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Cardiac MRI of small animal models of cancer radiation therapy (RT) is a valuable tool for studying the effect of RT on the heart. However, standard cardiac MRI exams require long scanning times, which is challenging for sick animals that may not survive extended periods of imaging under anesthesia. The purpose of this study is to develop an optimized, fast MRI exam for comprehensive cardiac functional imaging of small-animal models of cancer RT. Ten adult female rats (2 non-irradiated and 8 irradiated) were scanned using the developed exam. Optimal imaging parameters were determined, which minimized scanning time while ensuring measurement accuracy and avoiding imaging artifacts. This optimized, fast MRI exam lasted for 30 min, which was tolerated by all animals. EF was normal in all imaged rats, although it was significantly increased in the irradiated rats, which also showed ventricular hypertrophy. However, myocardial strain was significantly reduced in the irradiated rats. In conclusion, a fast MRI exam has been developed for comprehensive cardiac functional imaging of rats in 30 min, with optimized imaging parameters to ensure accurate measurements and tolerance by irradiated rats. The generated strain measurements provide an early marker of regional cardiac dysfunction before global function is affected.

Over half of all cancer patients receive radiation therapy (RT). However, radiation exposure to the heart can cause cardiotoxicity. Nevertheless, there is a paucity of data on RT-induced cardiac damage, with limited understanding of safe regional RT doses, early detection, prevention and management. A common initial feature of cardiotoxicity is asymptomatic dysfunction, which if left untreated may progress to heart failure. The current paradigm for cardiotoxicity detection and management relies primarily upon assessment of ejection fraction (EF). However, cardiac injury can occur without a clear change in EF.To identify magnetic resonance imaging (MRI) markers of early RT-induced cardiac dysfunction.We investigated the effect of RT on global and regional cardiac function and myocardial T1/T2 values at two timepoints post-RT using cardiac MRI in a rat model of localized cardiac RT. Rats who received image-guided whole-heart radiation of 24Gy were compared to sham-treated rats.The rats maintained normal global cardiac function post-RT. However, a deterioration in strain was particularly notable at 10-weeks post RT, and changes in circumferential strain were larger than changes in radial or longitudinal strain. Compared to sham, circumferential strain changes occurred at the basal, mid-ventricular and apical levels (p0.05 for all at both 8-weeks and 10-weeks post-RT), most of the radial strain changes occurred at the mid-ventricular (p=0.044 at 8-weeks post-RT) and basal (p=0.018 at 10-weeks post-RT) levels, and most of the longitudinal strain changes occurred at the apical (p=0.002 at 8-weeks post-RT) and basal (p=0.035 at 10-weeks post-RT) levels. Regionally, lateral myocardial segments showed the greatest worsening in strain measurements, and histologic changes supported these findings. Despite worsened myocardial strain post-RT, myocardial tissue displacement measures were maintained, or even increased. T1/T2 measurements showed small non-significant changes post-RT compared to values in non-irradiated rats.Our findings suggest MRI regional myocardial strain is a sensitive imaging biomarker for detecting RT-induced subclinical cardiac dysfunction prior to compromise of global cardiac function.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family which has been extensively studied for its roles in neural development, long-term memory, brain injury, and neurodegenerative diseases. BDNF signaling through tropomyosin receptor kinase B (TrkB) stimulates neuronal cell survival. For this reason, small molecule TrkB agonists are under pre-clinical develoment for the treatment of a range of neurodegenerative diseases and injuries. Our laboratory recently reported BDNF is secreted by pro-regenerative endothelial progenitor cells (EPCs) which support hematopoietic reconstitution following total body irradiation (TBI). Here we report BDNF-TrkB signaling plays a novel regenerative role in bone marrow and thymic regeneration following radiation injury. Exogenous administration of BDNF or TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following myelosuppressive radiation injury promoted faster recovery of mature blood cells and hematopoietic stem cells capable of multi-lineage reconstitution. BDNF promotes hematopoietic regeneration via activation of PDGFRα+bone marrow mesenchymal stem cells (MSCs) which increase secretion of hematopoietic cytokines interleukin 6 (IL-6) and leukemia inhibitory factor (LIF) in response to TrkB activation. These data suggest pharmacologic activation of the BDNF pathway with either BDNF or 7,8-DHF may be beneficial for treatment of radiation or chemotherapy induced myelosuppression.

