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).


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.


The proposal by Klier and colleagues (2002) was at odds with prevailing views of cervical dystonia, which focused on defects in the basal ganglia (Dauer et al., 1998) or some- times the cerebellum (Neychev et al., 2011) or propriocep- tive defects (Tempel and Perlmutter, 1990; Bove et al., 2004).

As a result, their idea was neglected until more recent studies resurrected the concept by characterizing the abnormalities of head movement in subjects with cer- vical dystonia, in relation to the neural integrator model (Shaikh et al., 2013).

Figure 3A-L depicts head positions for a subject with cervical dystonia, where the favoured head position (null) was 20 to the left of the centre position on the trunk (Fig. 3G-I).

Drifts of head position were minimal when the subject's head was voluntarily held at the null.

Coarse and jerky head movements were seen as the head turned 20 to the right (40 from the null) (Fig. 3A-C), or straight- ahead (20 from the null, Fig. 3D-F).


Typical of cervical dystonia, the jerky movements were present in all three rotational planes including the horizontal plane (torticollis), vertical plane (antero/retrocollis), and torsional plane (later- ocollis).

In such instances, the slow drift in head position was directed towards the null.

The rapid movements to- wards the desired head position corrected for the drifts toward the null.

Furthermore, in all three planes the drift velocity systematically changed with head position.

Downloaded from https://academic.oup.com/brain/article/139/10/2590/2196503 by McGill University Libraries - Serials Unit user on 15 May 2024 horizontal-torsional planes.

Qualitatively the drifts are dir- ected towards the null, minimal drifts are present at the null, and there is a further increase in drifts in the opposite direction as the head passes to the opposite side of the null.

These features of the drifts are quantitatively shown in Fig. 3O-Q. Such jerky, irregular, multidimensional head movements, composed of drifts and corrections in cervical dystonia, are often called 'dystonic tremor', a term that has generated considerable debate (Quinn et al., 2011; Elble, 2013).

It is interesting to note that the movements of dys- tonic tremor are analogous to the gaze-evoked nystagmus of the eyes.

Such a resemblance suggested an alternative label for the 'dystonic tremor', and that is 'head nystag- mus'.

Though 'nystagmus' is a term more commonly used in reference to various types of eye oscillations, it can also be applied more generally to any movement that has repeti- tive cycles of a slow drift followed by a rapid corrective movement in the opposite direction (i.e. a jerk nystagmus).


Interestingly, the term 'head nystagmus' was used more than three quarters of a century ago by Hyndman (1939) in his in descriptions of the jerky oscillations in cervical dystonia.

Regardless of the best terminology for these head movements, the results show that they can be inter- preted as deficits in a head neural integrator (Shaikh et al., 2013).


In addition to the coarse, jerky, oscillatory movements, which we have likened to gaze-evoked nystagmus of the eyes, some subjects with cervical dystonia also had smaller amplitude, sinusoidal, and more regular oscillations.

These oscillations, resembling 'essential tremor', were superim- posed on the coarse jerky movements (Fig. 3A and D).


It was subsequently shown that these sinusoidal head oscilla- tions had characteristics similar to the pendular eye oscil- lations that are thought to originate from instability in the ocular motor neural integrator (Das et al., 2000; Shaikh et al., 2015b).


Three characteristic features of these sinus- oidal head oscillations supported their origin in an abnor- mal neural integrator.

First, the amplitude of oscillation depended upon the head-on-trunk orientation; second there was no influence of head position on the frequency of oscillations; and finally there was a reset in phase of the oscillation after rapid head movements.


Thus, these quan- titative studies of head movements in cervical dystonia pro- vided strong support for a mixed disorder, combining at least two subtypes of oscillatory head movements, and both could be related to abnormal function of a head neural integrator (Shaikh et al., 2013, 2015b).


Proposals suggesting a relationship of the head neural inte- grators in the INC or its surrounding area to cervical dys- tonia do not imply that pathology in cervical dystonia must be intrinsic to these regions.

Major inputs to this area are from the cerebellum (Fig. 4), a region already implicated in cervical dystonia (Pelisson et al., 1998


These new concepts for the pathogenesis of cervical dys- tonia, based on the idea of an impaired neural integrator that receives multiple sources of feedback, are not mere academic exercises in anatomical localization or physi- ology.

They have direct implications for treatment.

