Effect of Two Mucoprotectants, Gelatin Tannate and Xyloglucan plus Gelatin, on Cholera Toxin-Induced Water Secretion in Rats

Round 1

Reviewer 1 Report (New Reviewer)

 

This paper by Eutamene and Theodorou assesses the effect of several compounds that have been shown to be mucoprotectants in a model for cholera toxin (CT)-induced intraloop water secretion in rats. The findings aim to highlight the beneficial properties of specific combinations of mucoprotectants when combatting water loss during diarrheal disease, an important cause of dehydration. The compounds are tested in 4 different treatment regimens: 4 days pre-treatment with the mucoprotectants followed by CT, treatment 12 h before CT application, treatment 6 h before CT and concomitant administration of mucoprotectant and CT. The materials and methods are well-written and adequately referenced, and the controls are generally sufficient. However, I do not think the conclusions are fully supported by the results shown, and the structure is repetitive and lacks insight into the reasoning behind some of the decisions made, making the read a bit confusing at times. \my specific comments are detailed below:

1.       In the discussion the authors keep on reiterating that the results show that the compounds exert their mucoprotective effects through the formation of a mucoadhesive film (lines 267, 281-282), but no evidence of this is provided in the article. Have the authors done any  histology with staining to show this?

2.       Also regarding supporting conclusions with results, I think the authors should tone downthe overall conclusion that both gelatin tannate and xyloglucan+gelatin reduced  CT-induced water secretion, as this depends on the specific treatment regimen tested.

3.       I am not sure of how relevant the concomitant administration of the compunds and CT is, as one imagines the polymeric compounds are just trapping the CT and this is why all are effective (tannic acid alone would not have these properties).

4.       As one reads the results, you are faced with a series of paragraphs that all start in the exact same manner (line 132, 147, 162, 175, 191): “After the 2-hour CT challenge, mean intra-loop water secretion was…”, making the read rather monotonous. While this would not necessarily be a major issue, the monotony could be solved by explaining the rationale behind choosing different compounds to be tested in the different treatment regimes. For example, the authors start assessing gelatin tannate, gelatin and tannic acid (Fig 1), then forgo tannic acid and keep the rest adding xyloglucan (alone, with gelatin tannate and with gelatin 125 mg/kg) (Fig 2A), and then just test xyloglucan but with two different gelatin combinations (2B), to then end testing everything again (Fig 3). Is it because some show more promise as you progress? Or because of some technical obstacles or intrinsic difficulties associated with the use of the compounds? And why the two different gelatin concentrations in Fig 2B?

5.       Figures should have figure titles that summarise what they show.

6.       When it’s said that there are 8 rats per group, that doesn’t mean these are biological replicates, were the experiments on each group performed at the same time? Or separated into biological repeats? Statistic analysis should only be performed if several biologically independent repeats have been done.

7.       Why does the mean change in loop weight in the control and vehicle groups differ so much in figure 3A and figure 3B?

8.       In the materials and methods, the authors should be more clear in regards to what the vehicle group is administered (line 83) as the vehicle in this case I imagine would be the 0.25 ml NaCl 0.9%, as the CT in 1 mL NaCl 0.9% I would imagine was given to all groups (also test groups) except the control group mentioned in line 84.

9.       I miss the presence of alternative readouts of disease, as the article only shows one readout (weight change of loop).

10.   If the relevance of the testing of this compounds resides in their potential use as treatment of acute diarrhoea (lines 43-48 for example), once there is onset of disease, wouldn’t it have been more relevant to test their effects in a model where they can be administered after inducing diarrhoea?

