An update on non-thyroidal illness syndrome.

The non-thyroidal illness syndrome (NTIS) was first reported in the 1970s as a remarkable ensemble of changes in serum TH (TH) concentrations occurring in probably any severe illness. Ever since, NTIS has remained an intriguing phenomenon not only because of the robustness of the decrease in serum triiodothyronine (T3), but also by its clear correlation with morbidity and mortality. In recent years, it has become clear that (parenteral) feeding in patients with critical illness should be taken into account as a major determinant not only of NTIS but also of clinical outcome. Moreover, both experimental animal and clinical studies have shown that tissue TH concentrations during NTIS do not necessarily reflect serum low TH concentrations and may decrease, remain unaltered, or even increase according to the organ and type of illness studied. These differential changes now have a solid basis in molecular studies on organ-specific TH transporters, receptors and deiodinases. Finally, the role of inflammatory pathways in these non-systemic changes has begun to be clarified. A fascinating role for TH metabolism in innate immune cells, including neutrophils and monocytes/macrophages, was reported in recent years, but there is no evidence at this early stage that this may be a determinant of susceptibility to infections. Although endocrinologists have been tempted to correct NTIS by TH supplementation, there is at present insufficient evidence that this is beneficial. Thus, there is a clear need for adequately powered randomized clinical trials (RCT) with clinically relevant endpoints to fill this knowledge gap.

The Role of Thyroid Hormones in Heart Failure.

Cardiovascular diseases are the leading cause of death worldwide. Heart failure is the terminal manifestation of cardiovascular diseases, and its morbidity and mortality remain high. The prevalence of heart failure with preserved ejection fraction (HFpEF) among heart failure patients remains uncertain. However, recent studies have found that it ranged from 40 to 71%. There is still no effective treatment for HFpEF. Thyroid hormones (TH) have central regulatory actions in the cardiovascular system, particularly in the heart. Changes in plasmatic or tissue thyroid hormone levels are associated with significant alterations in cardiovascular function. A significant proportion of patients with heart failure presents some form of thyroid dysfunction including hypothyroidism, hyperthyroidism, and low T3 syndrome. Furthermore, thyroid hormones can vary at a local level independently of the serum TH levels. This may lead to local cardiac hypothyroidism in heart failure. Based on these findings and the role that TH play in cardiovascular regulation, they were proposed as a potential target for heart failure therapy. Several clinical and experimental studies have shown beneficial effects of TH supplementation. Data from epidemiological studies supports a higher risk of heart failure and a worse prognosis in heart failure patients with low levels of TH. In addition, animal studies and small clinical studies suggest that TH supplementation may improve cardiac function in heart failure. Although further studies are needed to evaluate the safety and efficacy of TH in this context, the available evidence suggests that TH modulation is a promising therapeutic approach to heart failure.

[ABNORMALITIES OF THYROID HORMONE METABOLISM DURING COMMUNITY‒ACQUIRED PNEUMONIA: NONTHYROIDAL ILLNESS SYNDROME AND INTRACELLULAR IODINE DISTRIBUTION IN CHILDREN].

The aim of the study ‒ to investigate the changes of thyroid function and to reveal the relationship in between intracellular distribution of iodine in the blood with the severity of the course of pneumonia in children. We investigated 70 patients in age 6-14 years with moderate and severe CAP and 35 healthy children. The levels of free thyroxine (fT4), free triiodo-thyronine (fT3) and thyroid stimulating hormone, thyroid gland ultrasound and urinary iodine were estimated. Inorganic iodine, total and organificated iodine was investigated. The article presents that severe pneumonia in children is characterized by a transient low level of fT3 2,89 pmol/L (p˂0,05). In a dynamics initially low levels of fT3 raise up to normal data. The general condition and clinical symptoms of the patients was improving after treatment of pneumonia and thyroid status was normalized. Mild iodine deficiency has been established in all children. The intracellular pool of iodine with severe pneumonia showed an inverse relationship between the levels of iodine distribution for organificated and inorganic iodine and a close connection between the levels of total and organic iodine (p<0,001). Nonthyroid illness syndrome developed for patients with severe community-acquired pneumonia. The revealed changes in indices of the intracellular pool of iodine and its distribution in the body are directly proportional to the severity of CAP.