Specifically, the new concept points to potentially novel stimulation targets for deep brain stimulation.

Traditionally, deep brain stimulation for cervical dystonia targets the internal segment of the globus pallidus or the subthalamic nucleus, but outcomes are not predictable (Vidailhet et al., 2005; Kiss et al., 2007).

The neural inte- grator concept suggests that stimulation of the INC or its cerebellar inputs may be a future strategy to treat cervical dystonia.


The concept of involvement of the midbrain in the con- trol of 3D head orientation is not new.

In the early 1950s Hassler and Hess applied monopolar electrical stimulation near the INC to evoke head movements in normal humans (Hassler and Hess, 1954).

Two decades later, during elec- trical stimulation of the INC, medio-superior red nucleus, and parts of medical longitudinal fasciculus posterior and inferior to the intercommisural line and lateral to the mid- plane, Sano noted vertical head movements accompanied by marked contraction and electromyographic discharges of posterior neck muscles bilaterally, such as the splenius capitis and trapezius in subjects with cervical dystonia (Sano et al., 1970).

Ablation of these regions reduced retro- collis (Sano et al., 1970).

Stimulation of an area superior, anterior and lateral to INC resulted in contraction of both sternocleidomastoids accompanied by anteroflexion of the neck (Sano et al., 1970).

Periaquaductal stimulation using a weaker electrical charge extinguished electromyographic discharges in bilateral posterior neck muscles followed by improvement in neck muscle tone (Sano et al., 1970).

Hassler later targeted the efferent projections of the INC, the ventro-oralis internus thalami, for the surgical treat- ment of torticollis (Hassler and Dieckmann, 1970) and the prestitial nucleus for retrocollis (Hassler et al., 1981).

These procedures were largely abandoned as a treatment for cervical dystonia because results were sometimes unpre- dictable.


This unpredictability is not surprising, because of the lack of a guiding conceptual model regarding how ma- nipulations of these regions might alter head positions.

However, in light of the neural integrator hypothesis, we can begin to understand the subtleties of these effects, such as the initial drift of head position after the inactivation of INC (Fig. 2A), and the holding of final head orientation after unilateral stimulation of this nucleus (Fig. 2B).

Moreover, these new hypotheses incorporating a neural in- tegrator in the pathogenesis of cervical dystonia, encourage us to revisit these target areas with more refined techniques that might offer therapeutic benefit to subjects with cervical dystonia.


Contemporary concepts emphasize the role of feedback in the pathogenesis of dystonias and also link to the most popular treatment of cervical dystonia, botulinum toxin in- jections.

Such therapy not only affects the neuromuscular junction, but it also modulates the afferent output of the cholinergic extra and intrafusal fibres (Filippi et al., 1993; Rosales et al., 1996, 2006).

In support of this idea about pathogenesis are the effects of botulinum toxin on the spinal and supra-spinal reflex pathways (Rosales et al., 1996).

Additional evidence comes from reduced central ex- citability with trans-cranial magnetic stimulation in humans after injection of botulinum toxin type A injection into the extensor digitorum brevis muscle (Kim et al., 2006).

It is also possible that the effects of botulinum toxin are com- bined with the secondary effects of muscle weakening on spindle activity and proprioception.

Clinical observations also support the idea that botulinum toxin affects the muscle spindle as there is a lack of a simple relationship between the required dose of the toxin and the observed clinical benefit.

It is often noted that the abnormal neck


In summary, we present a novel conceptual framework for the pathogenesis of cervical dystonia that emphasizes the role of abnormal feedback to the midbrain head neural integrator.

The idea that proprioception, the cerebel- lum, and basal ganglia are key sources of feedback to the head neural integrator is compatible with contemporary perspectives on cerebellar or proprioceptive abnormalities as contributors to dystonia, as well as conventionally sug- gested impairments in the function of basal ganglia.

This proposed framework for the pathophysiology of cervical dystonia departs from longstanding traditional concepts of dystonia that focused exclusively on the basal ganglia.

More importantly, this framework suggests novel therapies for cervical dystonia such as chronic electrical stimulation of the cerebellum or modulation of proprioception using vibration or electrocutaneous stimulation devices.


We read with great interest the article by Shaikh et al. ( 2016) presenting a pathophysiological explanation of cervical dystonia.

Cervical dystonia is a neurological disorder characterized by involuntary twisting and turning of the head in any of the three dimensions.

