Correlation between serum anti-TSH receptor autoantibodies (TRAbs) and the clinical feature of Graves' orbitopathy.

BACKGROUND: Graves' orbitopathy (GO) is the most common extrathyroidal manifestation of Graves' disease (GD). Several studies support the involvement of TSH receptor autoantibodies (TRAbs) in the pathogenesis of GO, and a correlation between GO features and TRAbs has been reported, but not confirmed by all studies. Thus, we conducted a cross-sectional investigation to determine whether there is a correlation between TRAbs and the clinical features of GO in an initial phase of the eye disease. METHODS: Ninety consecutive patients with untreated GO (67 women and 23 men, age 48.9 ± 12.6 years) were included. Patients who had received treatments other than anti-thyroid drugs for hyperthyroidism or lubricants for GO were excluded. All patients underwent an endocrinological and ophthalmological evaluation, the latter including exophthalmometry, measurement of eyelid width, clinical activity score (CAS), visual acuity, assessment of diplopia, and NOSPECS score. TRAb levels were measured by a third-generation competitive immunoassay. RESULTS: There was a statistically significant, direct correlation between serum TRAb levels and CAS by linear regression analysis (R = 0.278, P = 0.007). The correlation was confirmed by a multiple regression analysis (R = 0.285; P = 0.006) including age and FT3 levels, which also correlated with CAS. There were no relationships between TRAbs and exophthalmometry, eyelid aperture, degree of diplopia, visual acuity, and NOSPECS score. CONCLUSIONS: The levels of TRAb in subjects with a recent-onset, untreated GO are directly correlated with the clinical activity of the disease, confirming a possible role of these antibodies in the pathogenesis of GO.

Serum antibodies against the insulin-like growth factor-1 receptor (IGF-1R) in Graves' disease and Graves' orbitopathy.

BACKGROUND: A role of the insulin-like growth factor-1 receptor (IGF-1R) in the pathogenesis of Graves' orbitopathy (GO) has been proposed, but the existence and function of anti-IGF-1R-antibodies (IGF-1R-Abs) are debated. METHODS: We designed a cross-sectional investigation to measure serum IGF-1R-Abs by a commercial assay in consecutive patients with Graves' disease (GD) compared with healthy subjects and patients with autoimmune thyroiditis (AT). A total of 134 subjects were screened including 27 healthy subjects, 80 GD patients (54 of whom with GO), and 27 AT patients. The main outcome measure was the prevalence of positive serum IGF-1R-Abs in GO, compared with GD without GO and with the other study groups. RESULTS: Having established a cut-off value at 55.2 ng/ml for positive tests, positive IGF-1R-Abs were more frequent in GD (25%), than in AT (3.7%, P = 0.003) and healthy subjects (0%, P = 0.006). Within GD, there was no difference between patients with or without GO. Serum levels of IGF-1R-Abs differed across the study population (P < 0.0001), reflecting their higher concentrations in GD (P < 0.0001 vs both AT and healthy subjects), but with no difference between patients with or without GO. In patients with GO, there was an inverse correlation between serum IGF-1R-Abs and CAS (R = - 0.376, 95% CI: from - 0.373 to - 0.631; P = 0.005), the significance of which remains to be investigated. CONCLUSIONS: Serum autoantibodies against the IFG-1R are present in one-fourth of GD patients, regardless of the presence of GO. Further functional studies are needed to investigate the significance of their inverse correlation with GO activity.

Rehabilitative orbital decompression for Graves' orbitopathy: results of a randomized clinical trial.

PURPOSE: Orbital decompression (OD) is a consolidated procedure for the treatment of exophthalmos in Graves' orbitopathy (GO). The efficacy of the various procedures remains unclear due to the variability of the techniques used. To address this issue, we performed a randomized clinical trial to compare the efficacy of two surgical techniques. The primary endpoint was the reduction in proptosis. Secondary aims were the risk of post-operative diplopia (POD) in primary gaze and other surgical complications. PATIENTS: 38 patients (76 orbits) affected with GO were enrolled and randomized into single lateral decompression (LD) (n = 19) or balanced medial plus lateral wall decompression (MLD) (n = 19). Following surgery, patients were seen for a follow-up ophthalmological evaluation at 6 months. Pre-operative diplopia in secondary gaze was present in 13/38 patients (34.2%, 8/19 treated with LD and 5/19 treated with MLD). RESULTS: The reduction of exophthalmos was greater in patients treated with MLD (5.1 ± 1.5 mm, range 2-8 mm) than in those treated with LD (3.5 ± 1.3 mm, range 1-6.5 mm) (p = 0.01). The overall incidence of POD in primary gaze was 5/38 (13.2%) and all of these patients had pre-operative diplopia in secondary gaze (5/13, 38.5%, vs patients with no pre-operative diplopia p = 0.005). Two of 19 patients (10.5%) treated with LD and 3/19 (15.8%) treated with MLD, developed POD in primary gaze, with no statistical difference between the two techniques. CONCLUSION: MLD provides a better result in terms of proptosis reduction compared to LD. The two techniques used here appear to have a similar safety profile in terms of POD. Pre-operative diplopia in the secondary gaze remains a major risk factor for development of POD.