 

Comments for author File: Comments.pdf

Author Response

This paper by Eutamene and Theodorou assesses the effect of several compounds that have been shown to be mucoprotectants in a model for cholera toxin (CT)-induced intraloop water secretion in rats. The findings aim to highlight the beneficial properties of specific combinations of mucoprotectants when combatting water loss during diarrheal disease, an important cause of dehydration. The compounds are tested in 4 different treatment regimens: 4 days pre-treatment with the mucoprotectants followed by CT, treatment 12 h before CT application, treatment 6 h before CT and concomitant administration of mucoprotectant and CT. The materials and methods are well-written and adequately referenced, and the controls are generally sufficient. However, I do not think the conclusions are fully supported by the results shown, and the structure is repetitive and lacks insight into the reasoning behind some of the decisions made, making the read a bit confusing at times. \my specific comments are detailed below:

1. In the discussion the authors keep on reiterating that the results show that the compounds exert their mucoprotective effects through the formation of a mucoadhesive film (lines 267, 281-282), but no evidence of this is provided in the article. Have the authors done any  histology with staining to show this?

We thank the reviewer for this relevant question. In this study, we did not conduct any histological study demonstrating the mucoadhesive properties of the compounds tested. However, we suggest that their mucoprotective effects may result from interplay with the intestinal mucus network during both oral and local administration based on data from the literature. Indeed, as mentioned in the Discussion section, gelatin tannate interacts with mucins via electrostatic bonds which occur rapidly [Freli et al. 2013]. Further, Scaldaferri et al. (2014) evidenced by confocal microscopy the polymeric layer of gelatin tannate over the ulcerated colonic mucosa of colitic mice and, using atomic force microscopy, they showed that gelatin tannate treatment restored mucus elasticity (decreased in colitic mice) to a level similar to controls. Concerning xyloglucan, Piqué et al. (Int J Mol Sci, 2018 ; this study is cited in the Discussion section) using GOLD v5.2 and electrostatic potential calculation by Advanced Poisson–Boltzmann Solver (APBS) methodology nicely demonstrated that xyloglucan from the seed of the tamarind tree (the compound used in our study) exerts molecular docking to MUC1 and consistent binding to MUC1 via hydrogen bond interactions (also occurring rapidly), highlighting its mucoadhesive properties. Based on these data showing the biophysical interactions (electrostatic and hydrogen bonds) between the polymeric structure of the compounds tested and the intestinal mucus we can speculate that the mucoprotective effects of gelantin tannate and xyloglucan observed herein may relate to their mucoadhesive properties resulting in enhancement of the intestinal mucus barrier.

2. Also regarding supporting conclusions with results, I think the authors should tone downthe overall conclusion that both gelatin tannate and xyloglucan+gelatin reduced  CT-induced water secretion, as this depends on the specific treatment regimen tested.

The overall conclusion has been reworded to indicate that the conclusion applies to this series of experiments.

3. I am not sure of how relevant the concomitant administration of the compunds and CT is, as one imagines the polymeric compounds are just trapping the CT and this is why all are effective (tannic acid alone would not have these properties).

As mentioned above, the compounds tested can exert mucoadhesive properties due to biophysical interactions as polymers with the intestinal mucus network by, for instance molecular docking of xyloglucan to MUC1, hydrogen bonds with this mucin or electrostatic interactions of gelatin tannate with mucus and enhancement of mucus elasticity in pathologic conditions such as experimental colitis. These data underline the ability of the polymeric compounds tested in this study to exert an overall enhancement of the intestinal mucus barrier including the possibility of trapping CT toxin in the strong layer formed. 

4. As one reads the results, you are faced with a series of paragraphs that all start in the exact same manner (line 132, 147, 162, 175, 191): “After the 2-hour CT challenge, mean intra-loop water secretion was…”, making the read rather monotonous. While this would not necessarily be a major issue, the monotony could be solved by explaining the rationale behind choosing different compounds to be tested in the different treatment regimes. For example, the authors start assessing gelatin tannate, gelatin and tannic acid (Fig 1), then forgo tannic acid and keep the rest adding xyloglucan (alone, with gelatin tannate and with gelatin 125 mg/kg) (Fig 2A), and then just test xyloglucan but with two different gelatin combinations (2B), to then end testing everything again (Fig 3). Is it because some show more promise as you progress? Or because of some technical obstacles or intrinsic difficulties associated with the use of the compounds? And why the two different gelatin concentrations in Fig 2B?

