Marco Chianelli

Characterization of a New Form of Inherited Hypercholesterolemia Familial Recessive Hypercholesterolemia

We previously described a Sardinian family in which the probands had a severe form of hypercholesterolemia, suggestive of familial hypercholesterolemia (FH). However, low density lipoprotein (LDL) receptor activity in fibroblasts from these subjects and LDL binding ability were normal. The characteristics of the pedigree were consistent with an autosomal recessive trait. Sitosterolemia and pseudohomozygous hyperlipidemia were ruled out. A second Sardinian kindred with similar characteristics was identified. Probands showed severe hypercholesterolemia, whereas their parents and grandparents were normolipidemic. FH, familial defective apoprotein (apo) B, sitosterolemia, and cholesteryl ester storage disease were excluded by in vitro studies. We addressed the metabolic basis of this inherited disorder by studying the in vivo metabolism of LDL in 3 probands from these 2 families. 125I-LDL turnover studies disclosed a marked reduction in the fractional catabolic rate (0.19+/-0.01 versus 0.36+/-0.03 pools per day, respectively; P<0.001) and a significant increase in the production rate [20.7+/-4.4 versus 14. 0+/-2.4 mg. kg-1. d-1, respectively; P<0.01] of LDL apoB in the probands compared with normolipidemic controls. We then studied the in vivo biodistribution and tissue uptake of 99mtechnetium-labeled LDL in the probands and compared them with those in normal controls and 1 FH homozygote. The probands showed a significant reduction in hepatic LDL uptake, similar to that observed in the FH homozygote. A reduced uptake of LDL by the kidney and spleen was also observed in all patients. Our findings suggest that this recessive form of hypercholesterolemia is due to a marked reduction of in vivo LDL catabolism. This appears to be caused by a selective reduction in hepatic LDL uptake. We propose that in this new lipid disorder, a recessive defect causes a selective impairment of LDL receptor function in the liver.

Low-activity (2.0 GBq; 54 mCi) radioiodine post-surgical remnant ablation in thyroid cancer : comparison between hormone withdrawal and use of rhTSH in low-risk patients

OBJECTIVE (a) To compare the efficacy of low-activity (2 GBq; 54 mCi) (131)I ablation using l-thyroxine withdrawal or rhTSH stimulation, and (b) to assess the influence of thyroid remnants volume on the ablation rate. DESIGN Patients underwent neck ultrasound, (131)I neck scintigraphy and radioiodine uptake. Post-therapy whole body scan (WBS) was acquired after 4-6 days. Ablation was assessed after 6-12 months by WBS, Tg and TgAb following l-thyroxine withdrawal. METHODS Group A: preparation by L-T(4) withdrawal (37 days); 21 patients received (131)I (2.02+/-0.22 GBq; 54.6+/-5.9 mCi) and on the day of treatment, TSH, Tg, TgAb were measured; Group B: stimulation by rhTSH; 21 patients received (131)I (1.97+/-0.18 GBq; 53.2+/-4.9 mCi) 24 h after the second injection of rhTSH (0.9 mg) and TSH, Tg and TgAb were measured after 2 days. RESULTS At follow-up, 90.0% of patients from group A and 85.0% of patients from group B had Tg levels <1 ng/ml; no uptake was observed in 95.2% and in 90.5% of patients from group A or B respectively, with no statistical differences for both ablation criteria. Before (131)I treatment, small thyroid remnants (<1 ml) were detected by US in <25% of all patients. CONCLUSIONS The use of rhTSH for the preparation of low-risk patients to ablation therapy with low activities of (131)I (2 GBq; 54 mCi) is safe and effective and avoids hypothyroidism. The presence of thyroid remnants smaller than 1 ml at US evaluation had no effect on the ablation rate.

Radiopharmaceuticals for the study of inflammatory processes: a review.