The authors proposed that the abnormalities of head movements seen in the disease stem from a malfunctioning head neural integrator caused by either an intrinsic dysfunction or as a result of impaired cerebellar, basal ganglia, or peripheral feedback.

Several studies hypothesized that the head neural integrator relies on feedback using visual information, neck proprioception, and input from the cerebellum [1][2][3].

Starting from the hypothesis that visual feedback may play a crucial role in the functionality of the head neural integrator and, subsequently, in the pathophysiology of dystonia, we decided to analyse the straight-ahead preference in healthy human subjects and patients with cervical dystonia.

Several studies had previously demonstrated a dysfunction of visuo-spatial perception in cervical dystonia [4][5].

In particular, it had been suggested that egocentric space representation was impaired in cervical dystonia and that visual straight-ahead perception was shifted towards the trunk compared to normal subjects whose visual straight-ahead were claimed to coincide with the head midsagittal plane [4][5].


Our study was based on the observation that healthy human subjects gazing to the side had been reported to respond faster to objects appearing in the peripheral visual field when the objects were displayed at a location in the subjects’ straight-ahead direction compared to objects displayed at an eccentric location to the subjects’ position [6].

This effect was independent of the subjects’ direction of gaze and could be found even though the objects formed similar images on the retina and had the same distance from the fovea.

Recently, the described behavioural observations of a preferential processing for visual information coming from the straight-ahead direction could be confirmed by a functional MRI study in healthy subjects on stimulus-evoked blood oxygen level-dependent responses in V1 and V2 [7].

The authors provided evidence that visual stimuli elicited an enhanced response when presented closer to the straight-ahead direction.

The straight-ahead preference for visual information coming from the peripheral visual field may be part of the head neural integrator system, however, the exact underlying mechanism for this effect remains unclear.

Possible explanations could be a top-down or attentional modulation of visual cortex processing that allows the subject to select behaviourally relevant information, or a bottom-up modulation by extra-retinal signals, such as proprioceptive information encoding the eye position.


In their study with human subjects, Durand et al. used an attention-demanding test that required their participants to not only disengage their gaze but also their attention from the straight-ahead position [6].

As the results of this particular task showed the same preference for the straight-ahead perception of visual objects, the authors concluded that while an attentional explanation could not be excluded, the observed effect does not seem to require full attentional resources, and that there might be an automatic early integration of visual and postural inputs, which facilitate straight-ahead perception.

This explanatory model would be in line with several studies reporting on the influence of gaze direction or proprioceptive eye position signals on visual cortex processing [8][9][10].


When adapting the paradigm of Durand et al. to an experimental setting (see Supplementary material for method) including healthy subjects and patients with cervical dystonia, we found that patients with cervical dystonia did not exhibit any preferential reaction for targets displayed closer to their head or their trunk axis at the population level (see Supplementary material for results).

Our study may reveal a subtle perturbation of the visual information processing in cervical dystonia.

It is unclear whether or not the described impairment of visual information processing in cervical dystonia is part of the cause or of the result of the disease’s pathophysiology.

However, our findings underline that other sensory inputs and cortical processes are affected in cervical dystonia and that their impairment may contribute to the hypothesized dysfunction of the head neural integrator.

Additionally, they may broaden the spectrum of possible novel therapies mentioned by Shaikh et al. in that physical therapy using visual feedback techniques could be helpful in treating the disease [11].


We appreciate the thoughtful comments from Amlang and colleagues regarding our recent viewpoint [1]. They raise the important point that the neural integrator responsible for controlling head position relies on visual feedback in addition to feedback from the cerebellum, proprioceptors, and the basal ganglia.

To address these ideas, Amlang and colleagues analyzed the ‘straight-ahead preference’ in subjects with cervical dystonia and compared it with that of healthy subjects.

The ‘straight-ahead preference’ is a classic physiological phenomenon in which the reaction time to a novel image appearing in the peripheral visual field is longer if the eyes and the gaze are straight ahead, aligned with the trunk than if the eyes are turned to an eccentric location and the target appears straight ahead, in line with head or trunk [2].

This privileged visual processing of the straight-ahead direction in humans is of fundamental functional significance.

As multi-tasking creatures, we scan the environment while in motion.

As a result, gaze often is not straight ahead, but it is beneficial to be able to refixate promptly to targets that appear directly in front to avoid potential obstacles or accidents.

