Santosh Mishra I

Osteoarthritis (OA), the most common form of arthritis, affects ~ 27M Americans and is increasing in incidence. It occurs due to degeneration of tissues comprising joints, and is associated with pain. OA pain is a major contributor to the burden of chronic pain in society. Current treatment options are limited to steroid injections, nonsteroidal anti-inflammatory drugs (NSAIDS), opioids and non-pharmacological approaches (exercise, weight loss). Unfortunately, each of these therapeutic approaches are problematic. Exercise, which helps weight management, is difficult for patients due to ongoing pain. NSAIDS can cause gastrointestinal irritation and bleeding and increase risk of heart attack or stroke, and opioids are associated with addiction and abuse (and can actually worsen chronic pain). Clearly, there is a critical need to identify new therapeutic targets and/or treatments for individuals suffering from OA pain. Here, we propose that a heretofore unrecognized neural pathway is a critical component of OA pain. This pathway involves ARTN, its receptor GFR????????3, and ???????????????pain?????????????????? channels on nerves (transient receptor potential [TRP] channels). Activation of this pathway initiates and maintains OA pain. The central hypothesis (based on preliminary data in multiple species [mouse, dog, cat, human]) is that ARTN, released from synovium of the OA joint in response to injury, results in de novo increase in its receptor, GFR????????3, in local and distant sensory nerves, producing local and widespread pain and hypersensitivity via Proto-oncogene tyrosine-protein kinase receptor (RET)-mediated upregulation of multiple downstream transient receptor potential (TRP) receptors. In this proposal, we will use multiple OA models and clinically relevant outcome measures, and leverage our unique access to dogs with naturally occurring OA, to achieve the following aims: Aim 1: To test the hypothesis that ARTN expression is increased in OA and is responsible for pain. Aim 2: To test the hypothesis that ARTN/GFR????????3 signaling is responsible for behaviorally manifested OA pain both in early and late stage disease. Aim 3: To test the hypothesis that RET-dependent ARTN/GFR????????3 signaling results in changes in multiple TRP channel expression and activation. Aim 4: To test/validate involvement of the above-described key molecules in a naturally occurring large animal model of OA (dog). Overall, this will be the first work investigating the role and mechanisms of ARTN/GFR????????3/TRP channel in OA pain and sensitivity. Based on solid, clinically relevant preliminary data, and leveraging PI expertise from two different and complementary disciplines, successful completion of this proposed work has the potential to identify clinically relevant neural mechanisms leading to the development of novel, effective therapeutics for the treatment of OA-pain in humans."

Atopic dermatitis is an inflammatory skin condition that affects an estimated 30% of the US population, mostly children and adolescents. Atopic dermatitis is characterized by chronically itchy skin that can weep clear fluid when scratched, and patients with atopic dermatitis are susceptible to bacterial, viral and fungal skin infection. There currently are no effective treatments for the chronic itch other than temporary symptomatic relief with topical applications (e.g. corticosteroids), and specific neurological pathways associated with the generation of chronic itch have not been elucidated. Here, we propose that CHRONIC ITCH, as occurs in Atopic Dermatitis, involves a novel signaling pathway that ends in release of NPPB by specific neurons in the DRG. Central to this pathway leading to chronic itch are four molecules: a) thymic stromal lymphopoietin (TSLP); b) PERIOSTIN c) the (????????v????????3) integrin receptor on specific neurons of the DRG called transient receptor potential vanniloid-1 (TRPV1) neurons; and, as described above, d) natriuretic polypeptide precursor b (NPPB). The Specific Hypothesis to be addressed is propagation of chronic itch is is initiated by a Th2 type immune response in the skin related to atopic dermatitis. This causes localized release of the cytokine TSLP from skin keratinocytes (and perhaps other cell types in the skin) which then, in an autocrine/paracrine fashion, binds to these and other keratinocytes via the keratinocyte TSLP/IL7R-receptor complex. This binding activates the JAK-STAT pathway in the keratinocytes, leading to production and release of the protein PERIOSTIN. PERIOSTIN, released by these keratinocytes, then sets in motion the following itch circuit: Released PERIOSTIN binds to a PERIOSTIN - binding integrin receptor ????????v????????3 expressed on a subset of neurons in the dorsal root ganglia, called TRPV1 neurons. As a result of PERIOSTIN binding, these TRPV1 neurons then release the neuropeptide NPPB centrally in the spinal cord that in response sends itch signals to the brain. We will test this hypothesis through the following specific aims: Aim 1). To determine if TSLP binding to the specific TSLP receptor complex on keratinocytes provokes production and release of periostin through activation of the JAK-STAT pathway in these cells; Aim 2) To determine whether PERIOSTIN binds directly to the integrin receptor ????????v????????3 on TRPV1 neurons (NPPB/SST) in the DRG, and whether this generates an itch sensation in vivo; Aim 3) To demonstrate a direct role of PERIOSTIN and neuropeptide NPPB in the generation of chronic itch in vivo. This proposed research will identify fundamental mechanisms for neuronal responses during the generation of chronic itch secondary to inflammatory skin disease. PERIOSTIN, integrin receptor signaling, and/or NPPB ?????????????????? producing neurons may provide novel therapeutic targets to treat skin diseases manifested by chronic itch.

