The k statistic was used to determine the level of agreement in diagnoses made by ABP and HBP.

Multivariable linear regression models were applied to investigate independent predictors of the ABP-HBP difference.

Binary logistic regression models were used to identify independent factors associated with WCP and MH diagnosed by HBP or ABP monitoring.

Covariates included in the models were age, gender, body mass index, systolic/diastolic clinic BP, sex, number of HBP and awake ABP readings, cardiovascular disease, diabetes, smoking, alcohol intake and treatment for hypertension.

Current smoking was defined as daily smoking or smoking cessation for o1 year and alcohol intake as consumption of any alcohol amount per week.

A probability value Po0.05 was considered statistically significant.

Analysis was performed using the PASW 18 (SPSS release 18.0; SPSS Inc., Chicago, IL, USA).


A total of 710 cases were reviewed for inclusion in the analysis and 97 (13.7%) were excluded.

Forty-eight cases were excluded due to inadequate HBP and/or ABP readings, 34 because evaluation has been performed more than once (first assessment was used), five due to treatment change during the study, five due to acute disease during the study and five due to other reasons.

A total of 613 subjects were finally included in the analysis.

Participants' characteristics are pre- sented in Table 1, for the total study population and separately for untreated and treated subjects.


In Figure 1, participants were classified according to the diagnosis of normotension (clinic and out-of-clinic), sustained hypertension (clinic and out-of-clinic), white coat and MH phenomena by HBP and awake ABP method, respectively, for the total population (1a) and in treated (1b) and untreated subjects (1c).


Sustained hypertension was diagnosed in 50% by either ABP or HBP (diagnostic agreement 89%, k¼0.79), WCP in 14 and 15%, respectively (agreement 89%, k¼0.56) and MH in 16 and 15% (agreement 88%; k¼0.52) (Figure 1; Table 3).

MH tended to be more common in treated (23% by HBP; 20% by ABP) compared with untreated subjects (10% by HBP; 12% by ABP, Po0.05/NS compared with treated), and this was also the case for WCP in treated (16% by HBP; 17% by ABP) vs. untreated subjects (12% by HBP, 13% by ABP, P¼NS compared with treated).

One-third of the treated and 10% of untreated subjects had controlled clinic and out-of-clinic BP.


By taking ABP as the reference method, the sensitivity, specificity, positive and negative predictive value of HBP in detecting sustained, WCP and MH are presented in Table 3 for the total study population and separately in untreated and treated subjects.


Among the total of 613 cases, 65 (11%) had disagreement between HBP and ABP in the diagnosis of sustained hypertension.

In 27 of these 65 cases (42%), the BP difference away from the corresponding diagnostic threshold was within 5 mm Hg, which was considered as clinically uncertain discrepancy.

Disagreement between HBP and ABP in the diagnosis of WCP was found in 65 subjects (11%) and in the diagnosis of MH in 76 (12%).

This diagnostic discrepancy in WCP was considered as clinically uncertain (BP within 5 mm Hg from diagnostic threshold) in 27 subjects (42%) and in MH in 48 (63%).

Independent predictors of the difference between HBP and awake ABP awake derived from multivariable regression models are presented in Table 4.


The same potential predictors of white coat and MH phenomena were entered in binary regression models separately for HBP and ABP.

The independent predictors of white coat and MH detected by HBP or ABP are presented in Table 5.


The main finding of this study is the consistent and substantial agreement between HBP and ABP in the diagnosis of sustained hypertension, WCP and MH in both untreated and treated subjects.

HBP appeared to have high sensitivity and specificity when tested by taking ABP monitoring as reference in the diagnosis of sustained hypertension.

Lower but still satisfactory was the sensitivity of HBP in the diagnosis of white coat and MH phenomenon whereas the specificity was 490%.


Several The investigation for potential predictors of the ABP-HBP differ- ence showed the level of systolic BP to be inversely related with the difference, suggesting that for raised BP levels ABP and HBP tend to provide similar results.

On the other hand, in lower BP, values close the diagnostic threshold disagreement is inevitably increased, since a few mm Hg difference attributed to BP variability might lead to disagreement, which is more often arithmetic (within 5 mm Hg) rather than clinically important.

A significant influence of age on the ABP-HBP difference was also shown, with elderly patients show- ing smaller difference than the younger ones.

This finding is in line with a recent meta-analysis by Ishikawa et al., 15 which showed no difference between awake ABP and HBP in older subjects, whereas in the younger HBP was lower.


The Pamela general population study provided a direct ABP/ HBP comparison in 1529 subjects and showed that of patients with white coat hypertension diagnosed by ABP 70% were detected by HBP, yet only two HBP readings were obtained in that study.

16 Smaller studies, showed diagnostic disagreement of the two methods in the detection of WCP within a range of 13-25%.

17-19 In the current study, superior agreement between the two methods in diagnosing WCP was found which as men- tioned before might be attributed to the standardized measure- ment methods according to guidelines, which fully exhausted their potential.

The sensitivity of HBP in detecting WCP was superior in the treated subgroup than the untreated and inferior to specificity values.

This trend was identified in some other smaller studies, 17,18,20 with sensitivity settled around 50% and specificity around 80%.


Regarding the MH phenomenon, in the Pamela study, 16 HBP confirmed only 57%/45% (systolic/diastolic) of patients having MH by ABP.

A cross-sectional study of 438 subjects attending a hypertension clinic also found similar proportions of masked hypertensives diagnosed by ABP or HBP (14.2 and 11.9%, respec- tively), yet among subjects with MH, there was disagreement in the diagnosis between the two methods in 23% for systolic and 30% for diastolic BP.

21 In this study, in untreated hypertensives, the diag- nostic agreement exceeded 90%, whereas in treated ones was about 80%.

As for WCP, the sensitivity of HBP was lower than specificity in the detection of MH, yet appeared to be satisfactory for clinical practice.


The determinants of white coat and MH phenomena have been intensively investigated in previous studies.

In a recent meta- analysis of 27 studies (n¼12 127), Ishikawa et al. 15 showed that increasing age, female gender and not using oscillometric device for clinic BP, predispose to WCP detecting by ABP.

In a study of 5716 subjects, white coat hypertension detected by ABP was more common among older adults, females and non-smokers.

22 These data are in line with the current findings, suggesting that female gender and non-smoking are established determinants of the WCP, irrespective of the measurement method (ABP or HBP).

This was also the case with MH, where lower clinic BP was associated with MH, irrespective of the measurement method, and smoking which is an established predictor of MH diagnosed by ABP, 23 was also a predictor of MH detected by HBP.

Taken together, the available evidence suggests that the white coat and MH phenomena have consistent clinical predictors, irrespective of the method used for their detection (ABP or HBP), which supports the view that the two methods are interchangeable.


In conclusion, HBP showed substantial diagnostic agreement with ABP in the diagnosis of hypertension.

The between-methods discrepancy may be attributable to inherent methodological differ- ences.

The imperfect reproducibility of both methods 24,25 also contributes to diagnostic discrepancy, as well as the fact that most of the BP values are close to the diagnostic thresholds, which might lead to disagreement even in cases with BP difference of a few mm Hg only.

The recent evidence from meta-analysis of outcome HBP studies, 7 together with the consistent evidence on the diagnostic ability of HBP suggest that this method should no longer be regarded as a screening test that requires confirma- tion by ABP, as has been previously proposed, 26 but as a reliable alternative to ABP.

However, ABP remains a unique tool in the assessment of nocturnal BP and the detection of non-dippers, as well as for the evaluation of short-term reading-to-reading BP variability.

Preliminary evidence suggests that novel home moni- tors can also evaluate nocturnal BP, 27 whereas medium-term day-to-day BP variability assessed by self-home measurements has been shown to predict cardiovascular risk beyond the other risk factors.

28 Given the wide availability of home monitors, their relatively low cost and the good acceptance by users, HBP monitoring should be applied in almost all subjects with elevated BP for both the initial evaluation and the long-term follow-up.


The authors declare no conflict of interest.


A recently published study by Nasothimiou et al. [ 1] concluded that daytime at-home blood pressure (BP) self-measurement is a reliable alternative to ambulatory BP monitoring (ABPM) to identify isolated-office, masked, and sustained hypertension.

Current guidelines [2] define sustained hypertension as consistently elevated clinic and ambulatory BP measurements.

Discrepancies between clinic and ABPM-derived values have been defined as 'isolated-office' hypertension, that is, elevated clinic BP but normal ambulatory BP, and 'masked' hypertension, that is, normal clinic BP but elevated ambulatory BP [2].

These guidelines also recommend the diagnosis of hypertension be based on elevated ABPM-determined awake and/or asleep BP means, using separate specified threshold values [2].

However, past studies, including that of Nasothimiou et al. [ 1], categorized individuals, as erroneously suggested in the guidelines themselves, as isolated-office hypertension or masked hypertension based solely on a comparison of clinic and awake-time BP values, whether the latter were derived from at-home self-assessments [1] or ABPM [3], and disregarding entirely the asleep BP mean, which is also recommended by the guidelines for defining hypertension.

A major limitation of all these studies [1, 3] is the process by which patients are classified; a comparison of clinic and at-home or ABPM-derived awake or even 24-h BP means would include, in each of the four resulting categories, individuals with either elevated asleep BP means and, thus, abnormally high cardiovascular disease (CVD) risk [4].


The literature indicates that the asleep BP mean better predicts CVD risk than the awake or 24-h BP means [4], and that the progressive reduction of the asleep BP during follow-up in outcome trials is the strongest predictor of decreased CVD risk, in both people with normal and elevated BP at baseline [4].

Particularly relevant are the recent findings of the Monitorización Ambulatoria para Predicción de Eventos Cardiovasculares (MAPEC) Study.

They show that the progressive decrease during follow-up in the asleep BP mean is better achieved when the entire daily dose of one or more hypertension medications is ingested at bedtime rather than in the morning, the most typical time of treatment [5-7].

The MAPEC Study was designed to investigate whether a bedtime vs. conventional morning-time hypertension therapy regimen exerts different changes in the 24-h BP profile and reduces CVD risk [4-7].

A total of 3344 subjects with baseline BP ranging from normotension to sustained hypertension according to ABPM criteria were prospectively studied throughout a 5.6-year median follow-up.

Hypertensive participants were randomized to ingest their prescribed hypertension medications upon awakening or the entire daily dose of one or more of them at bedtime.

At baseline and thereafter annually (or more frequently if hypertension treatment was adjusted), all MAPEC Study participants were first divided into categories of normal BP, isolated-office hypertension, masked hypertension and sustained hypertension by comparing their clinic BP and awake BP mean values—as customary in previous studies [3]—utilizing the recommended thresholds of 140/90 mm Hg for clinic systolic (SBP)/diastolic BP (DBP) and 135/85 mm Hg for ABPM-derived awake SBP/DBP means [2].