Astrocyte Elevated Gene-1 (AEG-1) Contributes to Non-thyroidal Illness Syndrome (NTIS) Associated with Hepatocellular Carcinoma (HCC).

Non-thyroidal illness syndrome (NTIS), characterized by low serum 3,5,3'-triiodothyronine (T3) with normal l-thyroxine (T4) levels, is associated with malignancy. Decreased activity of type I 5'-deiodinase (DIO1), which converts T4 to T3, contributes to NTIS. T3 binds to thyroid hormone receptor, which heterodimerizes with retinoid X receptor (RXR) and regulates transcription of target genes, such as DIO1. NF-κB activation by inflammatory cytokines inhibits DIO1 expression. The oncogene astrocyte elevated gene-1 (AEG-1) inhibits RXR-dependent transcription and activates NF-κB. Here, we interrogated the role of AEG-1 in NTIS in the context of hepatocellular carcinoma (HCC). T3-mediated gene regulation was analyzed in human HCC cells, with overexpression or knockdown of AEG-1, and primary hepatocytes from AEG-1 transgenic (Alb/AEG-1) and AEG-1 knock-out (AEG-1KO) mice. Serum T3 and T4 levels were checked in Alb/AEG-1 mice and human HCC patients. AEG-1 and DIO1 levels in human HCC samples were analyzed by immunohistochemistry. AEG-1 inhibited T3-mediated gene regulation in human HCC cells and mouse hepatocytes. AEG-1 overexpression repressed and AEG-1 knockdown induced DIO1 expression. An inverse correlation was observed between AEG-1 and DIO1 levels in human HCC patients. Low T3 with normal T4 was observed in the sera of HCC patients and Alb/AEG-1 mice. Inhibition of co-activator recruitment to RXR and activation of NF-κB were identified to play a role in AEG-1-mediated down-regulation of DIO1. AEG-1 thus might play a role in NTIS associated with HCC and other cancers.

Dynamic neuroendocrine changes in critically ill patients with polytrauma.

OBJECTIVE: Acute multiple-trauma induces activation of neuroendocrine system. Nonthyroidal illness syndrome (NTIS) is considered to be associated with adverse outcome in intensive care unit (ICU) patients. This study was aimed to assess dynamic changes of neuroendocrine hormones in patients with polytrauma and their association with the polytrauma score (PTS). METHODS: Blood samples from 24 critically ill patients with polytrauma were obtained on 1st, 2nd, 3rd and 7th day after admission to ICU for analysis of thyroid-stimulating hormone (TSH), total triiodothyronine (T3); free triiodothyronine (fT3), total thyroxine (T4), free thyroxine (fT4), growth hormone (GH), prolactin (PRL) and procalcitonin levels. RESULTS: Acute Physiology and Chronic Health Evaluation (APACHE) II score was 16±5 points on average at the admission to ICU. All patients had normal baseline TSH, T4, fT4, but low T3, and fT3 levels were found in 20% and 33% ICU patients, respectively. On the 7th day after admission to ICU TSH had tendency to increase (p=0.07) and fT4 significantly decreased (p=0.03). The PRL level significantly increased on the 3rd day after admission as compared to 1st day (p=0.04). PTS positively correlated with fT3 (r=0.582, p=0.004) and negatively with fT4 (r=-0.422, p=0.04) at the 1st day in ICU. CONCLUSION: Critical illness in patients with polytrauma leaded to trauma severity-dependent alterations of the thyroid axis response early after injury. Our findings suggest that detection of dynamic hormonal response is more appropriate than single measurement. However supplemental therapy for NTIS should be used after more detailed studies are completed.

Different Hypothalamic Mechanisms Control Decreased Circulating Thyroid Hormone Levels in Infection and Fasting-Induced Non-Thyroidal Illness Syndrome in Male Thyroid Hormone Action Indicator Mice.