More than 80% of patients undergoing treatment for head and neck cancer are prescribed opioid pain killers for management of radiation-associated pain, and unfortunately, 6 months after finishing radiotherapy, one-third of head and neck cancer patients will still be opioid-dependent. Chronic opioid use is associated with side effects such as constipation and decreased alertness; it can also lead to opioid abuse, misuse, addiction, and eventual overdose. Safer, more effective and less addictive pain relief strategies are desperately needed, and our proposed research addresses that need by investigating a novel radiation-activated pain signaling pathway that might serve as an excellent target that could be inhibited with new pain medications.

Chronic itch is the most common clinical symptom in numerous dermatological and systemic conditions in humans and animals, and it significantly reduces the quality of life of the patients and their families. Unfortunately, treatments selective for chronic itch are still limited due in large part to a lack of understanding of the mechanisms related to the neurological underpinning of itch. For more than decades, numerous studies have been performed to understand how itch is transduced and transmitted from the periphery via trigeminal (afferents projecting into head and face, TG) and dorsal root ganglia (afferents projecting into rest of the body, DRGs; a collection of cell bodies of cutaneous sensory neurons) into the central nervous system. The vast majority of these studies were performed in rodent models, but the limited success of translating findings from these rodents to the human disease situation has raised criticism about the benefit of such translational studies. Interestingly, recent comparative analyses of mice and humans demonstrated different expression patterns of nociceptors on DRGs in these two species, thus, indicating that different itch treatment strategies might be required in each species. These findings also may be applied to other animals where it is anecdotally said, ""cats are not small dogs"" or ""horses are large cats"" based on the clinical response to many drugs, including anti-itch medications. The value of translating data from mouse experiments to companion animal species remains largely unknown. Next-generation sequencing technologies have provided many valuable insights into complex biological systems. Currently, however, the majority of transcriptome analysis experiments use whole tissue samples that consist of a great variety of cell types, and it is based on the assumption that cells from a given tissue are homogeneous; thus, it does not give us any information regarding cell types and gene expression profile from same cell. Here, we proposed using a single cell multiome approach (ATAC-seq + gene expression) to elucidate the transcriptional landscape by systematically comparing the data obtained from sensory ganglia involved in itch detection and neurotransmission from multiple species and cell types and states in ganglia based on their location. Although single cell could be a good target but due to variation in cell sizes of the DRG neurons from largest being (>100 um in diameter) and the size of a single cell channel diameter is approx. 100 um), therefore, we focus on nuclei as a target for the multiomics analysis. Our proposed study will help elucidate the similarities and key differences of cellular and molecular pathways involved in multiple species and provide insight into trigeminal ganglia (TG) and DRG biology, which will guide the development of potential species-specific therapeutic targets, thus helping to improve itch treatment successfully.

