The size of the community rather than its geographical location better defines the risk of iodine deficiency: results of an extensive survey in Southern Italy.

AIM: The objective of this study was to establish the status of iodine nutrition in Southern Italy. MATERIAL AND METHODS: The survey was carried out on 11-14 yr old children attending primary school and living in urban and non urban areas of 8 regions of Southern Italy. Urinary iodine excretion (UIE) was measured in 23,103 urinary samples randomly collected. RESULTS: Median UIE in the whole studied population was 74 µg/l [interquartile range (IR) 34-139 µg/l]. UIE was significantly higher in chief towns compared to non chief towns (81 µg/l, IR 39-145 µg/l vs 73 µg/l, IR 33-138 µg/l, p<0.0001) and in areas with >500 inhabitants per km² (median 87 µg/l, IR 43-154 µg/l) compared to areas with 100-500 per km² (median 66 µg/l, IR 29-126 µg/l, p<0.0001) and with <100 per km² (median 61 µg/l, IR 25-121 µg/l, p<0.0001). Median UIE was significantly lower in inland mountainous/hilly areas (68 µg/l, IR 30-129 µg/l) compared to coastal mountainous/hilly areas (79 µg/l, IR 37-144 µg/l, p<0.0001) and lowland (79 µg/l, IR 37-146 µg/l, p<0.0001). According to a binary logistic regression model, population density was the only independent parameter significantly associated with UIE ≥ 100 µg/l. CONCLUSION: The results of the present survey indicate that: 1) in Southern Italy mild to moderate iodine deficiency is still present; 2) median UIE in non urban areas is lower than in urban areas and is related to the size of the community rather than to its geographical location, being higher in a larger community. This may be due to better diversification of dietary habits and the easier availability of iodized salt and processed food through commercial facilities, more common in larger communities. Future monitoring surveys should take into account these observations.

The effect of voluntary iodine prophylaxis in a small rural community: the Pescopagano survey 15 years later.

CONTEXT: Iodine deficiency disorders are a major public health problem, and programs have been implemented to improve iodine nutrition. OBJECTIVE: The objective of the study was to verify the effects of voluntary iodine prophylaxis in a small rural community (Pescopagano, Italy). DESIGN: The design of the study was the evaluation of the prevalence of thyroid disorders 15 years after a previous survey conducted before iodine prophylaxis. SETTING: The setting for this study was a general community survey. PARTICIPANTS: One thousand one hundred forty-eight residents were examined in 2010 and 1411 in 1995. RESULTS: In 2010, 757 of 1148 subjects (65.9%) routinely used iodized salt, urinary iodine excretion being significantly higher than in 1955 (median 98.0 µg/L, vs 55.0 µg/L, P < .0001). The prevalence of goiter was lower in 2010 than in 1995 (25.8% vs 46.1%, P < .0001), mainly due to the reduction of diffuse goiter (10.3% vs 34.0%, P < .0001). In 2010 vs 1995, thyroid autonomy in subjects younger than 45 years old (3 of 579, 0.5% vs 25 of 1010, 2.5% P = .004) and nonautoimmune hyperthyroidism in subjects older than 45 years old (8 of 569, 1.4% vs 18 of 401, 4.5%, P = .03) were less frequent. The prevalence of hypothyroidism was higher in 2010 vs 1995 (5.0% vs 2.8%, P = .005), mainly because of an increased frequency of subclinical hypothyroidism in subjects younger than 15 years old (7 of 83, 8.4% vs 0 of 419, 0.0%, P < .0001). Accordingly, serum thyroid autoantibodies (19.5% vs 12.6%; P < .0001) and Hashimoto's thyroiditis (14.5% vs 3.5%; P < .0001) were more frequent in 2010 than in 1995. CONCLUSIONS: In the present work, the role of voluntary iodine prophylaxis was assessed in a small rural community relatively segregated, in which genetic and other environmental factors have not substantially changed between the 2 surveys. Iodine intake strongly affected the pattern of thyroid diseases, but the benefits of correcting iodine deficiency (decreased prevalence of goiter and thyroid autonomy in younger subjects and reduced frequency of nonautoimmune hyperthyroidism in older subjects) far outweighs the risk of development of thyroid autoimmunity and mild hypothyroidism in youngsters.

