This thesis describes the clinical application of both 123I-labelled and 99mTclabelled Interleukin 2 (IL2) for the study of autoimmune diseases, cancer and atherosclerosis. Chapter 1, is an introduction to the research performed in the past 10 years. This is focused on the development of nuclear medicine techniques for imaging chronic and acute inflammation. While there are several approaches for the diagnosis of acute inflammation, the presence, severity and extent of chronic inflammation is much more difficult to evaluate. In particular, the possibility of diagnosing an ongoing autoimmune process during the preclinical phase may allow the early start of therapeutic intervention and possible prevention of the disease. Organ specific autoimmune diseases are characterized by chronic lymphomononuclear cell infiltration of the target organ, and studies reveal that 10% to 30% of infiltrating lymphocytes express the interleukin 2 receptor (IL2R) as a sign of cell activation. Therefore, the detection of sites of lymphocytic infiltration is an important goal for nuclear medicine. Radiolabelled human recombinant interleukin 2, which binds in vitro and in vivo to activated T-lymphocytes, may allow their identification in tissues. In chapter 2, it is briefly described the pathophysiology of inflammation and the concept that independently of the pathogenic noxae, tissues react to injuries with acute inflammation. If the inflammatory agent persists for several weeks or months, a chronic inflammatory process will develop, in which polymorphonuclear cells will not infiltrate the inflammation site, and lymphomononuclear cells are the main cell population in the infiltrate, organized in “granuloma like” lesions. It then follows an overview of the diagnostic techniques for imaging inflammation that have been developed in the last 25 years. Recent advances in the diagnosis of the inflammatory process have led to the development of specific radiopharmaceuticals aimed at specific, stage-related, diagnosis of disease: what is now called “molecular nuclear medicine”. I have classified these radiopharmaceuticals in six categories according to the different phases in which the inflammatory process develops. The “first mover” of an inflammatory process is a pathogenic noxa (phase I) that causes the activation of endothelial cells (phase II). An increase of vascular permeability then occurs, followed by oedema of tissue (phase III). The following phase is characterized by the infiltration of polymorphonuclear cells (PMN) (phase IV), and a self-limiting regulatory process called apoptosis is observed next (phase V). If the inflammatory process persists, a late chronic inflammation takes place (phase VI). In some pathological conditions, such as organ-specific autoimmune diseases, a chronic inflammation is present at the disease onset. In chapter 3, it is described in details the labelling of interleukin 2 with 123I (123I-IL2), its biochemical characterization, the in vitro binding assay and its use for in vivo detection of mononuclear cell infiltrated tissues. In vivo studies were performed 1 hour after administration of 2-3 mCi of 123I-IL2 in 10 newly diagnosed type 1 diabetes, 5 pre-diabetic, 10 Hashimoto’s thyroiditis, 10 - 137 – Celiac disease patients and in 10 normal volunteers. 123I-IL2 scintigraphy allowed in vivo detection and quantification of activated mononuclear cells in several affected tissues. In detail, a 123I-IL2 accumulation was detected in the thyroid of all patients affected by Hashimoto's thyroiditis, in the bowel of all Celiac disease and in the pancreas of all pre-type 1 diabetic patients. By contrast, in newly diagnosed type 1 diabetics, 123I-IL2 scan was positive in 5 out of 10 studied patients. I concluded that 123I-IL2 scintigraphy may be useful to study autoimmune phenomena in vivo and in diagnostic protocols to evaluate the presence of other tissue involvement in patients with an organspecific autoimmune disease. In chapter 4, 123I-Interleukin 2 was applied for the imaging of lymphocytic infiltration in Celiac disease patients and their response to diet. Indeed, Celiac disease is usually diagnosed by the presence of specific antibodies and jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Here I demonstrated the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. Ten Celiac patients were studied with 123I-IL2 scintigraphy at diagnosis and seven of them were also investigated after 12-19 months of gluten free diet. A significant correlation was found between jejunal radioactivity and the number of IL2R+ve lymphocytes per mm of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2=0.66; p=0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 binds only to activated Tlymphocytes infiltrating the gut mucosa. After a 1-year diet, bowel uptake of 123I-IL2 significantly decreased in 5 out of 6 bowel regions (p<0.03), although two patients still had a positive IL2 scintigraphy in one bowel region. It is concluded that 123I-IL2 scintigraphy is a sensitive non-invasive technique to assess in vivo the presence of activated mononuclear cells in the bowel of patients affected by Celiac disease. Unlike jejunal biopsy, this method provides information from the whole intestine and gives a non-invasive measure of the effectiveness of the gluten free diet. Activated mononuclear cells expressing interleukin 2 receptors heavily infiltrate the Crohn's Disease (CD) gut wall, and in chapter 5, it is described the application of 123I-IL2 scintigraphy to detect tissue infiltrating IL2R positive (IL2R+) in gut wall of CD patients. Fifteen patients with ileal CD (10 active and 5 inactive) and 10 normal subjects were studied by 123I-IL2 scintigraphy. Six active CD were also studied before and after 12 weeks of steroid treatment. After scintigraphy, patients were followed up for 29-54 months. Ex-vivo autoradiography was also performed to determine specificity of 125I-IL2 binding to IL2R+ cells. The results of autoradiography showed specific binding of 125I-IL2 to IL2R+ mononuclear cells infiltrating the CD gut wall. Intestinal 123I-IL2 intestinal uptake was higher in both active and inactive CD than in controls (p<0.0001 and p=0.03, respectively), positively correlated with the Crohn’s Disease Activity Index (CDAI) (p=0.01) and significantly decreased in steroid-induced remission CD (p=0.03). From this study, we concluded that 123I-IL2 accumulates in the diseased CD gut wall by specific binding to IL2R+ cells infiltrating the involved tissues. 