Background: Non-Thyroidal Illness Syndrome (NTIS) caused by infection or fasting is hallmarked by reduced circulating thyroid hormone (TH) levels. To better understand the role of local TH-action in the development of NTIS, we assessed tissue-specific changes of TH signaling in Thyroid Hormone Action Indicator (THAI) mice. Methods: NTIS was induced in young adult THAI mice by bacterial lipopolysaccharide (LPS)-administration or by 24 or 48 hours' fasting. Tissue-specific TH-action was assessed by the detection of changes of the Luciferase reporter of THAI mice with quantitative polymerase chain reaction along with tissue-specific examination of regulators of TH metabolism and signaling. Age dependence of revealed alterations of hypothalamic TH-action was also studied in 1-year-old male THAI mice. Results: LPS-treatment increased TH-action in the hypothalamic arcuate nucleus-median eminence (ARC-ME) region preceded by an increase of type 2 deiodinase (D2) expression in the same region and followed by the suppression of proTrh expression in the hypothalamic paraventricular nucleus (PVN). In contrast, LPS decreased both TH-action and D2 activity in the pituitary at both ages. Tshß expression and serum free thyroxine (fT4) and free triiodothyronine (fT3) levels decreased in LPS-treated young adults. Tshß expression and serum fT4 levels were not significantly affected by LPS treatment in aged animals. In contrast to LPS treatment, TH-action remained unchanged in the ARC-ME of 24 and 48 hours fasted animals accompanied with a modest decrease of proTrh expression in the PVN in the 24-hour group. Tshß expression and fT3 level were decreased in both fasted groups, but the fT4 decreased only in the 48 hours fasted animals. Conclusions: Although the hypothalamo-pituitary-thyroid (HPT) axis is inhibited both in LPS and fasting-induced NTIS, LPS achieves this by centrally inducing local hyperthyroidism in the ARC-ME region, while fasting acts without affecting hypothalamic TH signaling. Lack of downregulation of Tshß and fT4 in LPS-treated aged THAI mice suggests age-dependent alterations in the responsiveness of the HPT axis. The LPS-induced tissue-specific hypo-, eu-, and hyperthyroidism in different tissues of the same animal indicate that under certain conditions TH levels alone could be a poor marker of tissue TH signaling. In conclusion, decreased circulating TH levels in these two forms of NTIS are associated with different patterns of hypothalamic TH signaling.

Nutritional state alters the association between free triiodothyronine levels and mortality in hemodialysis patients.

BACKGROUND: Serum free triiodothyronine (fT3) level is suggested to be a risk factor for mortality in unselected dialysis patients. We investigated the prognostic value of serum fT3 levels and also low-T3 syndrome on overall survival in a large cohort of hemodialysis (HD) patients with normal thyroid-stimulating hormone levels. METHODS: A total of 669 prevalent HD patients were enrolled in the study. Serum fT3 level was measured by enzyme immune assay in frozen sera samples at the time of enrollment. Overall mortality was assessed during 48 months of follow-up. RESULTS: Baseline fT3 was 1.47 ± 0.43 (0.01-2.98) pg/ml, and low-T3 syndrome was present in 71.7% of the cases. During a mean follow-up of 34 ± 16 months, 165 (24.7%) patients died. fT3 level was a strong predictor for mortality in crude and adjusted Cox models including albumin or high-sensitivity C-reactive protein (hs-CRP). Further adjustment for both albumin and hs-CRP made the impact of fT3 on mortality disappear. The presence of low-T3 syndrome was associated with mortality in only the unadjusted model. CONCLUSIONS: Low-T3 syndrome is a frequent finding among HD patients, but it does not predict outcome. However, serum fT3 level is a strong and inverse mortality predictor, in part explained by its underlying association with nutritional state and inflammation.

Disorders in the secretory cycle of follicular thyrocytes and their correction with thyrotropic hormone in experimental non-thyroidal illness syndrome.

The influence of LPS on the thyroid gland leads to more intensive synthesis and release of thyroglobulin into the follicle lumen and inhibition of its resorption and proteolysis, which reduces the production of thyroxine. Treatment with thyroid-stimulating hormone normalizing the secretory processes in the follicular thyrocytes is a pathogenetically justified method for correction of non-thyroidal illness syndrome in acute endotoxicosis.

Selenium involvement in mitochondrial function in thyroid disorders.

