil regulation with potentially widespread therapeutic implications.

Specific Aim 1. Identify a role for Ly6G in the modulation of neutrophil activation

Specific Aim 2. Characterize the role of Ly6G in the inhibition of neutrophil migration

A. Recruitment to chemotactic stimuli in skin and peritoneum

B. Chemokinesis and chemotaxis in transwell assays

C. In vivo imaging of neutrophil adhesion to endothelium