Radiation therapy is received by over half of all cancer patients. However, radiation doses may be constricted due to normal tissue side effects. In thoracic cancers, including breast and lung cancers, cardiac radiation is a major concern in treatment planning. There are currently no biomarkers of radiation-induced cardiotoxicity. Complex genetic modifiers can contribute to the risk of radiation-induced cardiotoxicities, yet these modifiers are largely unknown and poorly understood. We have previously reported the SS (Dahl salt-sensitive/Mcwi) rat strain is a highly sensitized model of radiation-induced cardiotoxicity compared to the more resistant Brown Norway (BN) rat strain. When rat chromosome 3 from the resistant BN rat strain is substituted into the SS background (SS.BN3 consomic), it significantly attenuates radiation-induced cardiotoxicity, demonstrating inherited genetic variants on rat chromosome 3 modify radiation sensitivity. Genes involved with mitochondrial function were differentially expressed in the hearts of SS and SS.BN3 rats 1 week after radiation. Here we further assessed differences in mitochondria-related genes between the sensitive SS and resistant SS.BN3 rats. We found mitochondrial-related gene expression differed in untreated hearts, while no differences in mitochondrial morphology were seen 1 week after localized heart radiation. At 12 weeks after localized cardiac radiation, differences in mitochondrial complex protein expression in the left ventricles were seen between the SS and SS.BN3 rats. These studies suggest that differences in mitochondrial gene expression caused by inherited genetic variants may contribute to differences in sensitivity to cardiac radiation.

Purpose/Objectives: Radiation therapy is used by more than 50% of breast cancer patients, but radiation doses can be limited by normal tissue side effects. For example, breast cancer radiation therapy can improve breast cancer-specific survival, but increase cardiac deaths in those with left-sided cancers. Identifying genetic factors that can enhance tumor radiation sensitivity while decreasing normal tissue toxicities has the potential to improve the therapeutic ratio of radiation therapy – leading to more cures and less long-term toxicities. The use of animal models with differing genetic backgrounds to assess radiation toxicity, followed by genetic mapping of radiosensitivity phenotypes, has the potential to identify new targets that can predict cardiac toxicity from radiation therapy. This project examines how genetic host factors alter normal tissue toxicity risks from breast cancer radiation. Materials/Methods: Inbred female SS rats and SS.BN3 consomic rats, that are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. For this study, adult female SS and SS.BN3 rats received image-guided whole heart radiation to a dose of 21 Gy (3 fields, AP and 2 laterals). Cardiac troponin was serially measured at 2, 6, and 12 weeks, and echocardiograms with strain analysis were performed at baseline and 3 months. The Student's t-test was used to compare values. Results: The SS female rats exhibited enhanced cardiac toxicity compared to SS.BN3 rats, with cardiac troponin levels elevated at 12 weeks (0.32 ng/ml vs.0.08 ng/ml for SS vs. SS.BN3, p=0.01), and moderate to severe pericardial effusions seen in 6 of 9 SS rats vs. 2 of 7 SS.BN3 rats. At 3 months post-radiation, echocardiograms revealed increased left ventricular posterior wall thickness at end diastole (LVPWd) in SS vs. SS.BN3 rats (0.25 vs. 0.20 cm, p=0.002) and increased left ventricular mass (LVM) in SS vs. SS.BN3 rats (1.54 vs. 1.28 g, p Conclusions: These results demonstrate that genetic variant on rat chromosome 3 alter the radiosensitivity to single fraction cardiac radiation therapy. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). These models will also be expanded to test whether similar results are seen with fractionated cardiac radiation therapy. This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer. Citation Format: Bergom C, Schlaak R, Frei A, Fish BL, Harmann L, Gasperetti T, Schottstaedt AM, Flister MJ, Medhora M, Strande JL. The use of consomic animal models to identify genetic factors that modulate radiation-induced cardiac toxicity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-11-18.