Selenium (Se), an important oligoelement, is a component of the antioxidant system. Over the last decade, it has been ever more frequently discussed in the context of thyroid disorders. Graves' disease and Hashimoto's thyroiditis, differentiated thyroid cancer, and even endemic goiter may have common triggers that are activated by excess reactive oxygen species (ROS), which are involved in various stages of the pathogenesis of thyroid disorders. Most oxidative events occur in mitochondria, organelles that contain enzymes with Se as a cofactor. Mitochondria are responsible for the production of ATP in the cell and are also a major site of ROS production. Thyroid hormone status (the thyroid being the organ with the highest concentration of Se in the body) has a profound impact on mitochondria biogenesis. In this review, we focus on the role of Se in mitochondrial function in thyroid disorders with impaired oxidative stress, since both thyroid hormone synthesis and thyroid dysfunction involve ROS. The role of Se deficiency or its excess in relation to mitochondrial dysfunction in the context of thyroid disorders is therefore of interest.

Oxidative remote induction of type 3 deiodinase impacts nonthyroidal illness syndrome.

Imbalances in redox status modulate type 3 deiodinase induction in nonthyroidal illness syndrome. However, the underlying mechanisms that lead to D3 dysfunction under redox imbalance are still poorly understood. Here we evaluated D3 induction, redox homeostasis, and their interrelationships in the liver, muscle, and brain in an animal model of NTIS. Male Wistar rats were subjected to left anterior coronary artery occlusion and randomly separated into two groups and treated or not (placebo) with the antioxidant N-acetylcysteine. Sham animals were used as controls. Animals were killed 10 or 28 days post-MI induction and tissues were immediately frozen for biochemical analysis. D3 activity, protein oxidation and antioxidant defenses were measured in liver, muscle, and brain. Compared to those of the sham group, the levels of D3 expression and activity were increased in the liver (P = 0.002), muscle (P = 0.03) and brain (P = 0.01) in the placebo group. All tissues from the placebo animals showed increased carbonyl groups (P < 0.001) and diminished sulfhydryl levels (P < 0.001). Glutathione levels were decreased and glutathione disulfide levels were augmented in all examined tissues. The liver and muscle showed augmented levels of glutathione peroxidase, glutathione reductase and thioredoxin reductase activity (P = 0.001). NAC prevented all the alterations described previously. D3 dysfunction in all tissues correlates with post-MI-induced protein oxidative damage and altered antioxidant defenses. NAC treatment prevents D3 dysfunction, indicating that reversible redox-related remote D3 activation explains, at least in part, the thyroid hormone derangements of NTIS.

Nonthyroidal illness in critically ill children.

PURPOSE OF REVIEW: This review summarizes recent literature on nonthyroidal illness syndrome (NTI) and outcome of pediatric critical illness, to provide insight in pathophysiology and therapeutic implications. RECENT FINDINGS: NTI is typically characterized by lowered triiodothyronine levels without compensatory TSH rise. Although NTI severity is associated with poor outcome of pediatric critical illness, it remains unclear whether this association reflects an adaptive protective response or contributes to poor outcome. Recently, two metabolic interventions that improved outcome also altered NTI in critically ill children. These studies shed new light on the topic, as the results suggested that the peripheral NTI component, with inactivation of thyroid hormone, may represent a beneficial adaptation, whereas the central component, with suppressed TSH-driven thyroid hormone secretion, may be maladaptive. There is currently insufficient evidence for treatment of NTI in children. However, the recent findings raised the hypothesis that reactivation of the central NTI component could offer benefit, which should be tested in RCTs. SUMMARY: NTI in critically ill children can be modified by metabolic interventions. The peripheral, but not the central, component of NTI may be a beneficial adaptive response. These findings open perspectives for the development of novel strategies to improve outcome of critical illness in children.

Metabolic Dysfunction in Continuous-Flow Left Ventricular Assist Devices Patients and Outcomes.

Background Metabolic impairment is common in heart failure patients. Continuous-flow left ventricular assist devices (CF-LVADs) improve hemodynamics and outcomes in patients with advanced heart failure; however, the effect of CF-LVADs on metabolic status is unknown. This study aims to evaluate the changes in metabolic status following CF-LVAD implantation and measure the correlation of metabolic status with outcomes. Methods and Results Prospective data on CF-LVAD patients were obtained. Metabolic evaluation, including hemoglobin A1C, free and total testosterone, thyroid-stimulating hormone (TSH), and free T4, was obtained before and at multiple time points following implantation. Patients with nonelevated thyroid-stimulating hormone and normal hemoglobin A1C and testosterone levels were defined as having normal metabolic status. Baseline characteristics, hemodynamics, and outcomes were collected. One hundred six patients were studied, of which 56 had paired data at baseline and 1- to 3-month follow-up. Before implantation, 75% of patients had insulin resistance, 86% of men and 39% of women had low free testosterone, and 44% of patients had abnormal thyroid function. There was a significant improvement in hemoglobin A1C, free testosterone, and thyroid-stimulating hormone following implantation (P<0.001 for all). Patients with normal hemoglobin A1C (<5.7%) following implantation had higher 1-year survival free of heart failure readmissions (78% versus 23%; P<0.001). Patients with normal metabolic status following implantation also had higher 1-year survival free of heart failure readmissions (92% versus 54%; P=0.04). Conclusions Metabolic dysfunction is highly prevalent in advanced heart failure patients and improves after CF-LVAD implantation. Normal metabolic status is associated with a significantly higher rate of 1-year survival free of heart failure readmissions.