The subjects comprising these four categories were further divided into two additional classes according to whether they had a normal or elevated asleep SBP/DBP mean, utilizing the recommended thresholds of 120/70 mm Hg [2].

The hazard ratio (HR) of each group—adjusted according to the confounding variables of patient sex and age, diagnosis of diabetes or chronic kidney disease, sleep duration and hypertension treatment time (all medications upon awakening vs. one or more medications at bedtime)—was obtained through a Cox regression analysis using subjects with normal clinic SBP/DBP and normal awake and asleep SBP/DBP as reference.


A. Subjects with a normal asleep BP mean had an adjusted HR comparable to those of the normotensive reference group, regardless of their classification according to the awake BP mean; isolated-office (HR = 0.87, 95%CI (0.59–1.29); P = 0.501) or masked hypertension (0.71 (0.17–2.91); P = 0.629; Figure 1).

B. Subjects with an elevated asleep BP mean had a significantly higher adjusted HR than those of the normotensive reference group, regardless of their classification according to the awake BP mean; isolated-office (HR = 2.08 (1.33–3.24); P<0.001) or masked hypertension (1.77 (1.03–3.05); P = 0.039; Figure 1).

C. Subjects from the groups that were labeled either isolated-office or masked hypertension, according to the awake BP mean only, with a normal asleep BP mean had a significantly lower HR of CVD events than those with an elevated asleep BP mean (P<0.001; Figure 1).


Apart from the marked difference in CVD risk assessment when subject classification disregards the asleep BP mean, the MAPEC Study [8] indicates that the prevalence of the four considered patient categories, that is, normal BP, isolated-office, masked and sustained hypertension, is incorrect and, thus, misrepresentative of the actual clinical dimension of isolated-office and masked hypertension.

Specifically: (i) 58.2% of subjects with normal office and ambulatory awake BP, who should have been labeled with masked hypertension based on an elevated asleep BP, were in the past labeled as normotensive and, thus, mistakenly included in the reference group to calculate the comparative CVD risk of the other categories; (ii) 38.2% of subjects with normal office and elevated ambulatory awake BP, but normal asleep BP—which is indicative of low CVD risk—were labeled with masked hypertension; and (iii) 26.3% of subjects with sustained hypertension—that is, elevated office BP, normal ambulatory awake BP, and elevated asleep BP—have consistently been labeled with isolated-office hypertension.

Other reports also found attenuation by about 50% of the real prevalence of isolated-office hypertension and a significant two-fold increase in the real prevalence of masked hypertension, mainly in high-risk cohorts involving a preponderance of elderly, type 2 diabetic, chronic kidney disease, and resistant hypertension patients, who are prone to having an elevated asleep BP mean and a 24-h non-dipper/riser BP pattern [9, 10].

In conclusion, the asleep BP mean, determined by ABPM, must be used to properly identify out-of-office hypertension and cannot be omitted in the definition of isolated-office and masked hypertension.

Accordingly, these two conditions cannot be defined by comparing clinic BP with awake-time self-measurements at home, as is frequently done [3] and misleadingly recommended as an alternative to ABPM [1].


We have recently published the largest cross-sectional study to date that provides a direct comparison of ambulatory blood pressure (ABP) with home blood pressure (BP) measurements in terms of their ability to detect white coat and masked hypertension phenomena among 613 untreated or treated subjects attending a hypertension clinic [1].

Hermida et al. [ 2] criticized our paper for ignoring night time ABP measurements, and using only day time (awake) ABP for defining white and the masked hypertension phenomena.

This criticism is valid given the accumulating evidence on the prognostic ability of night time ABP, which in some studies has exceeded that of day time measurements [3].

In addition, it appears reasonable that, as far as possible, all of the information provided by 24-h-ABP monitoring should be used in diagnosing hypertension phenotypes.


We looked at the definitions applied for white coat and masked hypertension phenomena in published papers.

White coat and the masked hypertension phenomena have been extensively investigated in cross-sectional diagnostic trials.

In line with our study [1], that cross-sectional studies evaluated white coat and masked hypertension phenomena have been based almost exclusively on day time or awake ABP measurements [4, 5].

This was also the case in long-term outcome trials that assessed the prognostic significance of these phenomena, which also used day time or awake ABP measurements [6-10], with a few exceptions that used average 24-h-ABP values [11, 12].


Several guideline documents and statements by hypertension societies have provided recommendations on the evaluation of white coat and the masked hypertension phenomena in clinical practice.

The 2007 European Society of Hypertension guidelines defined white coat hypertension on the basis of day time or 24-h-ABP [13].

On the other hand, the American Heart Association recommended the use of awake ABP for the diagnosis of white coat hypertension [14, 15].

More recently, the UK National Institute for Health and Clinical Excellence, in its recommendations for the management of hypertension, defined white coat and masked hypertension on the basis of day time ABP [16].


The above mentioned data on the definitions used for white coat and masked hypertension in short-term and long-term trials, and in hypertension guidelines suggest that nocturnal ABP levels are largely ignored.

By defining white coat hypertension on the basis of day time ABP measurements, there might be cases with low day time ABP but elevated night time ABP that remain undetected.

On the other hand, by defining masked hypertension on the basis of day time measurements, there might be cases with low day time ABP but elevated night time ABP that also remain undetected.


We investigated this issue raised by Hermida et al. [ 2] by performing additional analysis of our data, with the aim of evaluating (i) the proportion of subjects who had elevated nocturnal ABP, but have been labeled as white coat hypertensive on the basis of low day time ABP and, (ii) the proportion of subjects with masked hypertension due to isolated nocturnal ABP elevation, who have been missed (classified as normotensives) because of low day time ABP.


According to our initial analysis, the white coat hypertension phenomenon was present in 88 of the 613 study subjects (14.4%) when night time (awake) ABP was ignored [1].

When night time (asleep) ABP was also evaluated, one quarter of these subjects (n = 23, 3.8%) were not classified as white coat hypertensives because of elevated night time ABP (Table 1).

As expected, these subjects tended to have higher 24-h-ABP levels than those with low day time and night time ABP or normotensives (low clinic, day time and night time ABP, Table 2).

It is important to note, however, that only 5 out of these 23 subjects had an elevated 24-h-ABP (systolic and/or diastolic >130/80 mm Hg).

Moreover, when a 5 mm Hg gray zone of diagnostic uncertainty was applied to define isolated nocturnal hypertension (5 mm Hg above the diagnostic threshold for night time ABP), only 10 subjects (1.6%) changed classification as a result of nocturnal hypertension (Table 1).


We also looked at the classification of the masked hypertension phenomenon, which was observed in 94 study subjects (15.3%) in our initial analysis that ignored night time ABP [1].

When night time ABP was taken into account, there were 27 more cases of masked hypertension due to isolated nocturnal hypertension (Table 3).

Subjects with isolated nocturnal hypertension tended to have lower 24-h-ABP levels than those with masked hypertension due to day time ABP elevation only, or day and night time ABP elevation (Table 4).

It is important to note that average 24-h ABP was normal in 24 of the 27 subjects with isolated nocturnal masked hypertension.

Moreover, when a 5 mm Hg gray zone of diagnostic uncertainty was applied to define isolated nocturnal hypertension (5 mm Hg above the diagnostic threshold for night time ABP), only seven subjects (1.1%) changed classification because of isolated nocturnal hypertension (Table 3).


The above analyses suggest that in our study taking into account night time ABP in the detection of white coat and masked hypertension phenomena, there are few cases which the diagnosis changes as a result of isolated nocturnal hypertension.

More importantly, in most of these cases, individuals have a normal average 24-h-ABP, which makes the diagnosis of ABP hypertension and the need for treatment questionable.


In our cross-sectional study, the vast majority of the participants attending a hypertension clinic had uncomplicated hypertension and low to moderate cardiovascular risk [1].

There are three important issues that cannot be investigated in our data set.

The first is the prevalence of isolated nocturnal hypertension in other populations, such as patients with established cardiovascular disease, nephropathy, diabetes or sleep apnea, in whom the prevalence of non-dipping is higher than in those with uncomplicated hypertension.

Second, we could not investigate whether individuals with isolated nocturnal hypertension have a higher cardiovascular risk than normotensives.

Third, we also could not investigate whether the selective reduction of night time BP with drug dosing before bedtime is more effective in preventing cardiovascular events than the usual morning dosing.


The International Database on Ambulatory Blood Pressure In Relation to Cardiovascular Outcomes included 8711 individuals from 10 populations, and identified 577 untreated individuals (6.4%) with isolated nocturnal hypertension (low office and day time ABP).

After a follow-up of 10.7 years, this condition was found to be associated with a higher overall mortality (hazard ratio 1.29, P = 0.045) and cardiovascular event risk (1.38, P = 0.037) [17].

Several studies in diabetic [18, 19] and non-diabetic subjects [20, 21] have suggested that elevated nocturnal ABP should be taken into account in decision making, even when day time ABP levels are low.


The usefulness of bedtime dosing of antihypertensive drug treatment aimed at improving nocturnal BP control, and thereby providing more effective protection against cardiovascular events has been inadequately investigated [22].

A recent study in hypertensive patients with nephropathy who were followed for 5.4 years showed that taking at least one antihypertensive drug at bedtime improved the control of nocturnal and 24-h-ABP and reduced the risk of cardiovascular events compared to taking all drugs in the morning [23].

On the other hand, the African American Study of Kidney Disease showed that once-daily dosing of antihypertensive treatment or the addition of drugs taken at bedtime did not significantly reduce nocturnal BP compared to morning dosing [24].


Despite the considerable number of studies that have demonstrated the prognostic relevance of nocturnal hypertension, there is still no conclusive evidence on whether a treatment strategy specifically targeting nocturnal BP can eliminate the associated cardiovascular risk [7, 22].

Although it seems reasonable that all aspects of the 24-h-BP profile should be normalized, randomized trials are still needed to demonstrate whether more complex and individualized treatment regimens can achieve more efficient cardiovascular protection than morning dosing with long-acting drugs.


Transferrin receptor-2 (TFR2) is a type II transmembrane gly- coprotein homologous to TFR1, 1 able to bind diferric-transfer- rin (holo-TF), although with lower affinity than TFR1.

In iron homeostasis TFR2 appears to have regulatory rather than transport functions, since TFR2 mutations in humans 2 and Tfr2 inactivation in mice 3-5 cause iron overload with low hepcidin.

6 TFR2 is mainly expressed in the liver 1,7 where it is essential for hepcidin control.

Its expression is up-regulated during hepatic development but is not modulated by iron; indeed TFR2 mRNA has no detectable iron-responsive elements in its untranslated regions.

8 TFR2 encodes an 801 amino acid protein with a short cytosolic tail that contains a potential internalization signal, a transmembrane domain and a large C-terminal ectodomain.