Modified-release recombinant human TSH (MRrhTSH) augments the effect of (131)I therapy in benign multinodular goiter: results from a multicenter international, randomized, placebo-controlled study.

BACKGROUND: Recombinant human TSH (rhTSH) can be used to enhance (131)I therapy for shrinkage of multinodular goiter (MG). OBJECTIVE, DESIGN, AND SETTING: The objective of the study was to compare the efficacy and safety of 0.01 and 0.03 mg modified-release (MR) rhTSH as an adjuvant to (131)I therapy, vs. (131)I alone, in a randomized, placebo-controlled, international, multicenter study. PATIENTS AND INTERVENTION: Ninety-five patients (57.2 ± 9.6 yr old, 85% females, 83% Caucasians) with MG (median size 96.0, range 31.9-242.2 ml) were randomized to receive placebo (group A, n = 32), MRrhTSH 0.01 mg (group B, n = 30), or MRrhTSH 0.03 mg (group C, n = 33) 24 h before a calculated activity of (131)I. MAIN OUTCOME MEASURES: The primary end point was a change in thyroid volume (by computerized tomography scan, at 6 months). Secondary end points were the smallest cross-sectional area of the trachea; thyroid function tests; Thyroid Quality of Life Questionnaire; electrocardiogram; and hyperthyroid symptom scale. RESULTS: Thyroid volume decreased significantly in all groups. The reduction was comparable in groups A and B (23.1 ± 8.8 and 23.3 ± 16.5%, respectively; P = 0.95). In group C, the reduction (32.9 ± 20.7%) was more pronounced than in groups A (P = 0.03) and B. The smallest cross-sectional area of the trachea increased in all groups: 3.8 ± 2.9% in A, 4.8 ± 3.3% in B, and 10.2 ± 33.2% in C, with no significant difference among the groups. Goiter-related symptoms were effectively reduced and there were no major safety concerns. CONCLUSION: In this dose-selection study, 0.03 mg MRrhTSH was the most efficacious dose as an adjuvant to (131)I therapy of MG. It was well tolerated and significantly augmented the effect of (131)I therapy in the short term. Larger studies with long-term follow-up are warranted.

Early abnormalities of left ventricular myocardial characteristics associated with subclinical hyperthyroidism.

The aim of the present study was to analyze heart function in subclinical hyperthyroidism (sHT) in otherwise healthy subjects by new methods using intramyocardial ultrasonic techniques. Twenty-four newly diagnosed and untreated sHT patients (20 women, 4 men; mean age: 42+/-4 yr) and 24 sex- and age-matched healthy volunteers were studied. All subjects were submitted to conventional 2D color-Doppler echocardiography, pulsed wave tissue Doppler imaging (PWTDI) for the analysis of diastolic function, color Doppler myocardial imaging (CDMI) for the analysis of regional strain and strain rate (SR) expression of regional myocardial deformability, and to integrated backscatter (IBS) for the evaluation of intrinsic contractility and tissue characterization. Regional myocardial systolic strain findings were significantly higher in sHT patients when compared with controls (p<0.001). Considering diastolic SR, the early phase of diastolic SR was compromised in sHT subjects as compared with controls (p<0.001). Cyclic variation index (CVI), expression of intrinsic contractility, was significantly higher in sHT subjects in comparison with controls (p<0.0001). IBS values were comparable between the 2 study groups. In conclusion, the present study suggests that in patients with sHT early systolic hyperdeformability and hypercontractility are present, together with impairment of both active and passive phases of diastole. On the contrary, no left ventricular hypertrophy or other structural alterations are documented.

The spectrum of thyroid disorders in an iodine-deficient community: the Pescopagano survey.