The Methods have been restructured to better explain the aim of each set of experiments. As each set of experiments informed the next set, the rationale for choice of compounds per experiment is clearer. The Results have been restructured to align with the Methods including relocating the figures. The Discussion follows the sequence of experiments.  

5. Figures should have figure titles that summarise what they show.

Figure titles have been amended accordingly to state what is shown in the figure.

6. When it’s said that there are 8 rats per group, that doesn’t mean these are biological replicates, were the experiments on each group performed at the same time? Or separated into biological repeats? Statistic analysis should only be performed if several biologically independent repeats have been done.

We fully agree with the reviewer’s comment and we apologize for the lack of this information in our manuscript. Indeed, our results come from two biological replicates of each experimental set (this is now added in the experimental design section, line 70).

7. Why does the mean change in loop weight in the control and vehicle groups differ so much in figure 3A and figure 3B?

In in vivo studies, we often encounter differences in biological responses between different series of experiments. However, the treated groups are always compared with the corresponding control/vehicle groups for each series.

8. In the materials and methods, the authors should be more clear in regards to what the vehicle group is administered (line 83) as the vehicle in this case I imagine would be the 0.25 ml NaCl 0.9%, as the CT in 1 mL NaCl 0.9% I would imagine was given to all groups (also test groups) except the control group mentioned in line 84.

The text has been revised for greater clarity:

“In all experiments, the effects of test compounds in treated groups of rats (test compound + CT in 1 mL NaCl 0.9% in the jejunal segment) were compared with those in the vehicle group (0.25 mL NaCl 0.9% + CT in 1 mL NaCl 0.9% in the jejunal segment). Each experiment also included a control group (0.25 mL NaCl 0.9% + 1 mL NaCl 0.9% in the jejunal segment).”

9. I miss the presence of alternative readouts of disease, as the article only shows one readout (weight change of loop).

As indicated in the title of our article, in this study, we focused on the cardinal readout of CT, which is water secretion and accumulation in the intestinal lumen. To evaluate this parameter we used intestinal loops commonly reported in the literature for evaluation of the CT secretory effect.

10. If the relevance of the testing of this compounds resides in their potential use as treatment of acute diarrhoea (lines 43-48 for example), once there is onset of disease, wouldn’t it have been more relevant to test their effects in a model where they can be administered after inducing diarrhoea?

This set of preclinical experiments was conducted with the specific aim of investigating the protective ability, protective duration, and mode of action of the test compounds. We agree that further research would be useful to test the effects after onset of diarrhea, although this has already been shown in clinical trials of acute diarrhea with gelatin tannate, xyloglucan and xyloglucan + gelatin.  

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

This MS reports the use of an in vivo animal model to assess the effects of combinations of muco-protective substances 

SPECIFIC COMMENTS

1. It would've been interesting/useful to have a further experimental arm whereby the substances were assessed for their treatment effect. This would bring the findings to be more clinically relevant. One does not take an anti-diarrhoeal agent for hours or days prior to catching Cholera: rather one has the treatment after exposure/after onset of symptoms

2. The various experiments (as in Figures 1 and 2 for example) utilised different treatment arms. In figure 1, all treatments were separate - in later experiments, a combination was employed. Why were the same doses/combinations not used for all experiments?