Such behavior would require the brain to have internal knowledge of the relative alignment of the position of the eye (retina) in the orbit, the head on the body.

Disorders affecting head movements, such as cervical dystonia, could interfere with the coordinate transformations necessary for optimal visual function.

The elegant experiments by Amlang and colleagues [1] provide proof of such a principle.


They found an overall increase in reaction time in subjects with cervical dystonia.

The reaction time to the peripheral target location that was in line with the sagittal plane passing through the head was still less compared to a peripheral target that was eccentric compared to the head.

It was unclear whether impaired visuospatial processing in cervical dystonia is the cause or consequence of these phenomena.

The authors now speculate that altered visual input can affect the function of a presumed head neural integrator.

We agree with this possibility as an additional mechanism for an impaired head neural integrator.

Here we offer another insight on how impaired neural integration affects visuospatial processing and reaction times.


These results can be explained given not only the head neural integrator hypothesis, but in general as a consequence of motor uncertainty or a mismatch between the efferent motor output and the efference copy being used for transformations.

There are two ways the brain receives head-on-trunk orientation.

One is via an efference copy from the neural integrator and the other is from proprioceptive afference.

Impairment in a head neural integrator could interfere with processing of both types of information.

As a result, the position signals necessary for coordinate transformations become less faithful, possibly more noisy and consequently increase the chances of inaccuracy.

To compensate for this potential for error the reaction time could be increased to give the brain more time to become more certain of the needed response.

It is also possible, however, that the head neural integrator has little role in such a phenomenon and that the increase in the reaction time is merely due to extraneous ‘noise’ in proprioceptive muscle output in cervical dystonia.

In such a situation, one might expect an indirect modulation of the output from the head neural integrator.


Subjects with cervical dystonia often present with a jerky head tremor [11][12][13].

These head oscillations and consequent eye oscillations (due to a tremor-induced but normal vestibulo-ocular reflex response) lead to a constantly changing head-on-trunk orientation and eye-in-orbit position.

As a result, there is a discrepancy as the target image on the retina remains stable, but the head-on-trunk and eye-in-head orientations are constantly in flux.

Such extraneous signals could also increase motor uncertainty and increase reaction time.

Several other studies suggested that visual-spatial orientation is disrupted in cervical dystonia [14][15][16] showing that the perception of visual-spatial orientation is referenced to the head in normal subjects, while in cervical dystonia it is shifted to the trunk.


In summary, we agree with the possibility that abnormal visuospatial processing could contribute to dysfunction of the head neural integrator.

However, it is also plausible that dysfunction of the head neural integrator itself can also affect visuospatial coordinate transformations.

This concept is consistent with the notion that cervical dystonia is not a disorder localized to a single brain region, but instead is a consequence of widespread impairment in a neural network responsible for sensorimotor integration.

With the considerable complexity of these neural networks controlling the position of the head, disentangling the core deficit causing cervical dystonia remains a challenge.

Nevertheless, the concept of a head neural integrator provides a conceptual model that may help to guide further studies of the pathophysiology of this disorder.


Employing advanced techniques in chemical synthesis consti- tutes an effective approach to realizing unconventional compounds, sometimes opening access even to new classes of materials featuring, e.g., novel bonding principles.

One such example concerns multinary silver oxides, which are thermally notoriously labile.

Using specially designed Bridgeman-type autoclaves made of distinctly scaling-resistant steel, 1 enduring conditions applied of up to 7 Â 10 8 Pa pressure of oxygen and up to 973 K temperature enables suppression of the thermal degradation of Ag 2 O and thus reaction of this oxide in all-solid state reactions with any other binary oxide in the periodic table.

In a preconceived view, one would expect the oxides attainable this way, Ag x M y O z (M ¼ nonmetal or metal), to represent analogues of respective alkali metal oxides.


However, systematic exploration of such systems has revealed that singular early observations of conspicuously short Ag + -Ag + separations in oxides are not strange exceptions, but manifestations of a general feature of the chemistry of silver.

2-4 Primarily in oxides with high silver contents, silver(I) ions tend to aggregate form- ing partial structures that are topologically reminiscent of elemental silver.

Furthermore, such structural motifs are asso- ciated with specic physical properties.

5, 12 for which precise dimensions have been determined here for the rst time, unaffected by disorder 13 or under-occupation of atomic positions.