Objectives: The tumor microenvironment (TME) can impact breast cancer tumor growth, progression, and treatment responses. Data suggests that genetic variants in not only breast cancer cells, but also in the TME, can also alter these processes. We have utilized a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel to identify genetic variants in the TME that affect radiation sensitivity. Materials/Methods: Human triple negative breast cancer MDA-MD-231 cells were implanted into immunodeficient (IL2RG KO) consomic rat strains that are genetically identical except for chromosome 3 is inherited from a separate strain (SS and SS.BN3 strains). On day 10, tumors were treated with 3 daily ionizing radiation (IR) treatments of 4 Gy or sham, and tumor growth was monitored. Tumors were also harvested for hypoxia staining using pimonidazole or for RNA-seq. RNA-Seq was performed and a custom SSRS protocol was used to align both rat and human transcripts. This yielded transcript and gene level estimated fold-change and adjusted p-values for human- and rat-derived transcripts separately. E077 mammary tumor cells were implanted into adult female immune competent C57/Bl6 mice. On day 5, tumors were treated with 5 daily IR treatments of 5 Gy or sham. Either vehicle or a mAb to the Notch ligand Dll4 (Genentech) was given twice weekly. Chi-square, Fisher’s exact, and Kolmogorov-Smirnov tests and empirical cumulative distribution plots for differential expression significance values were performed. Results: Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor IR sensitivity, as human breast cancer xenografts in the consomic strain (SS.BN3) were significantly more IR sensitive than SS rat strain tumors (supra-additive). Vascular gene pathways were differentially expressed, and tumor vascular phenotypes were distinct, with SS.BN3 tumors with increased but poorly functioning blood vessels. Hypoxia was similar at baseline, but increased in SS.BN3 tumors following IR. These results were consistent with less Dll4 expression in the SS.BN3 TME. The use of a Dll4-targeted mAb in mice demonstrated that targeting Dll4 enhanced mammary tumor IR responses. Conclusion: CXM demonstrated TME genetic variants can affect IR sensitivity of genetically identical tumor cells. Using SSRS, we identified candidate genes on rat chromosome 3 that may potentially influence IR sensitivity, and our studies ultimately led to identification of the Notch ligand Dll4 as a target to enhance breast cancer IR responses. Future studies will investigate the possibility of the Dll4 pathway as a therapeutic target, as well as interrogate other pathways responsible for changes in IR sensitivity seen in the CXM model. Determining TME factors that affect the IR sensitivity will allow more tailored and effective treatments. Citation Format: Carmen Bergom, Michael W. Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Rachel A. Schlaak, Shirng-Wern Tsaih, Michael J. Flister. Genetic variants in the tumor microenvironment alter radiation responses in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA004.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG&minus, /&minus, )) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p &lt, 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Background: The tumor microenvironment (TME) is known to impact tumor growth, metastatic potential, and treatment response. Nearly all studies of head and neck cancer (HNC) have focused on somatic mutations in the malignant cells. We hypothesized that genetic determinants limited to the microenvironment would influence HNC growth and metastatic potential. Approach: To demonstrate the impact of genetic differences in the TME on HNC cell line in vivo growth we utilized a novel tool, the consomic xenograft model (CXM). A consomic rat has an entire chromosome substituted into the isogenic background of another inbred strain by selective breeding. Use of immunodeficient (IL2Rγ-/-) consomic rats allows one to study the influence of stromal genetics on tumor biology without the confounding effect of differences in the immune system through the orthotopic implantation of cancer cells into different consomic rat strains. In this system, any differences in tumor growth or metastases are due to differences in the TME rather than cancer cells or immune response. We utilized SS and SS.BN3 consomic rat strains, previously shown to affect the growth of breast tumors, to study the effects of the TME on HNC tumor growth using two well-characterized HPV negative HNC cell lines, SCC-6 (base of tongue derived) and SCC-22b (derived from a hypopharyngeal cancer that had metastasized to lymph nodes). Both cell lines were modified to stably express luciferase. HNC cells were inoculated into the tongue of SS and SS.BN3 animals and tumor growth was monitored by biophotonic imaging after luciferin injection. Results: A significant difference in the tumor growth was seen between rat strains for both cell lines, with the SS.BN3 rats exhibiting less tumor growth