Relationship between serum cholesterol and Graves' orbitopathy (GO): a confirmatory study.

BACKGROUND: It has been suggested that high cholesterol represents a risk factor for Graves' orbitopathy (GO). In a recent cross-sectional study, a correlation between cholesterol and the presence of GO was found in patients with a Graves' disease (GD) of recent onset. To confirm this observation, we conducted a retrospective investigation in consecutive patients with GD. The primary outcome was the relationship between the presence of GO and low-density lipoprotein (LDL)-cholesterol. METHODS: The design entailed the inclusion of consecutive patients with a GD of recent onset, with or without GO, who came to our observation to receive radioiodine over a period of 6 months, and a stratification aimed at having two homogeneous group of patients in terms of thyroid function. A total of 86 patients fulfilled the inclusion and evaded the exclusion criteria. All patients underwent an ophthalmological assessment and serum lipids were measured. RESULTS: Serum levels of LDL-cholesterol were significantly higher in patients with GO (135.3 ± 41.3 mg/dL) compared with those without GO (106.6 ± 23.9 mg/dL, P = 0.0007). In a similar manner, serum levels of total cholesterol were higher in patients with GO (211.6 ± 44.0 mg/dL) than in those without GO (176.0 ± 27.2 mg/dL, P = 0.0001). There was no relationship between GO severity and activity and cholesterol. There was no relationship between GO and high-density lipoprotein-cholesterol or triglycerides. CONCLUSIONS: Our study confirms a relationship between the presence of GO and cholesterol in patients with GD of recent onset. Whether lowering of cholesterol ameliorates, GO remains to be established.

Use of low-dose radioiodine ablation for Graves' orbitopathy: results of a pilot, perspective study in a small series of patients.

OBJECTIVE: Elimination of thyroid antigens by total thyroid ablation (TTA), namely, thyroidectomy followed by radioiodine, may be beneficial for Graves' Orbitopathy (GO). TTA is usually performed with a 131I dose of 30 mCi. In Italy, this dose must be followed by a 24-h protected hospitalization, with increase in the waiting lists. In contrast, a 15 mCi dose can be given without hospitalization and with lower costs. Here, we investigated whether a lower dose of radioiodine can be used to ablate thyroid remnants in patients with GO, after thyroidectomy. METHODS: The study was performed in two small groups of consecutive thyroidectomized patients (six patients per group) with Graves' hyperthyroidism and GO. Patients underwent ablation with either 15 or 30 mCi of 131I following treatment with recombinant human TSH (rhTSH). The primary outcome was rhTSH-stimulated serum thyroglobulin (Tg) at 6 months. The secondary outcome was baseline Tg at 6 months. RESULTS: Baseline Tg and rhTSH-stimulated Tg after at 6 months did not differ between two groups, suggesting a similar extent of ablation. rhTSH-stimulated Tg was reduced significantly compared with rhTSH-stimulated Tg at ablation in both groups. GO outcome following treatment with intravenous glucocorticoids did not differ between the two groups. CONCLUSIONS: Our findings may provide a preliminary basis for the use of a 15 mCi dose of radioiodine upon rhTSH stimulation in thyroidectomized patients with Graves' hyperthyroidism and GO.

Statins are not a risk factor for liver damage associated with intravenous glucocorticoid pulse therapy for Graves' orbitopathy.