Reawakened interest in type III iodothyronine deiodinase in critical illness and injury.

Thyroid hormones influence gene expression in virtually all vertebrate tissues. Precise regulation of the active endogenous ligand, 3,5,3'-triiodothyronine (T(3)), is achieved by the sequential removal of iodine moieties from the thyroid hormone molecule. Type III iodothyronine deiodinase (D3) is the major inactivating enzyme terminating the action of T(3) and preventing activation of the prohormone, thyroxine (T(4)). Recent studies have revealed the induction of high D3 activity in diverse animal models of tissue injury including starvation, cryolesion, cardiac hypertrophy, infarction, and chronic inflammation. By analyzing serum and tissues taken from hospitalized patients at the time of death, investigators have also documented the robust induction of D3 activity in several human tissues that normally have none, including the liver and skeletal muscle, and shown clinically relevant consequences to systemic thyroid status. These studies reveal a novel role of D3 in the tissue response to injury and in the derangement of thyroid hormone homeostasis commonly observed during critical illness.

Is euthyroid sick syndrome a defensive mechanism against oxidative stress?

The body has a hierarchy of defence strategies to deal with oxidative stress. Among these arrays of defence mechanisms, the over expression and increased activity of glutathione peroxidases has been suggested as the first line of defence. The two main cofactors required for glutathione peroxidase activity are selenium and reduced glutathione. These two factors have been shown to be required for the deiodinase activity also. In vitro and in vivo studies have shown that oxidative stress decreases the activity of deiodinase. Thus, a decrease in deiodinase activity would facilitate the use of these cofactors by glutathione peroxidase in combating oxidative stress. Lowering of serum T3 is generally regarded as a valuable calorie-sparing economy. A decreased metabolic state of the cells as found in euthyroid sick syndrome indicates a decreased free radical generation from the mitochondria. For this reason, euthyroid sick syndrome could be considered as a physiological mechanism activated in response to oxidative stress.

Role of thyroid hormone in skeletal muscle physiology.

Thyroid hormones (TH) are crucial for development, growth, differentiation, metabolism and thermogenesis. Skeletal muscle (SM) contractile function, myogenesis and bioenergetic metabolism are influenced by TH. These effects depend on the presence of the TH transporters MCT8 and MCT10 in the plasma membrane, the expression of TH receptors (THRA or THRB) and hormone availability, which is determined either by the activation of thyroxine (T4) into triiodothyronine (T3) by type 2 iodothyronine deiodinases (D2) or by the inactivation of T4 into reverse T3 by deiodinases type 3 (D3). SM relaxation and contraction rates depend on T3 regulation of myosin expression and energy supplied by substrate oxidation in the mitochondria. The balance between D2 and D3 expression determines TH intracellular levels and thus influences the proliferation and differentiation of satellite cells, indicating an important role of TH in muscle repair and myogenesis. During critical illness, changes in TH levels and in THR and deiodinase expression negatively affect SM function and repair. This review will discuss the influence of TH action on SM contraction, bioenergetics metabolism, myogenesis and repair in health and illness conditions.

A potential role of activated NF-kappa B in the pathogenesis of euthyroid sick syndrome.