3. When one gives a longer or shorter period of the active agents, does this lead to particular changes in the mucous layer? It would've been helpful to have further focus on what happens with the agents at the epithelium after each type of exposure (e.g. histological analysis comparing baseline/control with each agent at relevant dose). Such information would augment the evaluation of fluid secretion alone

Author Response

SPECIFIC COMMENTS

1. It would've been interesting/useful to have a further experimental arm whereby the substances were assessed for their treatment effect. This would bring the findings to be more clinically relevant. One does not take an anti-diarrhoeal agent for hours or days prior to catching Cholera: rather one has the treatment after exposure/after onset of symptoms

This set of preclinical experiments was conducted with the specific aim of investigating the protective ability, protective duration, and mode of action of the test compounds. We agree that further research would be useful to test the effects after onset of diarrhea, although this has already been shown in clinical trials of acute diarrhea with gelatin tannate, xyloglucan and xyloglucan + gelatin. 

2. The various experiments (as in Figures 1 and 2 for example) utilised different treatment arms. In figure 1, all treatments were separate - in later experiments, a combination was employed. Why were the same doses/combinations not used for all experiments?

The Methods have been restructured to better explain the aim of each set of experiments. As each set of experiments informed the next set, the rationale for choice of compounds per experiment is clearer. The Results have been restructured to align with the Methods including relocation of the figures. The Discussion follows the sequence of experiments. 

3. When one gives a longer or shorter period of the active agents, does this lead to particular changes in the mucous layer? It would've been helpful to have further focus on what happens with the agents at the epithelium after each type of exposure (e.g. histological analysis comparing baseline/control with each agent at relevant dose). Such information would augment the evaluation of fluid secretion alone

We thank the reviewer for this relevant comment. In this study, we did not perform an evaluation of mucus changes (histology, microscopy, rheology) occurring from the different sets of experiments. However, we suggest that the compounds tested exert beneficial effects when administered orally or locally through mucus network enhancement based on previous data reported in the literature. Indeed, as mentioned in the Discussion section, gelatin tannate interacts with mucins via electrostatic bonds, which occur rapidly [Freli et al. 2013] conferring mucoadhesive properties. Further, Scaldaferri et al. (2014) evidenced by confocal microscopy the polymeric layer of gelatin tannate over the ulcerated colonic mucosa of colitic mice and, using atomic force microscopy, they showed that gelatin tannate treatment restored mucus elasticity (decreased in colitic mice) to a level similar to controls. Concerning xyloglucan, Piqué et al. (Int J Mol Sci, 2018 ; this study is cited in the Discussion section) using GOLD v5.2 and electrostatic potential calculation by Advanced Poisson–Boltzmann Solver (APBS) methodology nicely demonstrated that xyloglucan from the seed of the tamarind tree (the compound used in our study) exerts molecular docking to MUC1 and consistent binding to MUC1 via hydrogen bond interactions (occurring also rapidly), highlighting its mucoadhesive properties. Based on these data showing the biophysical interactions (electrostatic and hydrogen bonds) between the polymeric structure of the compounds tested and the intestinal mucus we can speculate that the mucoprotective effects of gelatin tannate and xyloglucan observed herein may relate to their mucoadhesive properties resulting in enhancement of the intestinal mucus barrier.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Many substances administered as pretreatments u edblock cholera toxin ,  but fail to have any effect when administered after CT has induced diarrhea;

such findings have little or no significance.

The authors should determine whether treatment given after CT has had its effect has any influence can diminish or reverse its effects.

Author Response

Many thanks for your review and critique of our manuscript.

In our study, because of the methodology of the two types of experiments conducted (oral administration of test compound(s) before CT inoculation; local administration of test compound(s) concomitantly with CT inoculation), we can conclude that 1) gelatin tannate and xyloglucan+gelatin attenuate CT-induced intra-loop fluid secretion; 2) the mechanism of protection is via the formation of muco-adhesive films. Determining whether test compound(s) can diminish or reverse CT-induced intestinal fluid secretion once the effect is established would require a separate set of experiments and could form the basis of future research.

Reviewer 2 Report

Overall the manuscript needs to expand the experimental design in order to fully support the conclusion. Each Figure has some flaws and need to fix. For example, why authors omit the effect of XG + gelatin on Figure 3. Do author check the electrolytes concentration before and after the experiments? Original design is fine but needs polish on detailed experiments.