14 The ndings shed light on the common effects of covalent bond length contractions caused by multiple bonding and/or superimposed polar contributions.

Comparison with isosteric P 4 O 10 reveals that the bond distances to the terminal oxygen atoms are signicantly less contracted in the borate, which does not feature any low lying orbitals that might mediate multiple bonding, while in accordance with the small difference in the electronegativities 15 of boron (2.051) and phosphorus (2.253), possible contractions due to polarities of the B-O and P-O terminal bonds are of comparable magnitudes.


The strange overall composition encountered is even more puzzling since such a kind of ternary oxide, a silver borate, is expected to follow heuristic rules of stoichiometry.

In the light of the electron counts given, which suggest the presence of itinerant excess electrons, it comes as a surprise that according to resistivity measurements the title compound is a small band gap semiconductor.

However, anal- yses of electron localizations using DFT based calculations comply with the experimental results.

Apparently, a new generalizable facet of the chemistry of silver becomes recog- nizable, which will complement text book knowledge of this element and bears potential for realizing interesting material properties due to the subtleties of electron localizations found.


Polycrystalline samples of Ag 16 B 4 O 10 were prepared by the solid state reaction of elemental Ag and H 3 BO 3 , or B 2 O 3 , in stainless- steel autoclaves at elevated oxygen pressures of 2 Â 10 7 to 5 Â 10 7 Pa and temperatures of 563-623 K. The starting materials were intimately mixed, pressed to pellets and placed in gold tubes, which were sealed at one end and crimped at the other.

Small amounts of H 2 O were added into the gold tubes to promote crystallization.

In a typical synthesis batch 0.03 mol of Ag and 0.01 mol of H 3 BO 3 were used, with the addition of 0.5 mL H 2 O.


Laboratory powder X-ray diffraction (PXRD) data (Fig. 1) were collected at room temperature on a Bruker D8 diffractometer with germanium monochromatized Cu-Ka1 radiation (l ¼ 1.5406Å), in steps of 0.01 over a 2q range of 10-90 degrees.

A Rietveld prole t was carried out with the TOPAS-4.2.0.2 (AXS) program.

16 The rened parameters were scale factor, sample displacement (mm), background as a Chebyshev polynomial of 5th degree, 1/x function, crystallite size, micro-strain (Stephens broaden model 17 ) and cell constants.

In addition, temperature dependent PXRD data were collected in the temperature range from room temperature to 773 K, in intervals of 50-100 K, as shown in the ESI, Fig. S1.†


A crystal suitable for single-crystal X-ray diffraction was selected in a drybox (M. Braun, Garching, Germany) under an argon atmosphere (<0.1 ppm O 2 , H 2 O) and mounted in a sealed glass capillary.

Diffraction data were collected at room temperature (298 K) with a SMART APEX-I CCD X-ray diffractometer (Bruker AXS, Karlsruhe, Germany), using Mo-Ka radiation.

The inten- sities of the Bragg reections were integrated with the SAINT subprogram in the Bruker Suite soware.

18 A multi-scan absorption correction was applied using SADABS.

19 The crystal structures were solved by direct methods and rened by full- matrix least-square tting with the SHELXTL soware package.

20,21 Experimental details and crystallographic data are given in Tables S1-S3.†


Magnetic properties were studied using a Quantum Design MPMS SQUID Magnetometer.

Zero-eld-cooled (ZFC) and eld- cooled (FC) magnetic susceptibility data were recorded in a 10 000 Oe eld while warming the sample from 5 to 300 K. Resistivities of polycrystalline bars (approximate dimensions 3 Â 3 Â 10 mm 3 ) of Ag 16 B 4 O 10 were recorded using a standard four-probe dc technique on a Quantum Design physical prop- erty measurement system.


Density functional (DFT) calculations, based on the experi- mental structure of Ag 16 B 4 O 10 , were performed using the CRYSTAL17 program package.

22 The bands in the semi-core and valence space, considering 19 valence electrons of each silver atom, 6 for O and 3 for B, are expanded in terms of local Gaussian basis functions.

The core electrons are represented by scalar relativistic pseudopotentials.

Details on the exponents and contractions are given in the ESI.† The integration in reciprocal space was based on 242 k-points in the irreducible part of the Brillouin zone.