Euthyroid sick syndrome, characterized by low serum 3,5, 3'-triiodothyronine (T(3)) with normal L-thyroxine levels, is associated with a wide variety of disorders including sepsis, malignancy, and AIDS. The degree of low T(3) in circulation has been shown to correlate with the severity of the underlying disorders and with the prognosis. Elevated TNF-alpha levels, which accompany severe illness, are associated with decreased activity of type I 5'-deiodinase (5'-DI) in liver, leading us to speculate that high levels of this factor contribute to euthyroid sick syndrome. Here we demonstrate that the activation of NF-kappa B by TNF-alpha interferes with thyroid-hormone action as demonstrated by impairment of T(3)-dependent induction of 5'-DI gene expression in HepG2 cells. Inhibition of NF-kappa B action by a dominant-negative NF-kappa B reversed this effect and allowed T(3) induction of 5'-DI. Furthermore, we show that an inhibitor of NF-kappa B activation, clarithromycin (CAM), can inhibit TNF-alpha-induced activation of NF-kappa B and restore T(3)-dependent induction of 5'-DI mRNA and enzyme activity. These results suggest that NF-kappa B activation by TNF-alpha is involved in the pathogenesis of euthyroid sick syndrome and that CAM could help prevent a decrease in serum T(3) levels and thus ameliorate euthyroid sick syndrome.

The kinetics of thyroid hormone transporters and their role in non-thyroidal illness and starvation.

Kinetic tracer studies show that thyroid hormones are transported into target tissues by stereospecific, high-affinity, low-capacity transporters, both in animals and humans. The K(d) of binding to the transporter varies within the nanomolar range. The different thyroid hormones (T(4), T(3), and rT(3)) are transported via different transporters, except in the pituitary, where they share the same transporter. The molecular mass of the transport proteins varies between 52 and 65kDa. The transport mechanisms are dependent on the energy charge of the cell and -- often -- the sodium gradient over the plasma membrane. A relationship exists with the transport systems of the aromatic amino acids. In non-thyroidal illness and starvation T(4) transport into T(3)-producing tissues is decreased, resulting in a low plasma T(3) concentration, by some considered to be an energy saving mechanism in situations of stress.

Hepatocyte 'priming' and increase in transforming growth factor-beta1 mRNA expression are delayed in hypothyroid versus euthyroid rats during liver regeneration.

Hypothyroidism decreases liver weight and delays the compensatory liver growth after partial hepatectomy (PH) as compared with the euthyroid condition. The aim of this study was to investigate, in hypothyroid rats, the mRNA expression of genes modulating these effects, focusing on c-fos and c-myc, hallmarks of hepatocyte 'priming', and on transforming growth factor-beta1 (TGF-beta1) and its receptor, the transforming growth factor-beta1 receptor-type II (TbetaR-II), negative regulators of liver growth. Euthyroid and hypothyroid male Wistar rats underwent 70% PH and total RNA was isolated from frozen liver samples removed at basal state and during regeneration, 0-144 h after surgery. In this study, we show for the first time that, in the basal liver state, hypothyroidism increased TGF-beta1 and TbetaR-II mRNA levels by 45% and 30%, respectively, as compared with the euthyroid condition and, after PH, resulted in a approximately 12-h delay in the activation of c-fos and c-myc mRNA expression. Moreover, the increase in TGF-beta1 mRNA levels, detected 24-48 h after PH in euthyroid rats, was delayed by 72 h in hypothyroid rats, occurring when a concomitant reduction in TbetaR-II was measured. These results suggest that, in hypothyroid rats, at the basal liver level, the increase in mRNA expression of genes that negatively regulate liver growth might be involved in the decrease in liver weight and that, after PH, the delay of hepatocyte 'priming' and coordinated changes in mRNA expression of negative regulators of liver regeneration might be involved in delaying the regenerative process.

Euthyroid Sick Syndrome.

In this review, we discuss the characteristics, pathophysiology, and therapeutic implications of the euthyroid sick syndrome. Multiple mechanisms have been identified to contribute to the development of euthyroid sick syndrome, including alterations in the iodothyronine deiodinases, thyroid-stimulating hormone secretion, thyroid hormone binding to plasma protein, transport of thyroid hormone in peripheral tissues, and thyroid hormone receptor activity. The euthyroid sick syndrome appears to be a complex mix of physiologic adaptation and pathologic response to acute illness. The underlying cause for these alterations has not yet been elucidated. Treatment of the euthyroid sick syndrome with thyroid hormone to restore normal serum thyroid hormone levels in an effort to improve disease prognosis and outcomes continues to be a focus of many clinical studies, although currently available data do not provide evidence of a clear benefit of treatment.