and metastasis. Median luciferase activity from baseline increased by 4.1-fold vs. 1.1 fold in SCC-6 tumors in SS vs SS.BN3 rats, respectively (p Conclusions: The use of the CXM model demonstrates an important role for the TME in the growth and metastatic spread of HNC cell lines. This model allows for future congenic mapping to identify the causative genetic variants in the TME mediating the HNC changes in tumor growth and metastasis. Citation Format: Michael W. Straza, Amy Rymaszewski, Kwangok P. Nickel, Anne Frei, Anirban Chatterjee, Rachel Schlaak, Amit Joshi, Michael Flister, Randy J. Kimple, Carmen Bergom. The consomic xenograft model identifies genetic changes in the tumor microenvironment that alter the growth and metastasis of head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5898. doi:10.1158/1538-7445.AM2017-5898

Progress in elucidating the molecular basis of breast cancer has allowed for treatment breakthroughs such as anti-estrogen and Her2-targeted therapy. It has also shaped the approaches to both surgical and systemic therapy. However, no similar use of molecular information has been utilized to better direct the use of radiation therapy. The development of predictive tools for the radiosensitivity of tumors could allow for personally tailored radiation doses, with treatment de-escalation for radiosensitive tumors, or dose escalation or the use of adjunct treatments in the case of radioresistant tumors. Communication between malignant tumor cells and the tumor microenvironment (TME) underlies most aspects of tumor biology, including chemotherapy and radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cell-sorting. Here we utilize these unique techniques to identify genetic variants in the TME that can affect radiation sensitivity. In CXM, human triple negative breast cancer MDA-MD-231 cells are orthotopically implanted into immunodeficient (IL2Rγ-/-) consomic rat strains, which are rat strains in which an entire chromosome is introgressed into the isogenic background of another inbred strain by selective breeding. Because the strain backgrounds are different but the tumor cells are not varied, the observed changes in tumor progression are due to genetic differences in the non-malignant TME. We hypothesized that the tumors in SS.BN3 rats (identical to SS rats but with BN strain chromosome 3) would be more sensitive to radiation due to increased tumor vascularity via CD31 staining, and increased tumor blood volume capacity, as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Our studies demonstrate differential responses to radiation in the CXM model comparing parental SS (IL2Rγ) rats to SS.BN3 (IL2Rγ) rats treated with fractionated radiation therapy (4 Gray x 3), with altered tumor growth kinetics and tumor recurrence rates. A difference was seen in time to 5-fold increase in tumor growth, with 44 vs. >130 days for SS versus SS.BN3 rats (supra-additive, p130 days) in the SS versus SS.BN3 rats (p=0.02). These results suggest that genetic determinants in the TME affect the radiation sensitivity of genetically identical tumor cells. Using SSRS, we identified a number of candidates on rat chromosome 3 that may potentially influence radiation sensitivity by altering the tumor vasculature. Future studies will further dissect the pathways responsible for the changes in radiation sensitivity. Determining TME factors that affect the radiation sensitivity of tumors has the potential to allow for more tailored and effective radiation treatments in breast cancer. Citation Format: Carmen Bergom, Michael Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Shirng-Wern Tsaih, Howard Jacob, Michael J. Flister. Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B07.

A number of malignancies are promoted by the activation of Rho GTPases. Regulators of RhoA activity, including the RhoGEF, SmgGDS, can also promote cancer development. Both RhoA and SmgGDS have been shown to enhance NF-ĸB activation in a number of tumors. Utilizing mass spectrometry, we identified the tumor suppressive small GTPase DiRas1 as a novel binding partner for SmgGDS. DiRas1 is a tumor suppressor in malignant gliomas and esophageal squamous cell carcinomas. Examining DiRas gene expression using the ONCOMINE mRNA microarray database (www.oncomine.org) revealed that DIRAS1 and DIRAS2 are markedly decreased in central nervous system tumors, including anaplastic astrocytomas and GBMs, when compared to normal tissue. The role of DiRas1 in other cancers is largely undetermined, and the ways in which DiRas1 may antagonize pro-oncogenic small GTPase signaling is largely unknown. To define how DiRas1 expression may affect SmgGDS functions, we tested the association of DiRas family proteins with SmgGDS. SmgGDS robustly bound wildtype DiRas1, and its closely-related family member DiRas2, but only weakly bound DiRas3 in vitro and in cell lines. In addition, DiRas1, DiRas2, or DiRas3 protein expression decreased proliferation of HEK293T cells. DiRas1 expression could promote less activated