The results presented here are ob- tained with the short-range-separated hybrid functional HSE- sol 23 for the exchange and correlation terms in the Kohn-Sham equations.


Atomic charges were evaluated by analyzing the electron density topologically according to the QTAIM approach.

24 A search of the atomic basins was performed with the critic2 (ref.

25 and 26) program on the basis of a 321 Â 321 Â 201 grid of data points of the valence electron density, augmented by core densities.

Within the basins, the valence density was integrated in order to get the net charges.

Data grids This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.


In more detail, all silver atoms are engaged in octahedral homoatomic building units, which are linked by sharing edges and vertices to form a 3D framework.

Every silver atom is coordinated by either one or two oxygen atoms at a distance typical of this pair of atoms d(Ag-O) ¼ 2.20-2.35Å (see Table S3†).


The rare complex anion B 4 O 10 8À has been observed here as an "isolated", i.e. unbridged, entity for the rst time.

12 Two previous reports present this building block as part of an extended framework.

Moreover, in both cases the structural analyses performed suffer from disorder, where occupation factors of 1/2 for the intra-cage bridging oxygen atoms even question the connectivity, i.e. the presence of such an integral anion, at all, 13 or from rening split atom positions for the anion in two orientations, impairing the accuracy of the data obtained.

14 The molecular anion, see Fig. 5, is isostructural and (valence) isoelectronic to P 4 O 10 .


The bond lengths are in the expected range, see Tables 1 and S3,† and the variations reect the position of the oxygen atoms, terminal or bridging within the adamantane type of cage.

Drawing the chemical bonds of the cage anion in terms of the Lewis concept requires placing a formal negative charge on both, the boron and the terminal oxygen atoms.

Although a formalism, this tells that the bonding situation is special and suggests looking for the respective structural distinguishing features by comparing with the This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

View Article Online isosteric cage molecule P 4 O 10 .

Such an approach would imply comparison of the bonding of elements from the second and third row of the periodic table.


For the latter, contributions of d p -p p multiple bonding and superimposed polar interactions are factors of inuence, 34 while for the former such d p -p p , or p p -p p interactions are not relevant; consequently, the bonding within the borate anion, where all boron atoms are in tetrahe- dral coordination, has to be analyzed exclusively in terms of s bonding schemes.

Indeed, comparing the bond lengths in B 4 O 10 8À and P 4 O 10 (ref. 35) reveals signicant differences.

In the borate the terminal B-O bonds are only 4.4% shorter than the bridging ones, whereas the respective shrinkage amounts to 10.1% for the phosphorous oxide, see Table 1.

Obviously, for the P-O terminal bonds multiple bonding and polarity act in a syner- gistic fashion, while in the borate for the shrinkage of the B- O terminal bonds only polar inter-actions superimposing the s bond are relevant, resulting in a smaller net effect.


The B 4 O 10 8À cage like polyoxoanion is electron precise, and thus the excess electrons according to (Ag + ) 16 (B 3+ ) 4 (O 2À ) 10 Â 8e À have to be accommodated by the silver substructure.

As a consequence, one would expect itinerant electrons being present, giving rise to metallic conductivity.

Surprisingly, the compound is a small band gap semiconductor, and diamag- netic, as shown in Fig. 6.

By tting the resistivities in the high temperature range above 150 K to the Arrhenius equation (Fig. 6 inset), an experimental band gap of 4.71(1) meV was obtained.

This value is essentially identical to the calculated one, see below.

The diamagnetic susceptibilities are estimated to be À1147 Â 10 À6 cm 3 mol À1 , based on an analysis of the magnetic susceptibilities applying a Curie-Weiss, Pauli and diamagnetic term.

The upturn tail below 30 K is dominated by an unknown paramagnetic impurity (about 0.2% with S ¼ 1/2).

These results indicate the presence of localized and paired excess electrons.


However, no particularly shortened individual Ag-Ag bond- length can be identied that would provide evidence for normal 2e-2c bonds, although the interatomic silver separa- tions are on average smaller than those found for elemental silver.

As can be seen from Fig. 4 (red circle) the 'empty' octahedral silver units appear to cluster, forming blocks con- sisting of ve edge sharing octahedra.

Each such cluster entity features two tetrahedral voids, where three out of six edges formed by silver atoms are substantially shorter (2.80Å on average) than the separation in elemental silver (2.89Å).