Comments:

 

1. On Figure 1, more doses are needed for each treatment.

2. On Figure 2, how to determine the dose of XG, 12.5 mg/Kg? Any experiments suggest that?

3. Does the volume change indicate electrolytes change? Such as NaCl?

4. On Figure 4, can authors explain why Tannic acid increase volume?

5. On Figure 3, what is the effect of XG + gelatin?

Author Response

Comments:

Many thanks for your review and critique of our manuscript. 

1. On Figure 1, more doses are needed for each treatment. We have changed mg/kg to mg/kg/day on Figure 1 to clarify that the dose was administered daily for 4 days prior to CT inoculation.
2. On Figure 2, how to determine the dose of XG, 12.5 mg/Kg? Any experiments suggest that? The dose of oral xyloglucan was determined according to previous experiments conducted in our laboratory showing that xyloglucan at 12.5 mg/kg: (i) does not alter intestinal water and electrolyte movements when administered alone (unpublished data) underlying the innocuity of this compound regarding the absorptive/secretory physiological intestinal function; and (ii) prevents increased gut permeability induced by lipopolysaccharides (LPS) [Bueno L et al. In: Proceedings of the 22nd United European Gastroenterology (UEG) week, Vienna, Austria, 18–22 October 2014. Abstract P1675]. Methods has been updated accordingly.
3. Does the volume change indicate electrolytes change? Such as NaCl? We did not measure electrolytes in the collected fluid. Evaluation of CT-induced fluid secretion in this model involves weighing the jejunal loops before and after emptying their contents.
4. On Figure 4, can authors explain why Tannic acid increase volume? We have not explored the mechanism of volume increase induced by tannic acid in this study. However, in the Discussion, we propose a possible mechanism through its toxic effect exerted on porcine intestinal cells IPEC-J2 via a mitochondrial pathway of apoptosis and S phase arrest of the cells at a concentration comparable to the dose used in our study [Wang J et al. Toxins (Basel) 2019;11(7), .
5. On Figure 3, what is the effect of XG + gelatin? This is now reported in section 3.1.3. Pretreatment with xyloglucan 12.5 mg/kg + gelatin 250 mg/kg 12 hours before CT challenge significantly (p < 0.05) reduced mean CT-induced intra-loop fluid secretion relative to vehicle. Pretreatment with xyloglucan 12.5 mg/kg + gelatin 125 mg/kg also reduced mean CT-induced intra-loop fluid secretion, but the difference relative to vehicle was not statistically significant (p < 0.07).

Reviewer 3 Report

General comment: The authors presented an interesting and original work concerning to the effect of two mucoprotectants in a rat model of diarrhea induced by cholera toxin. 

The manuscript is written in a comprehensive way. 

Title: The title is adequate. 

Abstract: It is adequate. 

The keywords should be different from those used in the title. 

Introduction: It is adequate. The authors provided an adequate overview of the thematic. 

Materials and methods: The methods are adequately described.   

The authors could provide a schematic representation of the experimental protocol. It would facilitate the comprehension of the experiments. 

How were the doses of tannate and xyloglucan defined?

Results: They are clearly presented and supported by the Figures. 

Discussion: It is adequate. 

Conclusion: The conclusion is based on the results. 

Recommendation: The manuscript should be accepted for publication after a Minor revision.

Author Response

The manuscript is written in a comprehensive way. 

Many thanks for your review and critique of our manuscript.

 

Title: The title is adequate. Thank you.

Abstract: It is adequate. Thank you.

The keywords should be different from those used in the title. 

We consider the current key words to be most reflective of the manuscript content. We have also added ‘mucoprotectant’ and ‘muco-adhesive film’ to the list.

Introduction: It is adequate. The authors provided an adequate overview of the thematic. Thank you.

Materials and methods: The methods are adequately described.   Thank you.