Recent advances in our understanding of the pathophysiology of inflammatory processes at the molecular level, combined with progress in radiopharmaceutical sciences, has boosted the development of nuclear medicine techniques for the diagnosis of infection/inflammation. The use of radiolabelled white blood cells has been studied and evaluated in several pathologies and is still the reference method. Several alternative approaches, however, have been developed that may, in the future, improve the specificity and the ease of use of the technique. For the first time, a radiopharmaceutical that may distinguish between sterile and septic inflammation, 99Tcm-Infecton, has been developed. Also, monoclonal antibody fragments, cytokines and a variety of new synthetic peptides that bind specifically to granulocytes have been prepared. Particularly promising appears to be the detection of the expression of adhesion molecules by activated endothelium as a first-line technique for the detection of inflammatory foci. For the diagnosis of autoimmunity and chronic inflammatory processes, important progress has also been made. Autoimmunity can now be studied by in vivo detection of tissue-infiltrating activated lymphocytes by radiolabelled interleukin-2. A radiopharmaceutical for the diagnosis of monocyte infiltration, J001X, is also available, and the commercially available Octreoscan holds promise in autoimmune and chronic inflammatory diseases. The efforts of the scientific community have given us new perspectives in diagnostic nuclear medicine: easier techniques that promise better sensitivity and specificity are now also being tested for the study of new disease conditions. The results of the clinical trials now in progress will determine the future of this challenging and fascinating field and the role of nuclear medicine in the management of patients with infection/inflammation.

A radiopharmaceutical for imaging areas of lymphocytic infiltration : 123I-interleukin-2. Labelling procedure and animal studies

The labelling of interleukin-2 (IL-2) with 123I and its in vivo application for imaging chronic pathological lymphocytic infiltrations are described. The lactoperoxidase/glucoseoxidase technique was the labelling method of choice leading to immunoreactive IL-2 with high specific activity. Labelled IL-2 was injected in diabetes-prone non-obese diabetic (NOD) mice with pancreatic lymphocytic infiltration. As control animals, Balb/c mice were used. As specificity control, monoclonal antibodies AMT13 and UCHT1, bovine serum albumin and alpha-lactalbumin were radioiodinated and injected in mice. Eighteen NOD mice and four control Balb/c mice were used for gamma camera imaging experiments. Fifty-four NOD and 20 Balb/c mice were used for time course single organ counting and autoradiography. Gamma camera images showed that radioactivity accumulated in the pancreatic region from the 10th minute onwards in NOD mice injected with 123I-IL-2 but not in Balb/c mice, or in NOD mice injected with control radiopharmaceuticals. These findings were confirmed by counting the radioactivity present in single organs. Autoradiography of NOD pancreas, after injection of labelled IL-2, showed that radioactivity was specifically associated with infiltrating lymphocytes. In conclusion, this technique is highly specific and easy to perform and we suggest its application in humans for in vivo detection of areas of lymphocytic infiltration.

The development of technetium-99m-labelled interleukin-2: A new radiopharmaceutical for the In vivo detection of mononuclear cell infiltrates in immune-mediated diseases

We describe here a new method for labelling interleukin-2 (IL-2) in high specific activity with 99mTc for in vivo studies in man. Labelling was performed via a two-step reaction using an N3S bifunctional chelating agent. To optimise the reaction, factors affecting the incorporation of 99mTc into the N3S ligand were studied. The conjugation of the preformed N3S chelate ligand to IL-2 was then similarly optimised. Various strategies for purifying the 99mTc-IL-2 were explored including size-exclusion, ion-exchange, and several modes of reversed-phase chromatography. The radiochemical purity of the purified protein was determined by HPLC, ITLC, TCA precipitation, and SDS-PAGE. The receptor binding capacity of 99mTc-IL-2 was studied. Biodistribution studies in normal mice were performed with 99mTc-IL-2 purified using different techniques or labelled after prolonged storage and compared to 125I-IL-2.

Ultrasound-Guided Laser Ablation of Incidental Papillary Thyroid Microcarcinoma: A Potential Therapeutic Approach in Patients at Surgical Risk