This last has a protease-associated domain, a peptidase M28- like domain and a dimerization domain with two RGD motifs, important for protein-protein interactions and for transferrin binding.


Mutations of TFR2 cause type 3 hemochromatosis: all reported mutations 2 are rare, often private.

Mutations cause loss of function and include frameshift, premature stop, small deletions and missense mutations prevalently affecting the protein C-terminus, especially the peptidase-like and the dimerization domains.

9 Binding to holo-TF stabilizes TFR2 on the cell surface 10 and this interaction redirects TFR2 towards the recycling instead of the lysosomal degradation pathway.

4 Experiments in cultured hepatoma cell lines suggest that TFR2, bound to holo-TF, may simultaneously bind HFE.

While the HFE- and the TF-binding sites on TFR1 overlap, in TFR2 they are different.

10,11 The HFE- TFR2 complex has been proposed to act as a sensor of circulat- ing iron and as an activator of hepcidin expression.

12 However, recent data suggest that the two proteins may have separate functions and their interaction is also controversial.

13 Iron over- load is more severe in Tfr2 −/− than in Hfe −/− mice and Tfr2-Hfe double knock-out mice show the most severe disease.

14 Studies in patients indicate that TFR2 plays a prominent and HFE a minor role in hepcidin activation in response to an acute increase of transferrin saturation after a single dose of oral iron.

15 As a further level of complexity, the TFR2 gene, which has two consensus sequences for the erythroid transcription factor GATA-1 in its promoter, 12 is expressed in immature ery- throid cells 16 where it is a component of the erythropoietin receptor (EPOR) complex.

17 The TFR2-EPOR association is required for the efficient transport of EPOR to the cell surface.


Although neither TFR2-hemochromatosis patients nor Tfr2 −/− mice have evident hematologic abnormalities, the erythroid progenitors from Tfr2 −/− young mice appear less sensitive to erythropoietin (EPO) and have increased serum Epo levels.

Moreover, TFR2 silencing in human erythroid progenitors delays their terminal differentiation.

17 TFR1 sheds a soluble counterpart (sTFR1), in vitro and in vivo, which is measurable in serum from normal individuals.


TFR1 is highly expressed on the surface of maturing ery- throblasts and, when unbound to holo-TF, is released into plasma in high concentrations.

This process occurs in iron deficiency anemia, in erythropoiesis expansion and in some hematologic malignancies.

19 In contrast, sTFR1 lev- els are reduced in aplastic and hypoproliferative anemias.

Although its function remains unexplained, sTFR1 has been proposed as a marker of iron deficiency and/or of total erythropoietic activity.

The shedding of sTFR1 is reg- ulated by its ligand holo-TF and, as recently shown, is mediated by the proprotein convertase subtilisin/kexin type 7 (PCSK7).

20 Interestingly, PCSK7 genetic variants are associated with sTFR1 quantitative trait in genome-wide association studies.

21 Here we characterize a previously unrecognized soluble form of TFR2 (sTFR2) that is shed from the plasma mem- brane both in transfected cell lines and in TFR2-expressing erythroid cells.


We show that the process of TFR2 release is regulated by the ligand holo-TF, a regulation lost in a TFR2 mutant (TFR2 G679A ) unable to bind the ligand.

Because of its low affinity for holo-TF, we suggest that the shedding of TFR2 signals iron deficiency simultaneously in hepatic and erythroid cells.


A TFR2 wild-type cDNA was cloned in pCMV-TAG4 vector with a FLAG-tag at the C-terminus (TFR2 WT-C-FLAG ).

TFR2 wild-type (TFR2 WT-N-FLAG ) and TFR2 G679A constructs in pcDNA3.1(+) have a FLAG-tag at the N-terminus.

A "soluble" artificial isoform of TFR2 (sTFR2*) was generated by amplification of the coding region from nucleotide 313 (Arg105) to nucleotide 2403 (Phe801) and in-frame- cloned in pSEC-TAG (B), with a MYC-HIS-tag at the C-terminus, using the following oligonucleotides: sTFR2-KpnI-fw: 5'-GGGGTACCCGAGGGTCCTGCCAGGCG- 3' sTFR2-EcoRI-rev: 5'-GGAATTCCAGAAGTTGTTATCAAT- GTTCC-3' Mammalian expressing vectors encoding for wild-type dynamin (Dyn WT ) and the dominant negative variant Dyn K44A are described elsewhere.

22 Hemojuvelin (HJV)-expressing vector and the hep- cidin promoter luciferase reporter construct (Hep-Luc) were as described previously.

23 FURIN and PCSK7 expressing vectors are described in the Online Supplementary Methods.


HeLa, HEK293, CHO-Trvb-0, the erythroleukemic cell line UT7 and the hepatoma cell lines HuH7 and Hep3B, culture conditions and reagents are described in the Online Supplementary Methods.


CD34 + cells were obtained from human donors who gave informed consent in accordance with the principles of the Declaration of Helsinki .

Approval for their use was obtained from the La Pitié-Salpétrière Hospital Institutional Ethic Committee.

CD34 + cells were purified from the peripheral blood after cyta- pheresis.

CD34 + and CD36 + cells were isolated by positive selec- tion as described in the Online Supplementary Methods.

CD36 + cells were cultured in the presence of 2 U/mL EPO, 100 ng/mL stem cell factor and 10 ng/mL interleukin-3 for up to 12 days for erythroid differentiation.


When indicated, cells were treated with different concentra- tions (0.5, 1.25, 5 and 25 mM) of human or bovine diferric-trans- ferrin (H-holo-TF and B-holo-TF, respectively) and human apo- transferrin (apo-TF) (Sigma-Aldrich, St. Louis, MO, USA) in serum-free media; erythroid cells were treated with 25 mM of human apo-TF or holo-TF.


Brefeldin-A (Sigma-Aldrich), a fungal antibiotic that alters intra- cellular trafficking by blocking protein maturation across the post- endoplasmic reticulum compartment, was used at a concentration of 100-500 ng/mL.


Procedures for pull-down of membrane and soluble TFR2, west- ern blot analysis of conditioned cell culture media and total cell lysates, and the luciferase reporter assay for the effect of sTFR2 on the hepcidin promoter are described in the Online Supplementary Methods.


UT-7 cells were suspended in binding medium (alpha-MEM containing 20 mM Hepes, 10% fetal calf serum, 0.1% sodium azide, pH 7.2).

Twenty-five microliters of the cell suspension, con- taining 4x10 6 cells, were incubated with 25 mL of conditioned medium from HEK293 cells transfected with the empty or sTFR2* expressing vectors.

Samples were equilibrated at 37°C for 60 min.

Twenty-five microliters of binding medium alone or 25 mL of a 100-fold excess of unlabeled EPO were added to the binding assay.

Then the chosen amount of 125 I-EPO was added in a volume of 25 μL and the cells were incubated for a further 30 min at 37°C.

The content of each well was carefully layered over 900 mL of a cold solution of phosphate-buffered saline and centrifuged at 4°C for 10 min at 600 g. Unbound radioactivity was counted and after three washes in phosphate-buffered saline, cell-bound radioactivity was counted in the pellet.

The non-specific binding of 125 I-EPO, deter- mined by adding a 100-fold excess of unlabeled EPO to the binding assay, was subtracted from the total binding to give the specific binding.


Two independent experiments were performed using two dif- ferent batches of 125 I-EPO and control conditioned medium or sTFR2*-containing conditioned medium from two independent experiments.


To study the cell localization and processing of TFR2, HeLa cells, which do not express endogenous TFR2 (data not shown), were transiently transfected with an expressing vec- tor that encodes for a N-FLAG tagged form of TFR2 (TFR2 WT- N-FLAG ).

Since TFR1 and other iron-related proteins present on the cell membrane release soluble forms, we investigated whether this would also occur with TFR2.


In the media of TFR2 transfected cells we identified a single band (sTFR2) of about 80 kDa, which migrated faster than the cell-associated isoforms, suggesting that it was the result of a cleavage mech- anism.

Since sTFR2 can be detected using an anti-TFR2 anti- body, recognizing residues from amino acid 150 to 250, but not with the anti-FLAG antibody when using an N-terminal flagged construct, we concluded that the cleavage occurs at the N-terminal part of the protein, close to the transmem- brane domain, as occurs for sTFR1.

24 To confirm this finding, sTFR2 from cells transfected with TFR2 WT-C-FLAG , which has a FLAG-tag at the C-terminus, was indeed detected with the anti-FLAG antibody (Figure 1A).


The size of sTFR2 is com- patible with that of a protein lacking intracellular and trans- membrane regions.

Indeed the artificial form of sTFR2 (sTFR2*), generated by cloning the TFR2 ectodomain (amino acids 105-801) downstream of the Ig k-chain leader sequence, migrates as the isoform shed from the full length TFR2 transfected cells (Figure 1A).


sTFR2 is not an artefact due to cDNA overexpression since it is also released by TFR2-expressing cells, such as the erythroleukemic cell line UT7 and human primary ery- throblasts.

Both UT7 cells and primary erythroblasts release a band of sTFR2 of approximately 80 kDa in culture media (Figure 1B), compatible with the size of sTFR2 released by TFR2-transfected cells.

In the total lysates of erythroid cells, TFR2 migrates as a doublet of about 102 and 92 kDa, as pre- viously observed.


It was previously reported that the receptor exposed on the cell surface is stabilized in the presence of holo-TF with a maximum effect at 25 mM holo-TF.

10 We confirmed the same stabilization both in transfected cells and in TFR2- competent cells.

HeLa cells transfected with TFR2 WT-C-FLAG construct were treated with human holo-TF and apo-TF (25 mM) in serum-free media and were biotinylated to label membrane proteins.

sTFR2 was pulled-down using anti- FLAG Sepharose-beads to avoid holo-TF interference with gel migration of concentrated culture media.

In these condi- tions, as a consequence of holo-TF treatment, we observed a reduction of sTFR2, mirrored by an increase in mem- brane-exposed TFR2 (Figure 2A).


A substantial increase in the expression of membrane TFR2 was also detected in biotinylated plasma membranes from erythroid UT7 cells and primary erythroblasts when cultured in the presence of holo-TF, while endogenous sTFR2 production was decreased (Figure 2B).

This confirms that the inverse regulation of membrane and soluble TFR2 in the presence of the ligand is also maintained in cells expressing endogenous TFR2 (Figure 2A).

We asked whether sTFR2 originates from the plasma membrane or is released during receptor internalization.

To this aim cell surface proteins were labeled using a cell- impermeable biotin and were treated with holo-TF.

sTFR2 was detected in the culture media of transfected cells after streptavidin pull-down of biotin-labeled proteins, demon- strating that it originates from membrane-exposed TFR2 (Online Supplementary Figure S1B).


To further prove the membrane origin of sTFR2, we treat- ed HeLa cells with brefeldin-A, a fungal antibiotic that alters intracellular trafficking by blocking protein maturation across the post-endoplasmic reticulum compartment.