We carefully assessed thyroid status and goiter by ultrasound in 1411 subjects virtually representing the entire resident population of Pescopagano, an iodine-deficient village of Southern Italy. Median urinary iodine excretion was 55 microg/L. The prevalence of goiter was 16.0% in children and 59.8% in adults. Thyroid nodularity was 0.5% in children and progressively increased with age to 28.5% in the 56- to 65-yr-old group. The prevalence of present or past hyperthyroidism was 2.9%, including 9 cases with toxic diffuse goiter and 20 with toxic nodular goiter. Functional autonomy was rare in children, progressively increased with age up to 15.4% in the elderly, and was related to nodular goiter. The prevalences of overt and subclinical hypothyroidism in the adults were 0.2% and 3.8%, respectively. Serum autoantibodies to thyroglobulin and thyroperoxidase were detected in 12.6% of the entire population. The prevalence of diffuse autoimmune thyroiditis was 3.5%, being very low in children. Thyroid cancer was found in only 1 case. In conclusion, in the present survey of an iodine-deficient community, a progressive increase with age of goiter prevalence, thyroid nodularity, and functional autonomy was observed. Hyperthyroidism was twice as high as that reported in iodine-sufficient areas, mainly due to an increased frequency of toxic nodular goiter. Although low titer serum thyroid antibodies were relatively frequent, the prevalences of both overt and subclinical autoimmune hypothyroidism were not different from those observed in iodine-sufficient areas.

[Epidemiology of endemic goiter in Italy]. / Epidemiologia del gozzo endemico in Italia.

Epidemiological surveys on goiter endemia carried out in Italy in the two last decades were recently reviewed. Iodine deficiency and endemic goiter were documented in the entire country. In recent studies, urinary iodine excretion was higher and prevalence of goiter was lower than that documented in the past, in absence of a national program of prophylaxis. Nevertheless, in a recent study carried out in Basilicata, in schoolchildren a goiter prevalence of 16% was documented. In conclusion, iodine deficiency and endemic goiter are still present in Italy; despite the beneficial effects of "silent prophylaxis", a iodine prophylaxis program is mandatory.

[Iodine prophylaxis legislation in Italy]. / Legislazione sulla iodoprofilassi in Italia.

Italian laws allow production and selling of iodized salt but this is still consumed on a voluntary basis. Thus, the use of iodized salt amounts to 3% of the used salt. Selling iodized salt instead of common salt would be the most effective iodine prophylaxis method. The Consiglio Superiore di Sanità has recently voted a document which asks the authorities to make the selling of iodized salt compulsory. Selling common salt to the people who explicitly ask for it could be a good alternative to the compulsory prophylaxis. The food education campaign promoted by the Italian Ministry of Health could provide an important contribution for the success of the iodine prophylaxis.

[Epidemiology of endemic goiter in Tuscany]. / Epidemiologia del gozzo endemico in Toscana.

In the present study, the data of epidemiological surveys carried out in Tuscany from 1981 to 1993 in schoolchildren population (6-14 years) residing in extraurban hilly and mountain areas are reported. Mild iodine deficiency and high prevalence of goiter were documented in the epidemiological surveys carried out up to 1990. An increase in urinary iodine excretion with a dramatic decrease in goiter prevalence was documented in the more recent surveys, independently from iodine prophylaxis. The results observed in Garfagnana and in Val Tiberina after the implementation of iodized salt confirmed the necessity to promote prophylaxis programs extended to the entire population.

[Effects of iodine prophylaxis on a voluntary basis on the thyroid volume of a young population in Val Tiberina]. / Effetti della iodoprofilassi su base volontaria sul volume tiroideo della popolazione giovanile in Val Tiberina.

In the present study, the thyroid volume of children residing in an area (Val Tiberina in Tuscany) characterized by moderate iodine deficiency in the past, was evaluated after iodized salt prophylaxis. In children born before the institution of iodine prophylaxis, thyroid volume was significantly higher than that in controls, both considering the entire population and only the nongoitrous children. In children born after iodine prophylaxis, no difference in thyroid volume was found with respect to controls. The data of the present study indicate that the exposure to iodine deficiency causes an enlargement of thyroid volume in schoolchildren. The iodized salt prophylaxis is able to prevent the development of goiter in the children born after prophylaxis and to keep the further increase of thyroid volume in older children.