The goal of this proposal is to advance our understanding of whether radiation-associated pain signaling pathways are co-opted to promote aggressive tumor behavior. The objective is to generate a rationale for future studies that will investigate specific mechanisms responsible for these effects, and determine the translational relevance of this phenomenon in both veterinary and human cancer patients. The proposed work is important because a common consequence of definitive-intent full-course radiotherapy is acute pain. This pain is often severe and difficult to control with current analgesics. That is especially true for certain anatomic sites, such as the oral cavity and perineum. Severe unmitigated radiation-associated pain can necessitate unplanned treatment breaks; these breaks negatively affect local tumor control and overall survival time. The common explanation is that treatment delays counteract the effectiveness of fractionated radiotherapy by allowing for accelerated proliferation of tumor cells during the course of radiation. We propose that these detrimental effects may arise directly from interactions between cancer cells and molecular components of the radiation-associated pain signaling cascade. This theory is based on our recent discoveries that: (1) lung tumor growth is accelerated in mice with severe oral radiation-associated pain; and (2) master regulators of that pain are neurons which express the TRPV1 ion channel. We have also found that radiation causes oral keratinocytes to secrete a neurotrophic factor called artemin, and oral irradiation also causes upregulation of artemin??????????????????s neuronal receptor, GFR????????3. The literature indicates that ARTN/GFR????????3 enhances expression of, and sensitizes, TRPV1 receptors; and ARTN, GFR????????3, and various TRP ion channels can each promote tumor growth and metastasis. Together, this knowledge from the literature, plus our preliminary data, give rise to the hypothesis that the same TRPV1-mediated neural pathway which gives rise to oral radiation-associated pain, also provokes aggressive tumor behavior. We will test that hypothesis with two specific aims: (AIM 1) to determine whether the tumor-promoting effects of oral irradiation are directly attributable to pain; and (AIM 2) to determine whether the tumor-promoting effects of radiation-associated pain are generalizable across tumor model systems. This contribution is significant because it will generate a clear understanding of whether radiation-associated pain signaling pathway components contribute to tumor growth and metastasis in our primary animal model. The results will pave the way for the next phase of our research, which will take a deep dive into understanding the underlying mechanisms by which pain pathway components alter tumor behavior, and comprehensively evaluate the relevance of these phenomena in clinical (veterinary and human) populations. The proposed research is innovative because our hypothesis challenges the dogmatic view that acute radiation-associated pain is a short-lived problem, ending with resolution of clinically-evident acute tissue injuries. Instead, we view radiation activation of pain signaling pathways as a process which may be independent of tissue injury, and which can have lasting and detrimental effects on patient prognosis; for this reason, the proposed research represents a critical first step toward identifying new treatments that serve the dual purposes of improving comfort and enhancing radiotherapeutic efficacy. Importantly, while we begin this work using a mouse model of oral irradiation, there is evidence that the pain pathways which are activated by radiation are conserved across anatomic sites, and thus it is possible that our results may extend well beyond the realm of head and neck cancers, and positively impact the care of millions of pets and people who undergo radiation for different malignancies. Additionally, because these pain pathways can be activated very soon after irradiation and even in patients without clinically overt acute effects or pain, these results coul

he aims of the experiments proposed herein are to 1) confirm the presence of a reciprocal amplification loop involving TSLP, IL-31 and periostin using both in vitro and in vivo assays and 2) to assess the therapeutic potential of inhibiting this pathway.

"Atopic dermatitis (AD) is chronic illness of childhood, adult, and is often a lifelong disease worldwide. AD poses second-highest disability rank of all non-malignant skin diseases, and is characterized by relapsing skin inflammation and itch. The other disease with similar conditions (skin inflammation and itch) is psoriasis. Numerous immune and non-immune cells have been implicated in the pathogenesis of AD and psoriasis including mast cell, neutrophils, basophils and T helper type 2 (Th2) cells. Emerging evidence from other study suggests that myristoylated alanine-rich C kinase substrate (MARCKS) protein regulates pro-inflammatory NF-kB in macrophages. The role of keratinocytes that constitute a solid physical skin barrier representing the skin first line of defense express MARCKS protein but the role of MARCKS in keratinocytes remain unknown. We hypothesize MARCKS regulates cytokines in the skin keratinocytes are key susceptibility pathways involved in the skin AD pathogenesis. The work from this proposal will allow us to pursue our long-term goal of better understanding how MARCKS may be targeted for new AD therapies in patients. Thus, the overall objective of our proposal is to test the mechanisms by which MARCKS involved in the regulation and contribution to AD pathogenesis. Currently, the role of MARCKS in AD remains completely unknown. Thus, there is an urgent need to correct this knowledge gap to develop novel therapies for AD. The rationale for this proposal is that once it is understood how MARCKS regulates skin inflammation, novel therapies can be developed to target AD and psoriasis. We will perform the following aims to test our hypothesis: AIM 1: To assess the role of MARCKS inhibitor (BIO11006) in skin inflammation and itch AIM 2: To evaluate the expression of MARCKS and other cytokines in mouse models of AD and psoriasis. "