123I-IL2 scintigraphy may represent an - 138 – objective tool for the “in vivo” assessment of intestinal activated mononuclear cell infiltration. After developing a method for labeling IL2 with 99mTc we described, in chapter 6, a study that compared the efficacy of 99mTc-interleukin 2 (99mTc- IL2) scintigraphy and 99mTc-HMPAO granulocyte (99mTc-WBC) scintigraphy in detecting the presence and the extent of bowel inflammation in patients with inactive Crohn’s Disease (>12 months) and in assessing the accuracy of these techniques in predicting future disease relapse. We studied 29 patients with ileal and/or colonic CD in stable clinical remission (CDAI <150 for at least 12 months) both with 99mTc-IL2 and with 99mTc-WBC scintigraphy in order to evaluate the extent of acute and chronic inflammation in the bowel. Results showed that, despite the absence of symptoms, 18 patients (62%) showed a positive 99mTc-IL2 and 18 (62%) a positive 99mTc- WBC scan. Only 11 of these patients (37.9%) were positive to both scintigraphies, but the site of IL2 and granulocyte bowel uptake was different in most areas, indicating that in CD, acute and chronic inflammation can be located in different sites. Both scans showed a high Negative Predictive Value (1.00 and 0.91, respectively), but a weak Positive Predictive Value (0.44 and 0.39, respectively) of predicting future disease relapse. Nevertheless, Kaplan- Meier curves generated between scintigraphic finding and time free from disease relapse were statistically different only for the 99mTc-IL2 scintigraphy (log-rank test, p=0.013). These results indicate that 99mTc-IL2 scintigraphy can be useful to select CD patients in clinical remission who could benefit from a preventive therapy to avoid disease relapse. In chapter 7, it is reported a new clinical application of 99mTc-Interleukin 2 scintigraphy: the evaluation of tumor-infiltrating lymphocytes in melanoma lesions. Cutaneous melanoma is an immunogenic tumour that is often characterized by the presence of tumor infiltrating lymphocytes (TILs). The degree of such infiltration together with the cell activation state are considered significant prognostic factors representing the immune reaction against the tumour. Patients with peri-tumoral infiltration may have better prognosis and may also achieve a better response to IL2 immunotherapy. We studied 30 patients with cutaneous lesions suspect for melanoma. Planar gamma camera images over known tumour sites were acquired one hour after the injection of 3 mCi of 99mTc-IL2. Peri-tumoral uptake of 99mTc-IL2 was measured as Target/Background (T/B) radioactivity ratio. All patients underwent surgery and a histological evaluation was performed. The percentage of different peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD16, CD25) and the percentage of IL2R positive cells on tumour histological sections were also measured. The results of this study showed that T/B ratios significantly correlated with the number of IL2R in TILs, while they did not correlate with the number of low affinity IL2R expressed on melanoma cells. In conclusion, this study demonstrated that 99mTc-IL2 scintigraphy could allow to quantify in vivo the amount of IL2R+ cells infiltrating lesions, suggesting its use for prognostic purposes and to select patients who may benefit of IL2 immunotherapy. In chapter 8, it is reported the results of a pilot clinical study by using 99mTc- 139 – interleukin 2 scintigraphy for the in vivo imaging of vulnerable atherosclerotic plaques. Indeed, several histopathological studies have demonstrated that vulnerable plaques are enriched in inflammatory cells. The aims of this study were therefore: 1) to test the ability of 99mTc-labelled interleukin 2 to bind to IL2R+ cells in carotid plaques; 2) to correlate the plaque uptake of 99mTc-IL2, measured in vivo, with the number of IL2-R+ cells within the plaque, measured ex vivo by histology; 3) to evaluate changes in 99mTc-IL2 uptake in plaques, before and after treatment with a statin or a hypocholesterolaemic diet. Fourteen patients (16 plaques) eligible for endoarterectomy were recruited for aim 1 and 2 and underwent 99mTc-IL2 scintigraphy before surgery. Nine patients (13 plaques) were recruited for the longitudinal study (aim 3); these patients received atorvastatin or a standard hypocholesterolaemic diet and 99mTc-IL2 scintigraphy was performed before and after 3 months of treatment. The degree of 99mTc-IL2 uptake was expressed as plaque/background (T/B) ratios and correlated with the percentage of IL2R+ cells at histology (r=0.707; p=0.002) and the number of IL2R+ cells at flow cytometry (r=0.711; p=0.006). In addition, the mean 99mTc-IL2 uptake decreased in statin-treated patients (2.16±0.44 vs 1.75±0.50; p=0.012), while it was unchanged in the patients on the hypocholesterolaemic diet (2.34±0.5 vs 2.33±0.45). It is concluded that 99mTc-IL2 accumulates in vulnerable carotid plaques; this accumulation is correlated with the amount of IL2R+ cells and is influenced by lipid-lowering treatment with a statin. Thus, radiolabelled-IL2 exemplifies how new molecular approaches to disease diagnosis can be revolutionary for the management of patients.
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