25 Brefeldin-A induced a strong reduction of both membrane- associated and soluble TFR2 (Figure 2C), consistent with the finding that TFR2 is shed from the cell surface.


To investigate the potential contribution of endocytosis to the release of sTFR2, we exploited the effect of dynamin dead-mutant K44A (Dyn K44A ), which blocks the endocytosis process, thus "fixing" the proteins on the cell surface.

26 The amount of sTFR2 in cells transfected with Dyn K44A was strongly increased compared to that of cells transfected with wild-type dynamin (Dyn WT ), further strengthening the belief that the cleavage occurs at the cell surface (Figure 2D).

Under these conditions, holo-TF was still able to reduce the shedding process (Figure 2E).


TFR2 can bind holo-TF and is highly homologous to TFR1 and the release of sTFR1 is reduced by its ligand.

27 For these reasons we investigated whether sTFR2 release was affected by holo-TF in our in vitro system.

HuH7 (Figure 3A) and HeLa (Online Supplementary Figure S1B,C) cells were transiently transfected with the TFR2-expressing vector and then treated with increasing concentrations of human holo- A. Pagani et al. 460 haematologica | 2015; 100(4) To avoid holo-TF interference with gel migration of con- centrated culture media, we biotinylated cell surface pro- teins and pulled-down sTFR2 in the supernatants using streptavidin beads.

As shown in Figure 3A, human holo-TF inhibited the release of sTFR2 in a dose-dependent manner.

This inhibition was not mediated by iron, since treatment of TFR2-transfected cells with iron donors (ferric ammoni- um citrate) or iron chelators (deferoxamine) did not modu- late sTFR2 concentration in the culture media (Online Supplementary Figure S1A).


To understand whether the observed inhibition was a direct effect of the ligand binding, we used TFR2 G679A , an artificial variant unable to bind holo-TF since the mutation inactivates the RGD binding domain.

28 As shown in Figure 3B, TFR2 G679A released sTFR2 in high concentrations and the process was not modulated by the ligand.


To exclude that sTFR2 release might be influenced by binding of holo-TF to its high affinity receptor TFR1 we used two approaches.

First, we used bovine holo-TF, which specifically binds TFR2 but is unable to bind human TFR1.

28 As with human holo-TF, sTFR2 was strongly decreased in the presence of bovine holo-TF both in HeLa cells (Figure 4A) and in HuH7 cells (data not shown).

Second, we analyzed the shedding process in TFR2-transfected CHO-Trvb-0 cells that lack TFR1.

29 We confirmed that these cells did not Shedding of TFR2 haematologica | 2015; 100(4) 461


express TFR1 (Online Supplementary Figure S2) and we showed that also in these cells holo-TF inhibited the release of sTFR2 and stabilized TFR2 on the cell surface (Figure 4B).


Overall our results demonstrate that the shedding of TFR2 does not require TFR1 and that the inhibition of sTFR2 release results directly from ligand-receptor binding.


Because the TFR2-EPOR association is required for effi- cient transport of EPOR to the cell surface, 17 we studied whether sTFR2 could modify the number of cell surface EPOR or the affinity of EPO for its receptor by Scatchard analysis in UT7 cells.

To investigate this issue, we used sTFR2*, which is functional (see below) and more efficient- ly produced than the naturally cleaved TFR2 (Figure 1A).


The conditioned medium of HEK293 cells transfected with sTFR2* (Sn sTFR2*) or with empty vector (Sn mock) did not significantly modify the number of EPOR expressed on the cell surface or the affinity for its ligand (Figure 5A).

However, brefeldin-A treatment decreased the number of EPOR on the plasma membrane (Online Supplementary Figure S3).

To investigate the activation of EPO-mediated intracellular signaling, the phosphorylation status of STAT5, AKT and ERK, major targets of this pathway, was assessed in UT7 cells treated or not with sTFR2*. As illustrated in Figure 5B and quantified in Figure 5C, EPO signaling was not significantly affected by sTFR2*. These data were sup- ported by the observation that exogenous sTFR2* did not interact with EPOR when added to the culture medium (Online Supplementary Figure S4).


To evaluate the functional role of sTFR2 in the hepatic To analyze whether sTFR2 could interfere with BMP sig- naling we studied the response of the hepcidin promoter in Hep3B cells in the presence of both sTFR2* and BMP6 and we confirmed the same partial inhibitory effect observed both in basal and in HJV-mediated hepcidin activation (Online Supplementary Figure S5D).

However, an effect of sTFR2 on the hepcidin promoter assay was observed only in vitro.


From its discovery more than 10 years ago 1 and its iden- tification as the gene responsible for hemochromatosis type 3, 2 the function of the second transferrin receptor (TFR2) has remained obscure.

The role of TFR2 as a component of the liver iron-sensing machinery and hepcidin activator is well established, but the molecular mechanisms remain unclear.

The role of TFR2 as a partner of EPOR in immature erythroid cells has been clearly defined, and our recent results have shown that it negatively controls erythro- poiesis expansion in iron deficiency.

30 In this study we have expanded our knowledge on the structure of TFR2, identifying its soluble form (sTFR2).

sTFR2 was detected in the media of different cell lines transfected with TFR2 and most importantly was present in the culture media of both erythroid cell lines and primary human erythroid cells that endogenously express TFR2.

16,17 We showed that sTFR2 is shed from the membrane- exposed receptor.

This conclusion is supported by two lines of evidence: (i) the lack of sTFR2 shedding when the recep- tor does not reach the cell surface, as after brefeldin-A treat- ment; (ii) the increase of sTFR2 release in the opposite con- dition, when the receptor is stabilized on the membrane by a mutant dynamin.


TFR2 associates with different partners (EPOR in ery- throid cells, HJV and HFE in the liver) and likely has differ- ent functions in the two tissues in which it is highly expressed.

However, the shedding mechanism appears to be under a common iron-mediated regulation.

Indeed bind- ing of the holo-TF ligand to the receptor inhibits sTFR2 release, while in the absence of ligand binding the release is increased in both hepatoma and erythroid cells, providing a common iron sensing mechanism.


Holo-TF binds to the RGD motif in the TFR2 ectodomain.

Importantly, the RGD mutant TFR2 G679A , unable to bind the ligand, 28 is insensitive to the shedding regulation.

This phenomenon clearly indicates that the shedding process is directly controlled by the receptor-lig- and molecular interaction and, as previously reported for TFR1, couples the TFR2 shedding process with iron defi- ciency.


However, the release of TFR2 does not require TFR1.

It is reasonable that the shedding of the two recep- tors occurs separately, since their expression is maximal at different stages of maturation of the erythroid cells, TFR2 being expressed in more immature erythroid precursors than TFR1.

16,17 Also in the liver, Tfr2 expression increases during mouse development in parallel with the decrease of Tfr1 expression.

8 Furthermore, the two receptors are likely cleaved by different proteases.

The consensus cleavage site for PCSK7, the proconvertase that cleaves TFR1, is not conserved in TFR2 (Online Supplementary Figure S6).

The observed inhibition of TFR2 shedding by the proconver- tase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylke- tone (CMK) indicates that a furin-like proconvertase oper- ates the cleavage.

However, both furin and PCSK7, the proteases involved in sHJV 31 and sTFR1 20 cleavage are not involved in TFR2 shedding (Online Supplementary Figure S6).


Overall, our results indicate that removal of TFR2 from the cell surface is not only due to lack of receptor sta- bilization, but is dependent on an active cleavage process.

It is conceivable that membrane stabilization of TFR2 by holo-TF subtracts the receptor from the protease activity by changing its conformation or by hiding the cleavage site.

The definition of these molecular mechanisms requires further studies.

We cannot exclude that, as previ- ously reported, 4 decreased endocytosis and TFR2 lysoso- mal degradation might also occur in addition to the reduced shedding in the presence of the ligand.


Different proteins and receptors acquire distinct functions when present either as membrane-bound or soluble forms, as shown for HJV.

31-33 We cannot exclude a dual and oppo- site role also in the case of TFR2.

Sensing the increased con- centration of the ligand, membrane TFR2 is proposed to be involved in hepcidin activation to reduce the levels of circu- lating iron.

12 Its soluble counterpart might act as an inhibitor to attenuate the signaling pathway, in conditions of reduced iron availability, to increase iron absorption.

We consistent- ly showed a modest inhibition of hepcidin promoter activa- tion by sTFR2, but not of endogenous hepcidin in hepatic cell lines (data not shown).

In addition, when iron deficiency is induced in double knock-out Tfr2 -/- , Tmprss6 -/- mice, hep- cidin is inhibited, ruling out a significant contribution of sTfr2, which cannot be produced in these animals.

34 Thus a decoy function on hepcidin activation is unlikely.

However, irrespective of sTFR2 function, removal of membrane TFR2 in iron deficiency would negatively affect hepcidin activa- tion.


An erythroid role for Tfr2 in conditions of low iron has recently been suggested by the observation that iron defi- ciency caused by ablation of the hepcidin inhibitor Tmprss6 induces erythrocytosis in mice knock-out for Tfr2, but not in mice with liver-specific Tfr2 deletion (which preserves the Tfr2 erythroid function.

34 This event was directly con- firmed by the increased erythropoiesis observed in mice that had Tfr2 exclusively deleted in the bone marrow.

30 sTFR2 added to the culture media of transfected cells does not seem to modify the affinity of EPO for EPOR or the cell surface expression of EPOR.

Consequently, EPO- mediated signaling appears not to be affected.

Preliminary experiments show that the addition of sTFR2 to the culture medium of primary human erythroblasts does not affect their proliferation, survival or terminal differentiation (data not shown).

However, irrespective of sTFR2 function, shed- ding membrane TFR2 in iron deficiency might serve to decrease the receptor's interaction with EPOR and to increase EPO sensitivity of erythroid cells, as recently shown in mice with bone marrow-specific Tfr2 deletion.

30 The lower affinity for holo-TF as compared with TFR1 A. Pagani et al. 464 haematologica | 2015; 100(4) makes TFR2 an ideal sensor of iron deficiency in the bone marrow which could anticipate the iron needs of maturing erythroblasts.

It is possible that when holo-TF concentra- tion starts decreasing, TFR2 would be shed from the mem- brane, whereas TFR1, because of its higher affinity, would still be able to bind holo-TF.


In conclusion we provide in vitro evidence of TFR2 shed- ding, a mechanism regulated by holo-TF, in analogy with the process described for TFR1.

Although the in vivo role of sTFR2, as well as that of sTFR1, remains to be clarified, we suggest that while membrane TFR2 in the liver senses excess iron, TFR2 in the bone marrow has the opposite role of sensing iron-deficiency.

This would explain why the effect of lack of TFR2 shedding is not evident in TFR2- hemochromatosis in which iron-deficiency is never achieved.