Globally this year, there will be an estimated 650,000 new human head and neck cancer (HNC) diagnoses.1 Half of those people will be cured with intensive combinations of surgery, radiation therapy and chemotherapy. But along their path towards a cure, essentially all of those patients will experience oral mucositis (OM) and discomfort. Indeed, significant treatment-associated pain is reported by 70% of HNC patients undergoing definitive radiotherapy, and 60% of patients report persistent pain for at least six months following treatment.2,3 OM occurs in nearly all patients who receive radiotherapy, and, in 10-25%, an interruption or modification in treatment is required. These treatment delays worsen local tumor control and overall survival time.4 To improve comfort and avoid treatment delays, effective analgesics are needed. Development of effective and appropriately targeted analgesics will require deep knowledge of the underlying pain signaling mechanisms that are activated by HNC treatment; however, there has been essentially no research in this area. Anecdotally, some HNC patients report cold sensitivity or pain associated with the development of OM. This observation led to our preliminary studies and the discovery that a critical component of acute orofacial radiation-associated pain is a signaling pathway mediated by neurons that express the TRPM8 (transient receptor potential melastatin family member 8) ion channel. Previously, this pathway has been associated with ????????????????cold pain.??????????????? We have evidence from mouse models that: ???????????????? Oral irradiation causes local release of a neurotrophic factor called artemin (ARTN), ???????????????? ARTN binds its receptor (GFR????????3) on free nerve endings in the mouth, and ???????????????? GFR????????3 activates TRPM8 in trigeminal sensory neurons, thus resulting in cold sensitivity and pain.

Atopic dermatitis (AD) is a chronic condition that causes severe inflammation and itch and affects millions. Ambient temperature and pollutants are major factors that trigger AD and associated itch. Unusual temperatures and increased exposure to pollutants, common outcomes of climate change, are believed to have led to higher severity and number of AD cases. However, the underlying neuromodulatory processing of climate change on AD remains elusive. Precise monitoring of neuromodulatory processes requires innovative neurochemical sensors that seamlessly integrate with the three-dimensional central nervous system for continuous monitoring of neurotransmitters along the nervous system in freely moving animals. Unfortunately, present neurochemical sensors are either tethered or rely on bulky, battery-powered, head-mounted wireless electronics. Such approaches significantly affect animal behavior and cause erratic data. We propose to overcome this major issue to study the role of environmental factors on AD and itch by developing a new class of miniaturized, battery-free, wireless microsensors interfaced along the spinal cord of freely moving animals for capturing catecholamine levels in real-time. Specifically, we will study the implications of acute and chronic exposure to elevated ambient temperature and common pollutants (formaldehyde) on the underlying neurological processes associated with itch and inflammation in na????????ve and AD mouse models. The proposed idea builds upon Dr. Mishra??????????????????s experience in developing AD mouse models that closely mimic AD in humans to study itch-related signaling in the central nervous system and Dr. Bandodkar??????????????????s expertise in developing wireless, battery-free neural probes for optogenetics and neurochemical sensing.

Chronic itch associated with inflammatory skin diseases such as atopic dermatitis, can become both physiologically and psychologically debilitating and impart serious clinical consequences. We will study how an endogenous ligand that releases in the skin and then sets in motion the following itch circuit by binding to integrin receptor expressed in the dorsal root ganglia neurons, called TRPV1 neurons, as a result, to propagate the sensation of chronic itch by releasing neuropeptide NPPB in the spinal cord. Our proposed research will identify a molecular pathway involved in cutaneous-nerve responses in the dissemination of chronic itch and may provide potentially open new therapeutic targets for prospective treatment for chronic itch associated with atopic dermatitis.