Effect of iodized salt on thyroid volume of children living in an area previously characterized by moderate iodine deficiency.

It is well established that an adequate iodine intake prevents iodine deficiency disorders. Prophylaxis through iodized salt is able to correct urinary iodine deficiency and to prevent goiter endemia, but scanty data are available about its effect on decreasing the thyroid size in goitrous children born before prophylaxis. The prevalence of goiter was evaluated by ultrasound in the school-children population of an area of Eastern Tuscany (Tiberina Valley) characterized by moderate iodine deficiency in 1985. At present, after the implementation of voluntary iodized salt consumption, iodine urinary excretion was borderline sufficient (median, 98 micrograms/L). Goiter prevalence was higher at ultrasound (17%) than by palpation (10%). The median thyroid volume ranged from 3.1 mL in 7-yr-old children to 9.2 mL in 14-yr-old children. In the 7-10 yr age class (i.e. in children born after iodine prophylaxis), no statistical difference in thyroid volume was found with respect to controls. In older children (11-14 yr) born before the institution of iodine prophylaxis, the median thyroid volume was significantly higher than that in age-matched controls. Moreover, in this cluster of subjects, the median thyroid volume in nongoitrous children was higher than that in controls. In conclusion, the data of the present study indicate that the iodized salt prophylaxis is able to prevent the development of goiter in children born after the implementation of iodized salt consumption and to further control thyroid enlargement in older children, but is less effective (or rapid) in reducing goiter size in children exposed to iodine deficiency in the first years of life.

[Multinodular goiter. Epidemiology and prevention]. / Gozzo multinodulare. Epidemiologia e prevenzione.

Nodular goiter is the natural evolution of nontoxic goiter, that may be endemic, sporadic or familiar. Iodine deficiency is the cause of endemic goiter, while genetical defects, impairing the thyroid hormone biosynthetic efficiency or altering the number and/or activity of growth factor receptors, play the major role in the pathogenesis of sporadic and familiar nontoxic goiter. The prevalence of nodular goiter is directly related to the degree of iodine deficiency that is still present in several areas of the world. In iodine deficient areas such as some Italian regions, nodular goiter is present in 25-33% of the population, its frequency increasing with age. In iodine sufficient areas the prevalence of nodular goiter is comprised between 0.4 and 7.2% high in iodine deficient areas and about 4% in iodine sufficient countries, its frequency increasing with the age. Dysphagia, dyspnea and coarsening of the voice may occur for esophagous, tracheal or laryngeal nerve compression, respectively. Iodine deficiency has little if any effect on the frequency of thyroid carcinoma, while, with regard to the histological pattern, it leads to an increased ratio papillary/follicular. Thyroid function is normal in uncomplicated nontoxic goiter. However, the evolution of nodular goiter is toward the functional autonomy of nodules that may result in thyrotoxicosis. Hypothyroidism is rare and is usually the result of thyroid autoimmunity. All the cases due to iodine deficiency can be prevented by an adequate iodine prophylaxis that can be accomplished in industrialized countries by the use of sale enriched in iodine.

Mild iodine deficiency during fetal/neonatal life and neuropsychological impairment in Tuscany.