We read with great interest the recent article by Pagani et al. [ 1] in which the authors identified a soluble form of transferrin receptor-2 (TfR2) in the media of cultured human erythroid cells and transfected cell lines.

Although the authors indicated that the cleavage occurs at the cell surface and is regulated by diferric transferrin [1], they did not identify the sheddase involved in this process.


We think that the results of study by Pagani et al. [ 1] may help to predict which protease is involved in the TfR2 shedding.

Using a general inhibitor of proprotein convertases, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (RVKR-cmk) [2], the authors revealed that a proprotein convertase is implicated in the TfR2 cleavage.

Indeed, in HeLa cells transfected with TfR2, this inhibitor treatment resulted in a strong reduction of soluble TfR2 in the media [1].

It is known that the dec-RVKR-cmk inhibitor blocks the activity of the seven basic specific proprotein convertases, i.e. PC1, PC2, furin, PC4, PC5, PACE4 and PC7 [2].

But, some proprotein convertases are tissue-specific and can be ruled out, e.g., PC1 and PC2, which are specifically expressed in neural and endocrine cells, and PC4, which is exclusively expressed in germ cells.


The remaining members of the family (furin, PC5, PACE4 and PC7) are ubiquitously/widely expressed and share overlapping cellular localizations [2], but two of them are known to be involved in iron homeostasis.

Indeed, furin can cleave hepcidin [3] and hemojuvelin [4] whereas PC7 is the sheddase of transferrin receptor-1 [3].

Pagani et al. [ 1] demonstrated that both furin and PC7 are not involved in TfR2 shedding because their overexpression in HeLa cells transfected with TfR2 did not modify the soluble TfR2 release.

Consequently, PC5 and PACE4 are the only two proprotein convertases potentially involved in the cleavage of TfR2.


However, it is known that HeLa cells express PACE4 but not PC5 [5].

Moreover, it was observed that UT7 erythroleukemia cells released soluble TfR2 in the media [1] and that K562 cells, which are also erythroleukemic cells, express PACE4 but not PC5 mRNA [3].

It is also known that TfR2 is mainly expressed in the liver [6] and that PACE4 mRNA levels were approximately 40-fold higher than those of PC5 mRNA in mouse primary hepatocytes [7].

Finally, PACE4 is activated at the cell surface, where it binds heparin sulfate proteoglycans [8], and hence it cleaves its substrates at the cell surface.


Taken together, these data strongly suggest that PACE4 is the proprotein convertase involved in the cleavage of TfR2.

Interestingly, analysis of the extracellular domain of the human TfR2 sequence revealed a putative proprotein convertase-cleavage site, RQTSLR163↓ER with an Arg at the P1 (residue 163) and P6 (residue 158) positions [2].

Nevertheless, because PACE4 may activate other proteases, such as the metalloprotease ADAMTS4 [2], we cannot exclude that the TfR2 was cleaved indirectly by activation of a latent protease.


In conclusion, we suggest that PACE4 constitutes the third member of the proprotein convertase family implicated in iron homeostasis probably through TfR2 cleavage but possibly also via the activation of the bone morphogenetic protein-6, a key regulator of iron metabolism [9], which was reported to be cleaved by PACE4 but not by furin in the mouse embryonic carcinoma-derived cell line ATDC5 [10].


We thank the authors of this letter [1] for their interest in our paper [2] and for their detailed analysis of the evidence for and against the proteases responsible for the cleavage of transferrin receptor-2 (TFR2) from the cell surface in hepatocytes and erythroid cells.

Their hypothesis of PACE4 (PCSK6) as the most reasonable candidate protease responsible for cleaving TFR2 on plasma membrane and releasing its soluble component is intriguing.


After the identification of soluble TFR2 (sTFR2) in the media of transfected and primary cells [2] we made several attempts to identify the protease involved in TFR2 shedding.

As reported in the paper [2], and also discussed by the Guillemot et al. [ 1], we were able to exclude some potential candidates that are already known to have a role in iron metabolism, namely furin and PCSK7, but we did not reach definitive conclusions.

We, therefore, generated an artificial form of sTFR2 corresponding to the whole ectodomain of TFR2 from position 105 whose size, based on gel migration, overlaps the natural form (see Figure 1A in the original paper [2]).


Guillemot et al. propose the interesting hypothesis that the proprotein convertase PACE4 might be the protease involved based on its expression and inhibition by CMK: this hypothesis deserves experimental validation.

Still, the size of a product cleaved at position R163, the proposed cleavage site of PACE4, is difficult to reconcile with the size of the released form (see Figure 1A of the original paper [2]) since the product of the cleavage would be 60 amino acids shorter.

In addition we observed that in non-denaturing conditions sTFR2 shows a high molecular weight band compatible with dimer formation (Figure 1A).

This suggests that cysteines at positions 108 and 111, the only potentially involved in a disulfide bond in the stem region, are most likely preserved in sTFR2.

This would exclude a cleavage C terminal to such a position, including the presumptive RQTSLR consensus sequence of PACE4 at positions 158-163.


In addition a mutation of Arginine 158 to Alanine (R158A) that is partially defective in plasma membrane localization did not suppress TFR2 release.

Unfortunately, no conclusion can be drawn on R163A, because the corresponding protein does not reach the cell surface (Figure 1B).

Finally sTFR2 release was not inhibited by leupeptin (Figure 1C), which was reported to inhibit PACE4 [3].

For all these reasons we did not consider PACE4 as a possible candidate.

Obviously all these speculations do not exclude the possibility of indirect cleavage of TFR2 by other proteases that are targets of PACE4 (PCSK6) activation.

In brief, until verified experimentally this protease remains an interesting candidate.


Crohn's Disease (CD) is a chronic illness characterized by destructive transmural inflammation of the gastrointestinal tract with periods of flares and remission.

1 While CD is most commonly diagnosed among 15-35 year olds, a quarter of patients are diagnosed as children under the age of 18.


Corticosteroids are the most commonly used medication to induce remission in the United States, 4 but can pose particular risks in a pediatric population including: growth retardation, low bone mineral density, adrenal suppression, and body image dissatisfaction.

5 As an alternative, gastroenterologists sometimes elect to use exclusive enteral nutrition (EEN) as induction therapy in these patients 6 because adverse effects of enteral nutrition are generally limited to gastrointestinal tolerability (e.g. nausea, vomiting, diarrhea) though rare reports of refeeding syndrome exist.

7 This therapy is frequently used in Europe, but fewer than 4% of pediatric gastroenterologists use it in North America, though emerging data shows it is gaining more prominence in Canada.


The benefits of enteral nutrition in patients with Crohn's disease were auspiciously identified in the surgical literature when patients administered enteral nutrition to optimize their nutritional status preoperatively, improved unexpectedly, with some even avoiding the intended surgery.

9 The mechanism of the beneficial effects of enteral nutrition was hypothesized to be the avoidance of enteral antigens resulting in improvements in intestinal permeability, though recent studies have elucidated other possibilities 10 , including down regulation of pro-inflammatory cytokines.

11 Others have found beneficial changes to the gut microbiome among Crohn's patients treated with enteral nutrition.


There are three major categories of enteral nutrition formulas, and they are differentiated by the structure of their protein content.

Elemental diets contain no intact protein, only amino acids.

Unfortunately, elemental diets are distinctly distasteful and often require a feeding tube to administer.

Even semi-elemental diets (i.e. peptides of varying length) were found to be intolerable with 57% of patients withdrawing from a major study mostly due to poor palatability.

14 Polymeric diets (i.e. intact protein), which are more palatable and do not always require feeding tube placement for administration, have been shown to perform as well as elemental and semi-elemental diets in the induction of remission of CD.


Previous comparisons regarding the effectiveness of EEN versus CS in remission induction have yielded mixed results.

Five out of seven meta-analyses published over the last 20 years have found CS to be superior to EEN at inducing remission.

15-19 Of the seven meta- analyses, two have mixed populations of adults and children, three included only adult studies and two focused only on children.

EEN is hypothesized to work more effectively in children than adults, though the reasons for this are unclear.

6 The two pediatric meta- analyses focused only on induction of remission, 20,21 found no difference in remission induction comparing CS to EEN.


In the ten years that have elapsed since the most recent meta-analysis on this subject, several more studies 22-25,35 have been published, which have included new data on biomarkers and mucosal healing, two previously unexplored end points.

Therefore, we performed an Swaminath et al. Page 2 Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01. updated systematic review and meta-analysis on the efficacy of CS versus EEN in the pediatric population.

Our primary outcome was the induction of remission.

We distinguished the treatment effects between newly diagnosed and relapsed patients.

Because the field has moved towards recognizing the importance of more objective criteria to identify improvement and monitor disease status, 26 we conducted three hypothesis-generating analyses to examine the effect of EEN vs. CS on durability of remission, mucosal healing and biomarkers.


Types of studies-All randomized and observational studies with at least two comparator arms including at least one dietary intervention administered by any route and one non- dietary intervention in English were considered for inclusion.

Abstracts and conference proceedings were excluded, as data suggests that abstracts may be inconsistent with the final published article 27 and they have not had the benefit of peer-review.


Types of participants-Patients under the age of 18 with newly diagnosed or relapsed active Crohn's disease.


Types of interventions-Administration of EEN in the following formulations: polymeric formula, semi-elemental formula, and elemental formula to one group of patients and CS to the comparator arm in this systematic review and meta-analysis.

Patients in the EEN arm must not be receiving any other medication, and those receiving CS must only be treated with CS.

Though there has been no convincing evidence that there is any difference in benefit between the types of formulas used, and the Zachos et al. 15 Cochrane analysis combined all exclusive enteral nutrition analyses, we conducted both combined and stratified analyses.


Exclusion Criteria-Trials without an EEN arm and trials without at least one clearly defined CS comparator arm were excluded from this analysis.

Additionally, authors who did not define remission within the text of their manuscript were excluded.


Types of outcome measures-All of the potential studies that met inclusion criteria (Figure 1) measured disease activity with either the Lloyd-Still Index (LSI) or Pediatric Crohn's Disease Activity Index (PCDAI).

Because the cut off points for each scale is subjective, we defined our outcome variable as the percentage of patients who remained in remission, as defined by the study authors.

In cases where this information was not provided in the published manuscript, the additional information was sought directly from the authors.


Web of Science, The Cochrane Library, Open Grey, Grey Literature Report, Clinicaltrials.gov and the WHO Clinical Trials Registry Platform from inception to September of 2016.

All relevant subject headings and free text terms were used to represent Crohn's Disease and enteral nutrition, including exp Inflammatory Bowel Diseases/OR (Crohn$ adj (disease or enteritis)).tw AND Enteral Nutrition/ OR((enteral or enteric or Swaminath et al. Page 3 Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.


A research librarian (LF) designed and conducted the complete search strategy.