OBJECTIVE: Acute liver damage (ALD) is associated with high-dose intravenous (iv) glucocorticoid (GC) (ivGC) pulse therapy in ~1 % of patients for Graves' orbitopathy (GO). It has been proposed that statins may increase the risk of ALD. Here we investigated the frequency of ALD according to the assumption of statins in a large retrospective cohort study. METHODS: We studied 1076 consecutive patients with GO given ivGC. ALD was defined as an increase in alanine aminotransferase ≥300 U/l. RESULTS: At the time of ivGC, 62 patients were taking statins and 1014 were not. The frequency of ALD has been reported to be 1.2 cases/100,000 statins users and 1300/100,000 in GO patients given ivGC. Thus, the expected frequency of ALD in patients given both statins and ivGC is 1560/100,000. Transferring these data to our series, one would have expected at least 0.96 cases of ALD (~one case), in the 62 patients given both ivGC and statins. However, no cases of ALD were observed in patients given statins, and the previously reported 14 cases of ALD in this series were seen in patients who were not taking statins. CONCLUSIONS: The lack of observation of cases of ALD in patients given ivGC and statins is quite reassuring. Although caution should be applied to any patient candidate to ivGC treatment and this should be particularly accurate in patients given statins, our findings somehow justify the use of ivGC in patients under statins, although further studies in larger cohorts are needed to confirm our conclusions.

Long-term outcome of Graves' orbitopathy following high-dose intravenous glucocorticoids and orbital radiotherapy.

OBJECTIVE: Intravenous (iv) glucocorticoids (GC) (ivGC) and orbital radiotherapy (ORT) are commonly used in active Graves' orbitopathy (GO), with favorable outcomes in up to 80% of patients. However, little is known on the factors that may affect GO outcome in the long term, an issue that we investigated here. METHODS: We studied retrospectively 96 untreated patients with GO, identified out of 787 consecutive patients who came to our GO Clinic for a follow-up visit between September 2010 and June 2013. After the first observation, patients were treated with ivGC and ORT and were then re-examined after a median period of 55.5 months. The primary end-point was the possible relation between GO outcome and several individual variables. RESULTS: Exophthalmometry, eyelid aperture, CAS, diplopia and visual acuity (the latter only in patients with an initial reduction) improved significantly after treatment. Overall, 67.7% of patients had improved and were considered as responders, whereas the remaining (29.1% stable and 4.5% worsened) were considered as non-responders. Age, smoking, thyroid volume, thyroid treatment, serum anti-TSH receptor autoantibodies and individual GO features at first observation did not affect the outcome of GO, which, in contrast, was affected by gender and by the time elapsed between first and last observation. Thus, the prevalence of responders was higher in females (76.4 vs 48% in males, P = 0.02) and the time elapsed between first and last observation was greater in responders (58 vs 39 months in non-responders, P = 0.02). Whereas the prevalence of responders and non-responders was similar up to 36 months, there was an increase in responders beginning between 37 and 48 months and reaching a peak of ~80% between 61 and 72 months, to plateau thereafter. CONCLUSIONS: Given the limitations of retrospective investigations, our study confirms that the combination of GC and ORT is effective in GO and shows that females have greater chances to respond to treatment. The notorious tendency of GO to improve spontaneously with time most likely contributes the long-term outcome of the eye syndrome.

Thyroglobulin autoantibodies switch to immunoglobulin (Ig)G1 and IgG3 subclasses and preserve their restricted epitope pattern after 131I treatment for Graves' hyperthyroidism: the activity of autoimmune disease influences subclass distribution but not epitope pattern of autoantibodies.

The subclass distribution of thyroglobulin autoantibodies (TgAb) is debated, whereas their epitope pattern is restricted. Radioidine ((131)I) treatment for Graves' disease (GD) induces a rise in TgAb levels, but it is unknown whether it modifies subclass distribution and epitope pattern of TgAb as well. We collected sera from GD patients before (131) I treatment and 3 and 6 months thereafter. We measured total TgAb, TgAb light chains and TgAb subclasses by enzyme-linked immunosorbent assay (ELISA) in 25 patients. We characterized the TgAb epitope pattern in 30 patients by inhibiting their binding to (125-) (I) Tg by a pool of four TgAb-Fab (recognizing Tg epitope regions A, B, C and D) and to Tg in ELISA by each TgAb-Fab. Total TgAb immunoglobulin (Ig)G rose significantly (P = 0.024). TgAb κ chains did not change (P = 0.052), whereas TgAb λ chains increased significantly (P = 0.001) and persistently. We observed a significant rise in IgG1 and IgG3 levels after (131)I (P = 0.008 and P = 0.006, respectively), while IgG2 and IgG4 levels did not change. The rise of IgG1 was persistent, that of IgG3 transient. The levels of inhibition of TgAb binding to Tg by the TgAb-Fab pool were comparable. A slight, non-significant reduction of the inhibition by the immune-dominant TgAb-Fab A was observed 3 and 6 months after (131)I. We conclude that (131)I treatment for GD increases the levels of the complement-activating IgG1 and IgG3 subclasses and does not influence significantly the epitope pattern of TgAb. In autoimmune thyroid disease subclass distribution of autoantibodies is dynamic in spite of a stable epitope pattern.