The neuropsychological performance of schoolchildren living in areas with present and past iodine deficiency in Tuscany was investigated. Children were submitted to: a) block design subtest of the Wechsler Intelligence Scale for Children-Revised (WISC-R) and a modified version of the WISC-R coding subtest which evaluate the general neuropsychological and cognitive performance, independently from familial cultural background; b) simple reaction time (RT) session which evaluates the efficiency of the whole information processing and nervous transmission mechanisms. Neuropsychological performance was tested in 107 children living in Borgo a Mozzano, an area of mild iodine deficiency (IDA) with a median urinary iodine excretion (UIE) of 64 micrograms/L (mean +/- SD: 80.1 +/- 57). One hundred and six sex and age-matched children living in Marina di Pisa, an iodine sufficient coastal village of Tuscany (ISA) with a median UIE of 142 micrograms/L (mean +/- SD: 173 +/- 95) were used as controls. Tests for neuropsychological performance were performed in 57 children living in the village of Vagli, an area with past iodine deficiency (PIDA): 30 children born before iodine prophylaxis (Group 1), when the median UIE was 32 micrograms/L (mean +/- SD: 47 +/- 22), 27 children born after the institution of iodine prophylaxis (Group 2), when the median UIE was 109 micrograms/L (mean +/- SD: 130 +/- 73). Sex and age-matched ISA-children were used as controls for each group. RTs were significantly delayed (p < 0.05) in IDA than in ISA children, while block design and coding subtests showed no significant difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid volume measurement by ultrasound in children as a tool for the assessment of mild iodine deficiency.

Thyroid ultrasound was used to measure thyroid volume in children and compared with thyroid palpation for the assessment of the prevalence of goiter in an area of mild iodine deficiency. School children, 6-14 yr old, were from control areas (n = 2693; urinary iodine excretion, 110 micrograms/L) or from an area of mild iodine deficiency (IDA; n = 278; urinary iodine excretion, 72 micrograms/L). Thyroid volume determined by ultrasound in control children increased with age (r = 0.62; P < 0.0001) and was significantly correlated with height (r = 0.51; P < 0.0001) and body weight (r = 0.126; P < 0.0001). Both median and mean thyroid volumes were greater in IDA children than in controls. The prevalence of goiter determined by ultrasound was 68 of 268 children (25.3%) in IDA and 105 of 2693 children (3.9%) in the control area (chi 2 = 204; P < 0.0001). Thyroid enlargement, as assessed by palpation, was found in 59 of 268 children (22%) in the IDA group and in 165 of 2693 (6.1%) subjects in the control area (chi 2 = 88; P < 0.0001). Some subjects of the IDA who were judged goitrous by palpation (11.2%) had a normal thyroid volume at ultrasound, and 12.7% of subjects with an abnormal thyroid volume at ultrasound were judged nongoitrous by palpation. In conclusion, 1) thyroid volume in children, as assessed by ultrasound, increases with age and is closely related to the parameters of body growth; 2) in every age group, thyroid ultrasound shows greater thyroid volume in an IDA group than in controls; and 3) a discrepancy between palpation and ultrasound is found in 23.9% of children living in an IDA, confirming that palpation is relatively inaccurate for assessing the prevalence of goiter in mild iodine deficiency. These data indicate that thyroid volume measurement by ultrasound in children provides a useful tool for the assessment of goiter in mild iodine deficiency.

Iodized salt prophylaxis of endemic goiter: an experience in Toscana (Italy).

It is well established that iodine supplementation is effective in correcting iodine deficiency and reducing goiter prevalence. In Italy, legislation has allowed the production of iodized salt since 1972, but its consumption is on a voluntary basis. In the present study, the efficacy of legislative measures that made compulsory the availability of iodized salt in foodstores has been evaluated. Urinary iodine excretion and thyroid size, scored according to Pan American Health Organization recommendations, were determined prior to (1981) and 10 years after (1991) the introduction of legislative measures in the whole schoolchildren population residing in a restricted area of the Tuscan Appennines. Moreover, in 1991, thyroid volume was determined by ultrasonography. In 1981, mean urinary iodine excretion was 47.1 +/- 22.4 mg/kg creatinine (0.412 mumol/l) and goiter prevalence was 60%, indicating a moderate iodine deficiency. Eighty of the families subsequently used iodized salt on a regular basis; as a result of this excellent compliance, in 1991 the mean urinary iodine excretion increased to 129.7 +/- 73 mg/kg creatinine (1.24 mumol/l) and goiter prevalence dropped to 8.1%. The results of this study underline the effectiveness of iodine prophylaxis in correcting iodine deficiency and abating endemic goiter in schoolchildren, and suggest that implementation of measures that make compulsory the availability of iodized salt in foodstores overcomes the fact that there is no law governing the exclusive production and trading of iodized salt.