Articles were screened independently by three reviewers (SLF, AF, and AS).

Any disagreement at this stage of screening was remediated by acquiring the full text of the article, and discussion until consensus was achieved.

Of the 2795 papers identified through our search strategy, 43 articles potentially meeting inclusion criteria were identified and agreed upon by all three reviewers and the full text was reviewed.

Nine articles met inclusion criteria, but three articles (Sanderson 1987 28 , Papadopoulou 1995 29 , and Levine 2014 25 ) did not provide the necessary data for inclusion in the meta-analysis.

These authors were contacted for more complete data.

Two authors (Levine 2014 25 and Sanderson 1987 28 ) provided additional data, leaving a total of eight papers which were included in the meta- analysis (Table 1, Figure 1).

Justifications for excluded articles are included in Table 2.


Levine 2014 25 had 4 arms: receipt of only EEN, only CS, only.

Mesalamine and last a combined EN and mesalamine arm.

In our meta-analysis we only included the EEN and CS arm.

Sanderson et al. 1987 28 used corticosteroids and sulfasalazine in one arm, and an elemental diet only in the other arm.

Since sulfasalazine has not shown any benefit in inducing remission in small bowel Crohn's disease, 30 we included this study in the meta- analysis with the assumption that CS + sulfasalazine (in small bowel Crohn's disease) is equivalent to CS only treatment.


Three authors (SLF, AF, AS) independently reviewed the quality of the studies.

We evaluated the bias in observational studies using the New Castle-Ottawa scale 31 and randomized controlled trials using the Cochrane RoB tool 32 (Table 1, Table 3).

Observational studies, less rigorous than randomized controlled trials (RCT), inherently introduced uncertainty and heterogeneity into our meta-analysis.

As such, we conducted two sensitivity analyses.

First, we removed the study where the author admitted that the study team preferentially assigned patients to the enteral nutrition arm of the trial (Lambert et al. 2012 24 ).

Second, we analyzed the results for the RCTs, prospective cohort studies, and retrospective cohort studies separately-to determine if the results from the non-RCTs affected our heterogeneity calculation or produced a different result from the more robust RCTs.

Because there was not a heterogenous effect across strata when we examined each study type separately for our main outcome, we felt it was appropriate to combine study types as hypothesis generating rather than confirmatory analyses.


Data extraction forms were developed by two of the authors (AF, AS) and confirmed by a third (SLF).

Data were extracted jointly by two authors (AF, AS) and then a third author (SLF) independently extracted the data.

Disagreements were reconciled by discussion.

Extracted data included: participant age and disease characteristics, number of patients in each treatment arm, outcome measurement (PCDAI vs. LSI vs. "biochemical remission"), study design, study inclusion criteria, details of enteral nutrition formulation Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.


The percentages of patients in remission at the end of the first follow- up period from each study were pooled.

Cross-study variation due either to heterogeneity or chance was quantified using both I 2 and Chi 2 where P < 0.10 or I² > 50% indicates substantial heterogeneity.

Fixed-effect models were used.

If heterogeneity was present, a random effects model was used to calculate the most conservative confidence intervals.

Each study's weight was determined using Mantel-Haenszel methods.

All analyses were conducted using RevMan5 (version 5.3.5 Copenhagen, Denmark).


We evaluated bias in randomized controlled trials according to the methods described in the Cochrane Handbook 32 which includes consideration of the following: blinding, randomization, completeness of outcome data and method of reporting data (Table 1).

Observational studies were evaluated for bias using the Newcastle-Ottawa tool 32,33 (Table 3).

Data have reported the data according to MOOSE guidelines.


Of the nine trials that met our inclusion criteria (Table 1, Figure 1), eight study authors either included or provided the proportion of patients who entered remission in each arm which could be pooled in the meta-analyses.


Of the eight trials included in the quantitative meta-analysis, seven compared EEN to CS (Borreli et al. 2006 35 , Berni Canani et al. 2006 36 , Hojsak et al. 2014 22 , Kierkus et al. 2013 23 , Lambert et al. 2012 24 Levine et al. 2014 25 , Luo et al. 2015 37 ).


Sanderson et al. 28 compared EEN to sulfasalazine + CS.


Five studies used a polymeric diet.

Sanderson et al. 28 used an elemental diet, and Berni Canani et al. 36 had separate arms for elemental, semi elemental, and polymeric diets that were pooled together for our meta-analysis.

Luo et al. 37 did not specify the type of enteral nutrition that they used.


Three studies provided specific details about the steroid dosing arms.

Berni Canani et al. 36 used methylprednisolone (1-2mg/kg) up to max of 40mg/d for 4 weeks, and then tapered over the following 4 weeks.

Levine et al. 25 used prednisone 1-2mg/kg up to max of 60mg/d for 8 weeks.

Sanderson et al. 28 used adrenocorticotropic hormone (ACTH) 2 IU/kg administered intramuscularly for 5 days followed by oral prednisone 2mg/kg/d up to maximum of 30mg/d with a taper starting after 3 weeks.


The study participants in all trials included in the meta-analysis were children, and the diets were administered via nasogastric tube (elemental diets) or orally.

Of the eight studies, four studies (Borrelli 2006 35 , Lambert 2012 24 , Levine 2014 25 , Luo 2015 37 ) included only newly diagnosed patients, whereas the others included both newly diagnosed and relapsed patients, Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01. or relapsed only.

Disease activity and remission were based on PCDAI scores, though the cutoff for remission varied and some studies did include supplemental information from biomarkers or endoscopic scores (CDEIS).

Sanderson et al. 28 used the LSI along with biomarkers (CRP, ESR, albumin concentration) to determine remission.

Correspondence with the author was required in order to obtain patient level Lloyd Still scores levels at multiple time points.

Outcomes were assessed at 6-8 weeks except Borrelli et al. 35 assessed outcomes after 10 weeks of treatment.


One other paper met inclusion criteria, but was not included in the meta-analysis because data could not be abstracted from the paper and could not be obtained from the corresponding author.

Papadopoulous et al. 29 used an elemental diet arm (n=19) and oral prednisone (2mg/kg/d, max 60mg/d; n=17) with taper over 8 weeks, but did not provide individual patient status at end of induction.

Instead they describe successful treatment of flare episodes (each patient could have more than one episode of flare).

They found remission in 83% of episodes treated with EEN and 64% treated with prednisone.


A. Enteral Nutrition Therapy for Induction of Remission-This meta-analysis of eight trials included 226 pediatric Crohn's patients treated with an exclusive elemental diet and 225 pediatric Crohn's patients treated with corticosteroids for newly diagnosed or relapsed disease demonstrated no statistically significant difference between the treatment arms (OR 1.26; [95% CI 0.77-2.05]; Figure 2.

Comparison of Remission induction for EEN vs. CS).

Heterogeneity was not demonstrated (I 2 = 0).


Given the interest in possible treatment differences between newly diagnosed CD patients and those with relapsed disease, the studies were separately analyzed by type of patient.

Borrelli et al. 35 , Lambert et al. 24 , Lou et al. 37 and Levine et al. 25 included only newly diagnosed patients.

Though these studies were an RCT, 2 retrospective cohort studies and a prospective cohort study respectively, we combined them to do an exploratory analysis.

The sub analysis of these 4 trials included 100 patients treated with enteral nutrition and 171 patients treated with corticosteroids.

No statistically significant difference was demonstrated (OR 1.61, 95% CI 0.87-2.98, Figure 3a.

Comparison of EEN vs. CS in Newly Diagnosed Patients Only).


A subanalysis of the 3 studies including only relapsed patients (Sanderson et al. 28 , Kierkus et al. 23 , Hojsak et al. 22 ) included 89 patients treated with enteral nutrition and 44 treated with steroids found no statistically identifiable differences between induction of remission between the groups (OR 0.76, 95% CI 0.29-1.98, Figure 3b.

Comparison of EEN vs. CS in Relapsed Patients Only).


There was no statistically significant difference in remission odds distinguishing newly diagnosed patients and relapsed patients, though the trend was towards EEN being more effective in those who were newly diagnosed as opposed to relapsed patients.

Heterogeneity was not demonstrated (I 2 =1%).


B. Benefit of enteral nutrition therapy beyond end of induction-An exploratory subanalysis of 2 studies (Sanderson et al. 28 , Berni Canani et al. 36 ) was performed to ascertain if being induced into remission with EEN vs. CS had led to a more durable remission.


Although Sanderson et al. 28 was an RCT and Berni Canani et al. 36 was a Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.

retrospective cohort study, we combined their results in an effort to generate a hypothesis regarding the potential durability of remission under the two treatment regimes.


Sanderson et al. 28 provided data at 12wks (6 weeks after end of induction).

Patients who had achieved remission in the steroid group were maintained on prednisone 10mg daily.


Those who had achieved remission in the EN group were in a prespecified food reintroduction program with tapering doses of EN.


At 12 weeks, 6 of 7 patients in the steroid induction group remained in remission whereas, 7of 8 patients in the EN induction group remained in remission.


Berni Canani et al. 36 provided data at 12 months of follow-up (10 months after the end of induction).

All patients in both EN and CS groups who achieved remission were treated with mesalamine 50-75mg/kg daily.

At 12 months, 22 of 37 patients in the EEN induction group remained in remission while only 3 of 10 patients in the steroid induction group remained in remission.


Although there was no statically significant difference between the groups (OR 2.75, [95% CI 0.72-10.53], Figure 4, Durability of Remission, EEN vs. CS), the trend was toward benefit with EEN.

Heterogeneity was not demonstrated (I 2 =0%).


C. Polymeric formula and Elemental formula for remission induction-The majority of the included studies (Borrelli et al. 35 , Hojsak et al. 22 , Kierkas et al. 23 , Lambert et al. 24 , and Levine et al. 25 ), used a polymeric formula in their enteral nutrition arm.


When we compared only these five studies for remission induction, there was no difference between CS and the polymeric formula of enteral nutrition (OR 1.10, [95% CI 0.64-1.89], Figure 5.

Polymeric formula only for Remission Induction, EEN vs. CS).


Sanderson et al. 28 was the only study which used an elemental diet only arm and Lou et al 37 did not specify the type of formula used in their study.


D. Mucosal healing-Borrelli et al. 35 and Berni Canani et al. 36 provided mucosal healing data at the end of induction.

Though these were two different types of studies-an RCT and a retrospective cohort study-we conducted a hypothesis generating analysis to determine whether mucosal healing rates were higher among patients who received EEN compared to those who received CS.


Borrelli found 26 of 37 patients in the EEN group achieved mucosal healing where only 4 of 10 in the steroid group achieved the same endpoint.

Berni Canani found 14 of 19 patients induced with EEN achieved mucosal healing whereas only 6 of 18 patients treated with CS achieved the same endpoint.

Overall, patients who received EEN were 4.5 times more likely to demonstrate mucosal healing (OR 4.50, [95% Cl 1.64, 12.32]; Figure 6a.