BACKGROUND Incidental papillary thyroid microcarcinoma (PTMC), a frequent clinical problem, is usually associated with a favorable outcome. During long-term follow-up, only a minority of cases show aggressive behavior with either lymph node or distant metastases. Recently, we had an opportunity to evaluate the efficacy of nonsurgical, ultrasound (US)-guided percutaneous laser ablation (PLA) for local treatment of PTMC in an otherwise inoperable patient. PATIENT AND METHODS Neck US examination revealed an incidental, solitary, 8 × 7 × 7 mm hypoechoic nodule with microcalcifications of the right thyroid lobe. The patient suffered from decompensated liver cirrhosis, renal failure, and recent surgery followed by external beam radiation therapy for breast cancer. Cytologic diagnosis showed papillary thyroid carcinoma, but the patient declined surgery because of high risk of thyroid surgery. After local anesthesia with 2% xylocaine, PLA was performed according to the previously reported procedure with an Nd:YAG laser. SUMMARY The procedure was well tolerated, without side effects, and the patient required no analgesics. US-guided fine-needle aspiration biopsy and core-needle biopsy were performed at 1 and 12 months after PLA, which demonstrated necrotic material and inflammatory cells with no viable neoplastic cell. At the 24 months US follow-up examination, the area of necrosis further decreased, demonstrating a 4 × 4 mm hypoechoic zone and a small hyperechoic area due to fibrotic changes. A fine-needle aspiration biopsy confirmed the absence of malignant cells. CONCLUSIONS Laser-induced thermal ablation was a safe and effective ablative treatment for a patient with PTMC confined to the thyroid gland who was at high surgical risk. This approach should be considered only in elderly patients and/or in those with comorbidities that might expose the patients to an undue high surgical risk and only after the evaluation by neck US, computed tomography, magnetic resonance imaging, or positron emission tomography/computed tomography rules out lymph-node involvement or metastatic disease.

Imaging active lymphocytic infiltration in coeliac disease with iodine-123-interleukin-2 and the response to diet

Abstract. Coeliac disease is diagnosed by the presence of specific antibodies and a jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Mucosal cell-mediated immune response is considered the central event in the pathogenesis of coeliac disease, and untreated coeliac patients show specific features of T-cell activation in the small intestine. Here we describe the use of iodine-123-interleukin-2 scintigraphy in coeliac patients as a non-invasive tool for detection of lymphocytic infiltration in the small bowel and its use for therapy follow-up, and we demonstrate the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. 123I-IL2 was administered i.v. [74 MBq (2 mCi)], and gamma camera images were acquired after 1 h. Ten patients were studied with 123I-IL2 scintigraphy at diagnosis and seven were also investigated after 12–19 months of gluten-free diet. Results were expressed as target-to-background radioactivity ratios in six different bowel regions before and after the diet. At the time of diagnosis all patients showed a significantly higher bowel uptake of 123I-IL2 than normal subjects (P<0.003 in all regions). A significant correlation was found between jejunal radioactivity and the number of IL2R+ve lymphocytes per millimetre of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2=0.66; P=0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 only binds to activated T-lymphocytes infiltrating the gut mucosa. After 1 year of the diet, bowel uptake of 123I-IL2 significantly decreased in five out of six regions (P<0.03), although two patients still had a positive IL2 scintigraphy in one region. We conclude that 123I-IL2 scintigraphy is a sensitive non-invasive technique for assessing in vivo the presence of activated mononuclear cells in the bowel of patients affected by coeliac 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.

123I-Interleukin-2 Scintigraphy: A New Approach to Assess Disease Activity in Autoimmunity

Several human chronic inflammatory diseases, such as organ specific autoimmune diseases, are characterized by a chronic, slowly progressing, mononuclear cell infiltration of the target organ with little increase of vascular permeability. This infiltration can precede the onset of clinical symptoms by several months or years. Tissue biopsies may not be easily applicable to every organ and may poorly represent the condition of the whole organ particularly in inflammatory bowel diseases and in type 1 diabetes (IDDM). Thus, the possibility to detect the presence and extent of mononuclear cell infiltration in vivo by simple scintigraphy may be of considerable clinical utility for diagnosis and followup of several chronic inflammatory diseases. Immunohistochemical studies of tissue biopsies in several chronic inflammatory diseases have revealed that the target tissue is infiltrated by mononuclear cells (mainly T-lymphocytes) and that 10-50% of these cells express interleukin-2 receptors (IL2R) as a sign of cell activation /1-5Λ We previously described the labeling of IL2 with I and its specificity for the in vivo detection of pancreatic mononuclear cell infiltration in two animal models of autoimmune type 1 diabetes, the Bio Breeding/Worcester (BB/W) rat 161 and the non-obese diabetic (NOD) mouse 111. In this study we describe for the first time the use of I-IL2 scintigraphy for the non-invasive evaluation of disease activity and extent in patients with several different chronic inflammatory diseases.