This gives a rst qualitative hint to the fact that these are the regions in real space where the excess electrons might be accommo- dated.

Since these tetrahedral sites add up to four per formula unit, which in total may host four pairs of excess electrons, i.e. all of them, this view would comply with the experimentally found magnetic and transport properties.


We exclude the possible presence of hydride anions, lling the contracted voids, for chemical reasons.

Along any synthesis protocol yielding the title compound protons are essential constituents, and, moreover, applying an elevated oxygen pressure is an indispensable requirement.

The pronouncedly basic and strongly reducing hydride ion cannot exist under such conditions.


In order to back this explanation quantitatively, we per- formed a DFT based computational analysis on the density of states (DOS), electron densities and localizations.

DOS as well as the projections onto the atoms (PDOS) is shown in Fig. 7a.


Over the whole energy range, which is shied relative to the Fermi level (E F ¼ 0 eV), oxygen basis functions contribute to the DOS.

Below À7 eV, the silver PDOS is low.

Bands are at and can be associated with the B-O bonds in the B 4 O 10 8À unit.

The low contribution of boron to these bands points to the ionic char- acter of the B-O bonds.


The DOS between À7 eV and À3.2 eV is dominated by the 4d states of the silver atoms, however, it From À1.44 eV up to the Fermi level bands with higher dispersion are located.

The respective atomic contributions can be visualized by drawing isosurfaces of the electron density computed from these bands (Fig. 7b).


These bands are delo- calized over the whole crystal.

Note, for clarity reasons, only a cut-out of the isosurface is shown in Fig. 7b.

The bands are lled and the system is not metallic.

There is a distinct band gap in most parts of the Brillouin zone.


Only at k ¼ (1/2 1/2 1/6) it becomes as tiny as 15 meV (Fig. 7c).

The highest valence band and the lowest conduction band exhibit a parabolic dispersion.

Signicant effects of spin-orbit coupling are not expected, as the Ag 4d-shell is practically lled and the 5d-orbitals are only affected indirectly.


Moreover, the projected densities of states show the same character in both bands.

The band gap at k ¼ (1/ 2 1/2 1/6) can be seen only in calculations with hybrid func- tionals.

When using gradient corrected (GGA) functionals without fractional Hartree-Fock exchange the band gap is underestimated and vanishes, although still a minimum of the DOS can be seen at E F .


The partitioning of space resulting from a topological analysis of the total electron density 24 gives atomic volumes and net charges, which are quite robust with respect to the choice of the basis set.

The values computed for Ag 16 B 4 O 10 are summarized in Table 2.

The volumes and charges at the various atomic positions are clearly related to the connectivity.

Ag3 and Ag4, which have a lower positive charge, have only one short distance <2.4Å to the neighboring oxygen, whereas Ag1 and Ag2 with a higher positive charge have two of them.


Like- wise, the negative charge of the oxygen atoms varies, depending on whether they occupy a bridging or terminal position in the B 4 O 10 8À unit.

The high positive charge of the boron atom points to a substantially ionic character of the B-O bond.


Further information on the bonding characteristics in posi- tion space can be obtained by the inspection of the electron localization function 27,28 (ELF), which can take values between 0 and 1.

High ELF values h are related to atomic shells, covalent bonds and lone pairs.

The ELF in Ag 16 B 4 O 10 comprises three distinct features.

At rst, there are nearly spherical domains around each silver atom, representing the lled 4d 10 shells.

In Fig. 8, these domains are omitted for clarity reasons.


The domains associated with the B 4 O 10 8À anion are repre- sented in Fig. 8a showing the lone pairs of the oxygen ions and one attractor respectively along each B-O bond.

The fact that the attractor is close to the oxygen atom is another hint to the polar character of this bond.

A special feature of the ELF, an attractor at h ¼ 0.23 appears in each tetrahedral void of the silver network.

Fig. 8b shows the domain with h ¼ 0.23 around one of them.


h ¼ 0.23 is a rather low value, though it represents a clear maximum in the ELF.

Attractors with low ELF values may originate from the inuence of close-by lled d-shells, 36 nevertheless pointing to covalent interactions in the valence shell.

Indeed, the face of the tetrahedron closest to the attractor contains two of the shortest Ag-Ag bonds (<2.80Å) within the range from 2.78Å to 3.23Å of all Ag-Ag contacts (cf.

metallic silver 2.89Å).


Putting