Comparison of Mucosal Healing in EEN vs. CS) compared to those who received CS.

This result was statistically significant.


al. 25 , and Sanderson et al. 28 measured CRP at baseline and at the end of induction in patients receiving EEN and CS.

Again, though these were three different types of studies-a retrospective cohort study, a prospective study, and an RCT-we conducted a hypothesis generating analysis to ascertain whether patients who received EEN vs. CS had differed in rates of CRP


Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.

Hojsak found CRP normalized in 48 of 57 patients treated with EEN and all 17 patients treated with CS.

Levine found 22 of 38 patients treated with EEN had normalization of CRP compared to 60 of 103 patients treated with CS.

Sanderson found 7 of 8 patients treated with EEN and 5 of 7 patients treated with CS had normalization of CRP.

Overall, no difference was detected in CRP normalization between patients who received CRP or EEN (OR 0.85, [CI 95% 0.44, 1.67]; Figure 6b.

Comparison of CRP normalization in EEN vs. CS).

Again, heterogeneity was not demonstrated (I 2 =0%).


Our systematic review and meta-analysis of induction regimens in children with Crohn's disease, did not find a treatment benefit of corticosteroids over exclusive enteral nutrition.

This finding held true in the sub-analysis of newly diagnosed patients.

In our hypothesis generating analysis EEN also had superior mucosal healing odds (p<.05), suggesting that this is an end point to be explored in future trials.


There is also a possibility that the benefits of induction with enteral nutrition are more durable than induction with corticosteroids, but this finding is driven by one study, Berni Canani et al. 36 In fact, the two studies combined to study this question may not reflect current practice, namely using steroids or mesalamines as post-induction maintenance regimen in Crohn's disease.


Our findings are in contrast with the most recent Cochrane review on the subject 15 which found corticosteroids superior to enteral nutrition (OR 0.33).

This is potentially explained by the fact that we only used high-quality studies.

When the Cochrane authors restricted their analysis to only high-quality studies, they also found no difference between CS and EEN.

Another potential explanation for their contrary findings is that they combined data from studies of both adults and children.

Of the seven studies (n=352) they pooled, only one study (n=37, Borrelli et al. 35 ) was composed of pediatric patients.


Given the heterogeneity of the results when stratifying by population age, it would seem reasonable to avoid combining pediatric and adult data in future analyses as it may result in underestimating the benefit of enteral nutrition in the pediatric population.

Some physicians believe that the discordance in efficacy between adults and children is overstated.

In clinical situations where tolerability of the EN product is high, enteral nutrition can induce remission in adult populations, suggesting that improved tolerability should be an area of further research.


Two previous meta-analyses and two systematic reviews on the subject of treatments for induction of remission in children with Crohn's disease have been performed, and our results confirmed their findings.

Heuschkel et al. 21 (n=194), concluded that there was no difference in efficacy between enteral nutrition and corticosteroids to treat active Crohn's disease.

However, this study was underpowered (n=144) to identify differences less than/ equal 20% between the steroid and EEN groups.

6 Of the seven studies combined in their meta-analysis four did not meet our inclusion criteria.


Two studies 38,39 have only ever been published as abstracts, and have not undergone detailed peer-review.

It is not clear whether patients overlapped between the two studies by the same author and the decision to avoid Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.

including in their meta-analysis was shared by the Cochrane review group.

15 Thomas et al. 1993 40 , was excluded from our analysis because their outcome was "improvement", rather than remission.


Another, Breese et al. 1995 41 , included 8 different steroid dosing strategies (prednisone 5mg/d - 40mg/d) among 10 patients treated with corticosteroids.

The heterogeneous patient populations and an inability to evaluate details of trial mechanics, made outcomes for more than 50% of the included population impossible to assess.


An earlier meta-analysis, Dziechciarz et al. 20 (n=204), also suggested similar efficacy between EN and CS for treating active Crohn's disease in children.

Their meta-analysis combined 4 studies.


Three of these studies did not meet our inclusion criteria.


Like Heuschkel et al. 21 , Dziechciarz et al. 20 also included the same two abstracts from Seidman et al. 38,39 as previously described.


Terrin et al. 2 was published in an Italian journal with an English abstract and non-English manuscript.


Because the full manuscript was not in English, we excluded it from our meta-analysis.


Recently, Pegnani et al. 42 conducted a large systematic review of pediatric nutrition and CD with several end points, including the efficacy of enteral nutrition on the induction of remission in a pediatric population.

While they did not conduct a meta-analysis themselves, using evidence from Heuschkel et al. 21 and Dziechciarz et al. 20 , they concluded that enteral nutrition in a pediatric population can effectively induce remission.


However, they included Thomas et al.1993 40 and Terrin et al. 2002 2 , which we excluded for the reasons listed in the previous paragraph.

They also included Ruuska et al. 1994 43 , Grover et al. 2015 44 , and Soo et al. 2013 45 , which we excluded because the former two articles did not use remission as their primary end point and the latter one did not have an enteral nutrition only arm.


Day et al. 46 also conducted a broad-view systematic review without meta-analysis on enteral nutrition in the treatment of pediatric Crohn's disease with several end points.

While they also found that enteral nutrition can induce remission, they included the Seidman et al. abstracts 38,39 , the Thomas et al.1993 40 paper and the Terrin et al. 2 papers that we excluded for previously explained reasons.

While both of these systematic reviews contribute substantially to the understanding of pediatric CD and enteral nutrition, they were both broad in scope. Because we took a narrower view point and only looked at enteral nutrition and its' ability to affect remission induction, we were able to more completely examine the topic.


The goal of every meta-analysis is to include the best possible data and pool data from comparable patient populations.

Our rigorous inclusion criteria disallowed the majority of studies included in 2 previous meta-analyses.

Our meta-analysis included data from randomized controlled trials, prospective cohorts, and retrospective cohort studies.

Our systematic sensitivity analysis showed that when we stratified our results by study design, there was no difference across strata, meaning that the results from the prospective studies were similar to those from the retrospective studies and the RCTs.


By not including abstracts, we may have introduced publication bias into our study, but only including full manuscripts, we were able to more fully assess the limitations of each study and understand sources of bias in assignment of EEN vs. CS.

We have detailed these biases Aliment Pharmacol Ther.

Author manuscript; available in PMC 2018 October 01.


Current clinical guidelines, from both the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition and its European counterpart, consider enteral nutrition acceptable to use in all patients with Crohn's disease.

47,48 In fact, the European Society for Clinical Nutrition and Metabolism guidelines recommend EN as a first line therapy for children and adolescents with CD, with "strong consensus".

48 However, there are many persistent unanswered questions on this subject.

Specifically, the mechanism of action of enteral nutrition remains vague and there is still little guidance on which types of patients would be most likely to benefit, though weak evidence suggests effectiveness is greater with small bowel involvement.

7 With regard to disease duration, our analysis suggests that outcomes may differ between newly diagnosed and previously diagnosed patients.

Future guidelines may need to make that distinction when it comes to benefits of induction with these therapies 47 .

Challenges also remain in considering induction treatment duration and the best strategy to maintain remission as long-term exclusive enteral nutrition is unlikely to be acceptable to most patients.


In summary, our study suggests that exclusive enteral nutrition works equally as well as corticosteroids in inducing remission in pediatric Crohn's disease based on clinical symptom scores, but EEN could potentially be superior when assessing improvement by mucosal healing endpoints.

We believe these results suggest a need for further research in this area.

EEN may have the added benefit of minimizing growth failure, avoiding undesirable and difficult to reverse changes to body habitus, and can potentially result in a deeper and longer duration of remission.

As such, greater advocacy for this therapy among physicians from the United States may be warranted.


We read with interest the recent meta-analysis by Swaminath et al (2017) that reviewed studies on induction of remission with exclusive enteral nutrition (EEN) in paediatric Crohn’s disease (CD) [1].

We feel however that the title of the article is slightly misleading as the authors only include studies that directly compare the efficacy of EEN against corticosteroids (CS), excluding other studies where EEN was used, without a direct comparator arm with CS (table 2, page 1) excluding also points of secondary analysis like faecal calprotectin (FC).

We were encouraged to note the authors describe EEN to be as efficacious as CS, as shown in the previous AP&T meta-analysis; with the advantage of superior outcomes in terms of mucosal healing [2].

We are aware that FC associates with rates of mucosal healing [3].

Therefore, it seemed paradoxical to us that there was no difference in FC levels between EEN and CS, despite the significant differences in mucosal healing rates.


We looked for the FC data mentioned but found no evidence of the studies from which the abstract data was generated.

We performed our own literature search specifically for studies reporting changes to FC levels during EEN, independent of the necessity including a CS arm.

From an initial search using MEDLINE with the term “Exclusive enteral nutrition AND Crohn’s” we identified and reviewed 181 abstracts and were left with 18 relevant studies, including five studies from overlapping patient cohorts [4-10].

To prevent duplication of results, only one study from the group were considered for the analysis.

Using the mean or median FC as values presented from each study, we calculated the percentage decrease during the course of EEN and found that on average FC drops 48% on completion of an EEN course (figure 1 A and B).

All studies except Dunn et al report a significant decrease in FC concentration while on EEN.

This study reports FC as binary < or > 250 μg/g.

This may mask significant reductions in FC during EEN, that remains above normal reference values.


We identified only one study with paired longitudinal endoscopic mucosal healing and FC data.

However, the analysis performed in this study combined patients treated with either EEN or CS.

As they did not distinguish the treatment arms of the patients it was removed from our analysis [11].

While the exact mechanisms by which EEN works, and drives FC reduction, remain elusive; the gut microbiota plays a pivotal role [9,12].

It is well known that the composition and metabolic activity of the microbiota is altered during EEN.

The study from our group, Quince et al (2015), collected serial faecal samples and by using stepwise regression, we identified 14 operational taxonomic units that predicted 78% of the variation to FC.

This study and others have shown that EEN substantially modulates the microbiota, by reducing bacterial diversity, altering the relative abundance of metabolic pathways while altering the local microenvironment and major bacterial metabolites.

We suspect these mechanisms by extension would be important in the process of mucosal healing too although specific studies addressing this are needed.


We thank Drs.

Logan and colleagues for taking an interest in our paper and raising an important point regarding data on faecal calprotectin [1], [2].

Only 1 study in our systematic review contained information regarding faecal calprotectin.

Levine et al measured faecal calprotectin at baseline and at end of treatment in patients induced with exclusive enteral nutrition and corticosteroids [3].

Among those induced with exclusive enteral nutrition, 22 of 72 had normalisation of faecal calprotectin.

Only 3 of 23 treated with corticosteroids had normalisation of faecal calprotectin.

Although the difference was not statistically significant, the trend was towards greater improvement in the exclusive enteral nutrition group (OR 2.93, [CI 95% 0.79-10.90]).