RhoA, as a low level of DiRas1 expression caused markedly decreased RhoA-SmgGDS binding in a number of cell lines. DiRas1 expression also abrogated SmgGDS- and RhoA-mediated NF-ĸB activation in a concentration-dependent manner. SmgGDS may exhibit GEF activity toward DiRas1, as a dominant negative DiRas1 (S21N) binds to SmgGDS more than the wildtype protein. However, DiRas1 is predominantly GTP-bound in cells, likely due to a high rate of guanine nucleotide dissociation and low intrinsic GTPase activity. Thus, DiRas1 expression may in part promote tumor suppression by non-productively binding SmgGDS, resulting in less RhoA activity. Taken together, these results suggest that the tumor suppressive small GTPase DiRas1 can antagonize RhoA signaling by competing for binding of the RhoGEF SmgGDS. Further characterizing these actions may lead to novel therapeutic targeting of RhoA activation in cancer malignancies. Citation Format: Andrew D. Hauser, Kristen M. Barr, Anne C. Frei, Patrick Gonyo, Ellen L. Lorimer, Carol L. Williams, Carmen Bergom. The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4443. doi:10.1158/1538-7445.AM2014-4443

The acute painful event is the hallmark of sickle cell disease (SCD) in humans, accounting for the majority of hospitalizations. In addition, pain managed at home is underreported in both children and adults with SCD. Vaso-occlusion is intricately linked to the development of sickle cell pain, initiating tissue damage and numerous biochemical, electrochemical, and neurologic cascades which affect nociception (the perception of pain). It has been hypothesized that repeated painful events may lead to sensitization, which can manifest as allodynia (painful response to a usually non-painful stimulus) and chronic pain. However, the mechanisms of pain in SCD remain poorly understood. The objective of this study was to evaluate nociception in a mouse model of SCD both at baseline and after acute vaso-occlusion. The Berkeley mouse model of SCD exhibits many characteristics of human SCD, including vaso-occlusion. Acute vaso-occlusive events in the Berkeley SCD mouse can be induced by exposure to short periods of hypoxia followed by reoxygenation. We compared pain behavior responses to heat and mechanical force in female Berkeley SCD (SS) mice before and after exposure to 1 hour of hypoxia (FiO2 10%) followed by 1 hour of reoxygenation in room air. Pain behavior in SS mice was also compared to that in control female HbA (AA) mice, which solely express normal human hemoglobin and are maintained on the same mixed genetic background as the SS mice. Female mice expressing both normal and sickle human hemoglobin (AS mice) comprised an additional control group. Pain behavior was assessed by two methods: the Hargreaves test of thermal (heat) nociception and the von Frey test of mechanical allodynia. The Hargreaves test is conducted by applying a radiant heat stimulus to the plantar surface of the hind paw (Hargreaves, et al, 1988). The time to withdrawal of the paw from the heat stimulus is recorded as the paw withdrawal latency. Baseline data from the Hargreaves test of 12 AA, 12 AS, and 14 SS mice showed a subtle decrease in paw withdrawal latency in SS as compared to AA or AS mice; however, the differences in paw withdrawal latency between groups were not statistically significant. Exposure to hypoxia-reoxygenation did not affect paw withdrawal latency in response to heat stimulation in any of the three groups of mice. The second test for pain behavior used was the von Frey test for mechanical hypersensitivity (i.e., allodynia). Mice are tested for response to the application of increasing and decreasing force strengths of nylon monofilaments to the plantar surface of the hind paws. The paw withdrawal threshold was determined using the up-down method of Dixon (1980). Baseline data from 12 AA, 12 AS and 12 SS mice showed a significant difference in paw withdrawal threshold between groups (p=0.05); SS mice exhibited the lowest paw withdrawal threshold, reflecting increased sensitivity to mechanical stimuli. Exposure to hypoxia-reoxygenation did not alter the paw withdrawal threshold of AA and AS mice and tended to decrease paw withdrawal threshold of SS mice, consistent with vaso-occlusion causing pain in SS mice exposed to hypoxia-reoxygenation. These results suggest that Berkeley SS mice exhibit mechanical hypersensitivity at baseline compared to AA and AS mice, perhaps reflective of increased baseline vaso-occlusion in SS as compared to control mice. Exposure to hypoxia-reoxygenation, which induces RBC sickling, acute vaso-occlusion and tissue damage in SS mice, accentuates the sensitivity of the SS mice to mechanical stimuli. The results of these ongoing studies will expand our understanding of mechanisms underlying sickle cell-associated pain behavior in mice with SCD. Translationally, this may lead to improved pain management strategies for both acute and chronic pain experienced by individuals affected by this chronic debilitating disease.