Genotypes and haplotypes in the 3' untranslated region of the HLA-G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta-thalassemia.

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11.

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.

Neuromyelitis optica spectrum disorder and multiple sclerosis in a Sardinian family.

The coexistence of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) in the same family is a rare event. We report a familial case originating from Sardinia of two siblings: one with NMOSD and one with MS. Human leukocyte antigen (HLA) typing showed that the two affected siblings were HLA-identical, sharing risk-increasing alleles, while a younger unaffected sister was haploidentical to her siblings but she also carried protective alleles. Our findings confirm the role of HLA in raising the risk to develop CNS inflammatory diseases and provide further knowledge on the relationship between NMOSD and MS.

Impact of HLA-G polymorphism on the outcome of allogeneic hematopoietic stem cell transplantation for metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) is particularly sensitive to immune intervention. HLA-G, a non-classical HLA class I molecule with immunomodulatory properties, has been studied with regard to outcome after hematopoietic stem cell transplantation (HSCT), in particular the 14 bp insertion/deletion polymorphism in the 3' untranslated region. Here we analyzed n=56 patients affected by metastatic RCC who received an allogeneic HSCT between 1998 and 2006 in Milano, Marseille, Clermont-Ferrand and Stockholm. The 14 bp polymorphism was analyzed in correlation with overall survival (OS), PFS, acute and chronic GvHD. With a median follow-up of 13 years, a trend towards better outcome was observed when homozygosity for the 14bp-del allele was present: multivariate hazard ratio was 0.50 (95% confidence interval (CI): 0.23-1.13; P=0.10) and 0.57 (95% CI: 0.26-1.26; P=0.17) for OS and PFS, respectively, when 14bp-del/del was compared with 14bp-ins/X. Further exploratory analysis revealed a significant association between T/C at p3003 and improved OS (P=0.05) and PFS (P=0.006) compared with T/T. To our knowledge this is the first study on HLA-G and outcome after HSCT for a solid malignancy. After a coordinated multicenter study, we found that the more tolerogenic polymorphisms (14bp-del/del) is associated with better PFS and OS. The finding on p3003 deserves further investigation.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation.

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65-38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05-0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Functioning and nonfunctioning thyroid adenomas involve different molecular pathogenetic mechanisms.

The molecular biology of follicular cell growth in thyroid nodules is still poorly understood. Because gain-of-function (activating) mutations of the thyroid-stimulating hormone receptor (TShR) and/or Gs alpha genes may confer TSh-independent growth advantage to neoplastic thyroid cells, we searched for somatic mutations of these genes in a series of hyperfunctioning and nonfunctioning follicular thyroid adenomas specifically selected for their homogeneous gross anatomy (single nodule in an otherwise normal thyroid gland). TShR gene mutations were identified by direct sequencing of exons 9 and 10 of the TShR gene in genomic DNA obtained from surgical specimens. Codons 201 and 227 of the Gs alpha gene were also analyzed. At histology, all hyperfunctioning nodules and 13 of 15 nonfunctioning nodules were diagnosed as follicular adenomas. Two nonfunctioning thyroid nodules, although showing a prevalent microfollicular pattern of growth, had histological features indicating malignant transformation (a minimally invasive follicular carcinoma and a focal papillary carcinoma). Activating mutations of the TShR gene were found in 12 of 15 hyperfunctioning follicular thyroid adenomas. In one hyperfunctioning adenoma, which was negative for TShR mutations, a mutation in codon 227 of the Gs alpha gene was identified. At variance with hyperfunctioning thyroid adenomas, no mutation of the TShR or Gs alpha genes was detected in nonfunctioning thyroid nodules. In conclusion, our findings clearly define a different molecular pathogenetic mechanism in hyperfunctioning and nonfunctioning follicular thyroid adenomas. Activation of the cAMP cascade, which leads to proliferation but maintains differentiation of follicular thyroid cells, typically occurs in hyperfunctioning thyroid adenomas. Oncogenes other than the TShR and Gs alpha genes are probably involved in nonfunctioning follicular adenomas.