Because only 1 study met our inclusion criteria, we did not feel it met the threshold to be included in the final manuscript, but failed to update the abstract.


We agree that the absence of statistical benefit does not imply an absence of benefit, but simply that the literature we reviewed was not mature enough to have enough studies with which to base any conclusions.

Logan et al do provide data on improvement in faecal from studies which did not meet our inclusion criteria.

We agree that biomarkers have taken on a large importance in the management of Crohn’s disease, and that future rigorous comparison studies of Crohn’s nutritional therapy should include biomarker and mucosal healing data as part of their design.


The human brain requires a series of complex neural pro- cesses to accomplish routine tasks.

For example, looking at an object and reaching for it first uses the visual system to locate and compute the spatial coordinates of the object of interest (Sparks, 1989; Andersen et al., 1993; Crawford et al., 2011).

The eyes and head then turn towards the object and gaze is stabilized in its new position to capture more detailed visual information (Guitton et al., 2003).


The arm then reaches for the object and, once stabilized in its new orientation, forms a platform from which the fingers can grasp the object (Gosselin-Kessiby et al., 2008; Monaco et al., 2015).

Each of these steps is accompanied by a brief transmission of information encoded in a short-lasting pulse of neural discharge.

To use such ongoing bits of information to maintain each step, the brain must convert the transient pulses of neural discharge into sustained neural activity, both at the higher levels of cognition, e.g. working memory (Goldman-Rakic, 1996; Curtis and Lee, 2010), and at lower levels for the control and maintenance of posture.


David Hearley, in the early 18th century, proposed the process of converting the pulse of neural discharge into persistent neuronal activity and formation of a short-term memory.

Alexander Bain, later in the 1800s, further elabo- rated the process of short-term memory.

Subsequent inves- tigators posited that the conversion of the pulse of neural discharge to steady-state firing requires sustained synaptic activity generated by reverberation in a neuronal network (Lorente De No, 1938; Hebb, 1949).


In the 1970s David Robinson further developed the concept of mathematical integration for the ocular motor system, based on the fun- damental observation that vestibular inputs encode 'vel- ocity' whereas the ocular motor effector, 'plant', is a 'position' actuator (Robinson, 1974).

This idea was sup- ported by studies showing that pharmacological inactiva- tion of circuits in the medulla, responsible for neural integration in subhuman primates, led to horizontal drift of the eyes from a target location (Cannon and Robinson, 1987; Godaux et al., 1993; Arnold et al., 1999).

Other experiments showed analogous integrators for the vertical and torsional components of eye orientation in the mid- brain interstitial nucleus of Cajal (INC), where torsion is defined as rotation around the line of gaze at its primary position (King et al., 1981; Fukushima, 1987; Crawford et al., 1991).

Together, these integrators appear to control eye orientation about axes or planes similar to those defined by the semicircular canals and the eye muscles themselves (Crawford et al., 2003).


Theoretical constructs for neural integration were sup- ported by studies suggesting that continual feedback be- tween the neurons was fundamental to ensure the efficacy of the neural integrator (Cannon and Robinson, 1985; Arnold and Robinson, 1991; Aksay et al., 2001, 2007; Miri et al., 2011).

To ensure perfect neural integration, the synaptic strengths of all network neurons had to be optimally tuned.

Most biological systems, however, cannot maintain optimal tuning indefinitely, making neural integrators inherently imperfect.


They can become 'leaky', leading to velocity-decreasing drifts toward a null position, or 'unstable', leading to velocity-increasing drifts away from the null position.

Imperfect integrators can be restored to more faithful activity by appropriate feedback.

The cerebellum is one of the key sources of feedback to the neural integrator (Carpenter, 1972; Robinson, 1974; Zee et al., 1976, 1980, 1981; Baier and Dieterich, 2011).


A deficit in cerebellar feedback can lead to imperfect neural integration associated with characteristic centripetal drifts in the eyes from eccentric targets.

The drifts are followed by corrective saccades in the opposite direction that rapidly realign the eyes towards the target of interest.

These alternating drifts and corrective movements define gaze- evoked nystagmus (Robinson, 1974; Zee et al., 1976, 1980, 1981).


Figure 1A-C depicts an example of typical gaze-evoked nystagmus.

The eyes are relatively stable in the straight- ahead position (Fig. 1B).

The eyes drift to the left after they are turned 30 to the right (Fig. 1A), and are followed by corrective saccades, or quick phases, in the opposite direction (Fig. 1A).

Rightward drifts and leftward quick phases follow after the eyes are turned 30 to the left (Fig. 1C).


The drift velocity, or slow-phase velocity, changes systematically with the eye-in-orbit position, lessen- ing as the eye-in-orbit position approaches straight-ahead (Fig. 1D).

The direction of the drift reverses when the eye- in-orbit position passes beyond the straight-ahead to the contralateral side (Fig. 1D).


All these features of drifts of the eyes can be attributed to suboptimal neural integration, and are distinct from the constant-velocity drift associated with pure vestibular dysfunction (Robinson, 1974; Zee et al., 1980, 1981; Leigh and Zee, 2015).


Until recently the neural integrator concept was largely confined to the ocular motor system.

In 2002 Klier and colleagues injected muscimol in the midbrain of subhuman primates to inactivate the INC and surrounding structures such as the rostral interstitial medial longitudinal fasciculus and nucleus of Darkschewitsch (Klier et al., 2002).

Based on previous experiments showing that the INC houses part of the ocular motor integrator, and its projections to the spinal cord via the interstitiospinal tract (Fukushima et al., 1981), they expected additional deficits in head movements.


As predicted, monkeys developed position-dependent def- icits in holding the head still, especially in the vertical and torsional dimensions (Klier et al., 2002; Farshadmanesh et al., 2007).

Attempts to voluntarily straighten the head caused repeated drifting of the head towards a resting position (Fig. 2A).

This motor behaviour was analogous to gaze-evoked nystagmus of the eyes (Fig. 1).

Indeed, when eye and head movements were re- corded simultaneously, their drifts were highly correlated Downloaded from https://academic.oup.com/brain/article/139/10/2590/2196503 by McGill University Libraries - Serials Unit user on 15 May 2024 (Farshadmanesh et al., 2007).

Eventually, the corrective movements abated and the head settled into a laterally tilted position (Fig. 1A).

Finally, unilateral stimulation of the INC produced head rotations that often held their final position (until corrected), as expected by the neural inte- grator theory (Fig. 2B).

In other words, unilateral stimula- tion and inactivation simply produced head tilts in opposite directions, although the underlying patterns of muscular activation were much more complicated (Farshadmanesh et al., 2008, 2012).


Based on these findings it was proposed that the INC could function as a neural integrator for both eye and head movements (Klier et al., 2002; Farshadmanesh et al., 2007).

They further proposed that the postural symptoms of cervical dystonia in humans, for example, the fixed neck posturing in torsion dystonia, could be explained by severely imbalanced activity within such an integrator, either from intrinsic dysfunction (as in Fig. 2A) or from an imbalance of inputs (as simulated by stimulation in Fig. 2B), or a combination of these two.

For example, the head only drifted in the early stages of INC inactivation because the system was still trying to correct head torsion (Fig. 2A).

The eventual settling of head pos- ition toward the torsional null position could thus be ex- plained either as an additional deficit in surrounding phasic inputs like the rostral interstitial medial longitudinal fascic- ulus (obliterating the corrective movements) or as the system accepting a new set-point for torsion at the null position.


Other experiments further suggested that the INC con- trols head orientation about the planes sensed by the four vertical semicircular canals, again analogous to its role in eye position control (Klier et al., 2002, 2007).

The head neural integrator in the INC receives inputs from the ven- tral-caudal and rostral mesencephalic reticular formation.

Injections of muscimol into the nucleus subcuneiformis located ventro-caudally in the mesencephalic reticular for- mation, lateral to the oculomotor nucleus, and caudal to the posterior commissure, as well as into the peri-INC mes- encephalic reticular formation, produces head tilts but with less accompanying ocular drift (Waitzman et al., 2000a, b).

There must then also be a horizontal head integrator.

Head rotations produced by stimulation in the area near the hori- zontal eye movement generator suggest that the head move- ment integrator might be located in the pons or medulla (like the eye integrator), but this head integrator has not yet been directly identified (Gandhi and Sparks, 2007).


The neural integrator for head movements is also inherently leaky and feedback-dependent.

Recent studies hypothesized that the head neural integrator relies on feedback using visual information, neck proprioception, and input from the cerebellum (Chan-Palay, 1977; Noda et al., 1990; Fukushima and Fukushima, 1992).

Suboptimal calibration of any of these feedback pathways results in impaired function of the head neural integrator.


Healthy subjects with optimal visual and proprioceptive feedback can hold their head steady in eccen- tric positions (Supplementary Fig. 1A and B).

If visual feed- back is removed the head drifts from eccentric head positions towards a central null, where drifts are minimal (Supplemen- tary Fig. 1C and D).

This observation implies the neural in- tegrator depends at least in part on visual feedback.


The velocity of centripetal drift increases when the position of the head shifts farther away from the null (Supplementary Fig. 1D), as predicted by the neural integrator hypothesis.


Head movements are more complex than eye movements in the sense that they are controlled by numerous neck mus- cles; any given neck muscle can be activated during move- ments in more than one plane, and movements can be around more than one joint.

Hence it is advantageous to use multiple independent sources of proprioceptive feedback to 'advise' the brain about the state of the position of the head (Shaikh et al., 2013).

When the proprioceptive signal is distorted by vibrating paraspinal neck muscles, centripetal drift of the head increases during eccentric head holding (Supplementary Fig. 1E).

The relationship between drift vel- ocity and head on trunk position had a steeper slope suggest- ing a synergistic relationship between visual and proprioceptive influences to improve the fidelity of the head neural integrator (Supplementary Fig. 1F).


In another experi- ment, in which normal visual feedback was allowed but Downloaded from https://academic.oup.com/brain/article/139/10/2590/2196503 by McGill University Libraries - Serials Unit user on 15 May 2024 proprioception was distorted by vibration, drifts in head pos- ition still emerged and were followed by rapid corrective head movements (Supplementary Fig. 1G and H) (Shaikh et al., 2013).

These slow drifts and rapid corrections that appear when feedback is altered resemble the jerky oscilla- tions of 'dystonic tremor' in subjects with cervical dystonia, a neurological disorder characterized by involuntary twisting and turning of the head in any of the three dimensions of head rotation.


Indeed, in the original description of the head neural integrator Klier and colleagues (2002) proposed that the abnormal head movements shown by monkeys after in- activation of the INC resemble the abnormal torsional head movements in subjects with cervical dystonia (laterocollis).

Based on this idea, one would expect to also see chronic horizontal tilts if one could continuously activate one side, and thus one direction, of the (as yet to be localized) hori- zontal head integrator.