Detection of insulitis by pancreatic scintigraphy with 99mTc-labeled IL-2 and MRI in patients with LADA (Action LADA 10)

OBJECTIVE Pancreatic scintigraphy with interleukin-2 radiolabeled with 99mTc (99mTc-IL-2) is a technique used to image chronic inflammatory-mediated disorders. We used this method to detect a signal consistent with the presence of insulitis in patients with autoimmune diabetes. Positive and negative controls (patients with pancreatic carcinoma and type 2 diabetes, respectively) also were studied. RESEARCH DESIGN AND METHODS We examined 25 patients with autoimmune diabetes (16 with recently diagnosed type 1 diabetes, 9 with latent autoimmune diabetes in adults [LADA]), 6 with type 2 diabetes, and 7 with pancreatic carcinoma (the latter two groups were used as negative and positive controls, respectively). All patients underwent 99mTc-IL-2 scintigraphy and contrast-enhanced MRI of the pancreas. To validate positive controls, samples were taken from patients with pancreatic carcinoma during surgery for histological and immunohistochemical investigations. RESULTS Pancreatic accumulation of 99mTc-IL-2 was detected in patients with autoimmune diabetes (61% positive) and, notably, in 6 of 9 patients with LADA; semiquantitative evaluation of pancreatic uptake of 99mTc-IL-2 showed higher values in patients with autoimmune diabetes (both childhood and LADA) and pancreatic carcinoma than in those with type 2 diabetes (4.45 ± 1.99, 4.79 ± 1.1, and 4.54 ± 1.62 vs. 2.81 ± 0.63; P = 0.06, P = 0.01, and P = 0.04, respectively). In patients with pancreatic carcinoma, pancreatic interleukin-2 receptor expression correlated with pancreatic 99mTc-IL-2 uptake (r = 0.8; P = 0.01). In patients with LADA, 99mTc-IL-2 uptake inversely correlated with duration of disease (r = 0.7; P = 0.03). CONCLUSIONS Autoimmune diabetes in adults is associated with increased pancreatic 99mTc-IL-2 uptake, indicating the presence of insulitis, particularly within 1 year of the beginning of insulin therapy, similar to type 1 diabetes at diagnosis.

99mTc-EDDA/HYNIC-TOC in the management of medullary thyroid carcinoma

An early diagnosis of distant metastases or local recurrences of medullary thyroid carcinoma (MTC) can be achieved by several conventional radiological modalities (e.g., ultrasonography, computed tomography [CT], and magnetic resonance imaging [MRI] as well as by radioisotopic procedures, such as positron emission tomography (PET), scintigraphy with different types of radiopharmaceuticals, and radiolabeled receptor-ligands in particular. The aim of this study was to evaluate the clinical utility of 99mTc-EDDA/HYNIC-TOC, a new octreotide derivative, to detect recurrences of disease or distant metastases in MTC. Images obtained of 5 patients with high levels of serum calcitonin were compared to findings obtained with other diagnostic procedures: 111In-octreotide, 99mTc-DMSA-V, 18F-flouro-D-deoxyglucose-PET, and CT/MRI. 99mTc-EDDA/HYNIC-TOC was positive in all patients and showed 15 areas of pathological uptake in the cervical and mediastinal regions. 111In-octreotide was positive in 3 of 3 patients and showed 4 areas, compared to 8 of 99mTc-EDDA/HYNIC-TOC. 99mTc-V-DMSA was positive in 3 of 4 patients but showed 6 pathological areas, compared to 13 of 99mTc-EDDA/HYNIC-TOC. 18F-FDG-PET was positive in 5 of 5 patients but showed only 11 areas, compared to 15 of 99mTc-EDDA/HYNIC-TOC. The CT scan was positive in only 2 patients. In conclusion, 99mTc-EDDA/HYNIC-TOC detected more sites of pathological uptake than other modalities, showed better imaging properties than 111In-octreotide, and might be the radiopharmaceutical of choice for providing a rationale for radioisotopic therapy.