Comparison of the effectiveness and tolerability of intravenous or oral glucocorticoids associated with orbital radiotherapy in the management of severe Graves' ophthalmopathy: results of a prospective, single-blind, randomized study.

Eighty-two consecutive patients with moderate-to-severe and active Graves' ophthalmopathy were randomly treated with orbital radiotherapy combined with either oral (prednisone; starting dose, 100 mg/d; withdrawal after 5 months) or iv (methylprednisolone; 15 mg/kg for four cycles and then 7.5 mg/kg for four cycles; each cycle consisted of two infusions on alternate days at 2-wk intervals) glucocorticoids. The two groups did not differ for age, gender, duration of hyperthyroidism and ophthalmopathy, prevalence of smokers, thyroid volume, and pretreatment ocular conditions. Both groups of patients received radioiodine therapy shortly before treatment for Graves' ophthalmopathy. Follow-up lasted for 12 months. A significant reduction in proptosis (from 23.2 +/- 3.0 to 21.6 +/- 1.2 mm in the iv glucocorticoid group, P < 0.0001; and from 23 +/- 1.8 to 21.7 +/- 1.8 mm in oral glucocorticoid group, P < 0.0001) and in lid width (from 13.3 +/- 2.5 to 11.8 +/- 2.2 mm, and from 13.6 +/- 2.0 to 11.5 +/- 1.9 mm, respectively; P < 0.001 in both cases) occurred, with no difference between the two groups. Diplopia significantly improved in both groups: it disappeared in 13 of 27 (48.1%) iv glucocorticoid patients (P < 0.005) and in 12 of 33 (36.4%) oral glucocorticoid patients (P < 0.03). The degree of amelioration of diplopia did not significantly differ between the two groups (P = 0.82). Optic neuropathy improved in 11 of 14 iv glucocorticoid (P < 0.01) and only in 3 of 9 oral glucocorticoid (P = 0.57) patients, with no significant difference in these outcomes. The Clinical Activity Score decreased from 4.5 +/- 1.2 to 1.7 +/- 1.0 (P < 0.0001) in the iv glucocorticoid group and from 4.2 +/- 1.1 to 2.2 +/- 1.2 (P < 0.0001) in the oral glucocorticoid group; final Clinical Activity Score was significantly lower in iv glucocorticoid than in oral glucocorticoid patients (P < 0.01). By self-assessment evaluation, 35 (85.3%) iv glucocorticoid and 30 (73.2%) oral glucocorticoid patients reported an improvement of ocular conditions (P = 0.27). Overall, both treatments produced favorable effects in most patients, but responders in the iv glucocorticoid group (36 of 41, 87.8%) were more than in the oral glucocorticoid group (26 of 41, 63.4%) (P < 0.02). Moreover, iv glucocorticoid treatment was better tolerated than oral glucocorticoid treatment. Side effects occurred in 23 (56.1%) iv glucocorticoid and 35 (85.4%) oral glucocorticoid patients (P < 0.01); in particular, cushingoid features developed in 5 of the former and 35 of the latter patients. One iv glucocorticoid patient had severe hepatitis of undetermined origin at the end of glucocorticoid treatment, followed by spontaneous recovery. In conclusion, high-dose iv glucocorticoid and oral glucocorticoid (associated with orbital radiotherapy) are effective in the management of severe Graves' ophthalmopathy, but the iv route seems to be more effective and better tolerated than the oral route and associated with a lower rate of side effects.

Activating thyrotropin receptor mutations in histologically heterogeneous hyperfunctioning nodules of multinodular goiter.