Mild iodine deficiency in fetal/neonatal life and neuropsychological performances.

The effects of mild/moderate iodine deficiency during the fetal/neonatal life on neuropsychological performances are still poorly defined. In this research we analyzed some parameters of cognitive performance with sensitive psychometric tests in children living in an area with moderate iodine deficiency. Each subject was submitted to the following neuropsychological tests: 1) a reaction time (RT) session, 2) the block design subtest of the WISC-R, 3) the coding subtest of the WISC-R. No significant difference was found between children with mild iodine deficiency and controls, for block design and coding tests. On the other hand controls had significantly faster RTs. Our data show that exposure to mild iodine deficiency in fetal/neonatal life does not affect general cognitive performance but influences the velocity of motor response to visual stimuli, which may be due to an alteration of the efficiency of the information processing mechanism.

Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver.

The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.

Evaluation of thyroid function in patients with rapid-cycling and non-rapid-cycling bipolar disorder.

The problem of whether rapid-cycling (RC) bipolar disorder is more frequently associated than non-rapid-cycling (NRC) bipolar disorders with thyroid dysfunction was investigated in two groups of 11 women matched for age and therapy. Seven patients in each group were under chronic lithium therapy. Both RC and NRC patients, as compared to euthyroid controls, showed a reduction in mean total and free thyroid hormone concentrations, subnormal values of free thyroxine being found in four RC and three NRC patients. No patient had supranormal baseline thyroid stimulating hormone (TSH) values, but an exaggerated TSH response to thyrotropin releasing hormone was found in three RC and two NRC patients: all these patients had been receiving lithium therapy for more than one year. No differences in the prevalence of goiter and thyroid-directed autoantibodies were observed in the two groups. These data confirm that bipolar disorder, especially during treatment with lithium, is associated with at least subclinical hypothyroidism, and suggest that RC patients do not differ from NRC patients in the prevalence of spontaneous or lithium-induced thyroid hypofunction. Lithium-induced hypothyroidism is likely to be related to the length of treatment.

Lack of nocturnal serum thyrotropin (TSH) surge in patients with chronic renal failure undergoing regular maintenance hemofiltration: a case of central hypothyroidism.

Thyrotropin (TSH) secretion was evaluated in a group of patients with chronic renal failure (CRF) undergoing regular maintenance hemofiltration and in normal controls. The study group included 68 patients (39 males and 29 females, age range 39-73 years, mean: 53 years). In all patients blood was drawn at 08:30-09:00 h; in 20 patients the nocturnal (24:00-02:00 h) serum TSH peak was also evaluated; 12 patients underwent stimulation test with synthetic TSH-releasing hormone (TRH). TSH was measured by an ultrasensitive immunoradiometric assay. CRF patients showed a significant decrease in serum total and free thyroxine and triiodothyronine concentrations, which in a substantial proportion of subjects were below the lower normal limit. Serum reverse triiodothyronine and thyroxine-binding globulin values did not differ in the two groups. Despite this trend of thyroid hormones to decrease, no patient had supranormal TSH values as in primary hypothyroidism. While the mean morning TSH concentrations of CRF patients did not differ from those of controls, the mean nocturnal values were significantly reduced in CRF (1.0 +/- 0.2 vs 3.2 +/- 0.4 mU/l, p less than 0.0005) and the nocturnal serum TSH surge was not observed in 18 of the 20 patients (90%) in whom it was evaluated. The mean serum TSH peak value after TSH-releasing hormone (TRH) administration was also reduced in CRF patients, and the TSH response to TRH was blunted in 3 out of 12 patients (25%). The results of this study demonstrate a major impairment of TSH secretion in CRF, which baseline TSH measurements in the morning and the evaluation of the TSH response to TRH may not reveal.(ABSTRACT TRUNCATED AT 250 WORDS)