Activating thyrotropin (TSH) receptor mutations have been found in toxic adenomas and in hot nodules contained in toxic multinodular goiter. The typical feature of multinodular goiter is the heterogeneity in morphology and function of different follicles within the same enlarged gland. In this report we describe a patient with a huge multinodular goiter, normal free triiodothyronine (FT3) and free thyroxine (FT4) serum values, and subnormal TSH serum concentration. Thyroid scintiscan showed two hot areas corresponding to the basal and apical nodules of the left lobe. The right lobe was poorly visualized by the radioisotope. The patient underwent thyroidectomy, and histological examination of the tissue was performed. Genomic DNA was extracted from the tissue specimen and direct sequencing of the TSH receptor and Gs alpha genes was done. At histology, one hyperfunctioning nodule had the typical microscopic structure of thyroid adenomas, and the other contained multiple macrofollicular areas not confined by a capsule. In spite of this histological difference, both hyperfunctioning nodules harbored a mutation of the thyrotropin receptor (TSHr) gene: an isoleucine instead of a threonine in position 632 (T632I) in the first nodule and a methionine instead of an isoleucine in position 486 (I486M) in the second nodule. In conclusion, our findings show for the first time that gain-of-function TSHr mutations are not only present in hyperfunctioning thyroid nodules with the histological features of the true thyroid adenomas, but also in hyperfunctioning hyperplastic nodules contained in the same multinodular goiter.

Identification of thyroglobulin in orbital tissues of patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is thought to have an autoimmune pathogenesis because of its association with autoimmune thyroid disease, in particular with Graves' disease. Nevertheless, the nature of the autoimmune reaction is unclear, and a target orbital autoantigen has not been conclusively identified. A widely discussed hypothesis is that antigens constitutively shared by the thyroid and orbital tissues are targets of an autoimmune reaction. It has been also postulated that a thyroid-soluble antigen, namely thyroglobulin (Tg), is transported to orbital tissues through the lymphatics, where it accumulates and elicits autoimmune damages in susceptible individuals. Here we have investigated whether Tg is present in orbital tissues from patients with TAO. Retrobulbar tissue specimens were obtained from three patients with Graves' disease and TAO who underwent decompressive orbitotomy, and at autopsy from two patients with no thyroid or eye disease. All patients with TAO had been previously treated with radioiodine to control Graves' hyperthyroidism. Western blot analysis with a monoclonal anti-Tg antibody showed the presence of intact Tg, both in soluble and membrane-associated fractions of orbital tissue extracts from the patients with TAO, in amounts estimated to range from approximately 320 to approximately 900 pg/microg of tissue protein. In contrast, Tg was not detected in orbital tissue extracts from patients with no thyroid or eye disease. Tg was also demonstrated in orbital tissue extracts from two of three patients with TAO by enzyme-linked immunosorbent assay (ELISA), in amounts estimated to range from approximately 450 to approximately 1000 pg/microg of protein. In addition, Tg in orbital tissue extracts from patients with TAO was immunoprecipitated by a rabbit anti-Tg antibody, suggesting that it retained its native conformation. An anti-thyroxine (T4) antibody captured in solid-phase Tg from orbital tissue extracts, showing that it contained thyroid hormone residues and had therefore originated in the thyroid. Tg-anti-Tg immune complexes were not found in orbital tissues, suggesting that if an autoimmune reaction to Tg occurs in TAO, it is likely to be cell mediated.

The conundrum of HLA-DRB1*14:01/*14:54 and HLA-DRB3*02:01/*02:02 mismatches in unrelated hematopoietic SCT.

Uncertainty still exists on the role of polymorphisms outside the HLA-DRB1 binding site or inside the HLA-DRB3 binding groove in unrelated hematopoietic SCT (HSCT). The ideal model to solve the conundrum consists of the transplants mismatched for HLA-DRB1*14:01/*14:54 and/or for HLA-DRB3*02:01/*02:02. A task force was set up in Italy to recruit transplanted pairs defined as HLA-DRB1*14:01 before 2006, the year crucial for the proper definition of the HLA-DRB1*14:54 allele in molecular biology. Out of 2723 unrelated pairs, 189 transplanted in Italy from 1995 to 2006 were HLA-DRB1*14:01 positive; 103/189 pairs with good historical DNA were retyped for HLA-DRB1*14 and HLA-DRB3 at-high resolution level; 31/103 pairs had HLA-DRB1*14 and/or HLA-DRB3 mismatched; 99/103, having complete clinical data, underwent statistical analysis for OS, TRM, disease-free survival and acute and chronic GvHD. No significant involvement of HLA-DRB1*14:01/*14:54 or HLA-DRB3*02:01/*02:02 mismatches was found, either alone or combined. Our findings suggest that disparities at exon 3 of the HLA-DRB1 gene seem unlikely to influence the outcome after HSCT. The same may be envisaged for HLA-DRB3(*)02:01 and (*)02:02 alleles which, although differing in the Ag binding site, seem unable to modulate an appreciable immune response in an HSCT setting.