The authors could provide a schematic representation of the experimental protocol. It would facilitate the comprehension of the experiments. Although the experiments used an identical animal model, each series differed in terms of design (timing of treatment, type of treatment). Providing a schematic representation would require five separate figures which might be viewed as excessive.

How were the doses of tannate and xyloglucan defined? The doses of orally administered gelatin tannate and xyloglucan were determined according to previous experiments conducted in our laboratory showing that: (i) both compounds at doses used in our study did not alter intestinal water and electrolyte movements when administered alone (unpublished data) underlying the innocuity of these compounds regarding the absorptive/secretory physiological intestinal function and (ii) xyloglucan at the dose of 12.5 mg/kg protects the intestinal mucosa by preventing LPS-induced increase of gut permeability [Bueno L et al. In: Proceedings of the 22nd United European Gastroenterology (UEG) week, Vienna, Austria, 18–22 October 2014. Abstract P1675] and gelatin tannate at the dose of 250 mg/kg, through its ability to form a polymeric protective layer, leads to an overall decrease of fecal colonization by gut bacterial commensals such as E. coli and Enterococci resulting in protection against urinary tract infections [Olier M et al. Future Microbiol. 2017;12:505-513]. Methods has been updated accordingly. 

Results: They are clearly presented and supported by the Figures. Thank you

Discussion: It is adequate. Thank you

Conclusion: The conclusion is based on the results. Thank you

Recommendation: The manuscript should be accepted for publication after a Minor revision. Thank you

Round 2

Reviewer 1 Report

The authors have added a comment about "future research" to address the issue of the lack of any data on whether the proposed therapeutic agents would have any effect when given after cholera toxin has induced fluid accumulation, but have not addressed this key issue in the paper itself.

Many agents nonspecifically or specifically bind to cholera toxin(CT) and prevent its action when added to CT or to the gut lumen before CT, but NONE have been effective after CT has already bound to the mucosa and begun to cause intestinal fluid accumulation. These substances include kaolin, charcoal, cholera antitoxin, aspirin and others. The authors have failed to include any of the available references on this prior research.

There appears to be a misleading suggestion of clinical utility and efficacy by including a reference to use of the substances studies to treat pediatric "diarrhea" of unspecified etiology. This has nothing to do with cholera prevention or treatment.

It is not at all clear that the authors actually intend to test their substances for efficacy when given after CT has begun to induce diarrhea; there is no indication of funding for such a study, without which the current findings are of no clinical relevance to cholera treatment. The authors are best advised to complete such a study to clarify whether their findings have any clinical significance.

Author Response

Many thanks again for your constructive comments.

In reviewing your comments, we better understand your concerns. The paragraph in the Introduction beginning “Diarrheal infections are considered to be ...” may have inadvertently inferred some ability of mucoprotectants to treat cholera. That was not our intention. The paragraph has been deleted.

The purpose of this study was to investigate the ability of gelatin tannate, its constituents tannic acid and gelatin, xyloglucan, and combinations of these test compound(s) to protect against intestinal fluid accumulation. To this end, we used a validated animal model with CT as the secretagogue. We found that gelatin tannate (but not its individual constituents) and xyloglucan + gelatin attenuated CT-induced intra-loop fluid secretion through a mechanism that involves their ability to form mucoadhesive films.

The findings provide a rationale for the development of medical devices containing gelatin tannate or xyloglucan-gelatin to treat diarrhea. At the clinical level, these devices have been shown to be effective in treating acute diarrhea.

Reviewer 2 Report

No more comments

Author Response

Many thanks 

Round 3

Reviewer 1 Report

The latest version of this manuscipt still does not note the possibility, based on numerous past published studies, that many substances when given with or before cholera toxin (CT) in intestinal loops block its action, none of these has any effect when administered after CT fluid production has already started. This illustrates the inutility for predicting clinical potential efficacy of using the author's current study design.

The results reported do not rule out the possibility that, like activated charcoal, kaolin, antiserum, aspirin and other previously studies materials, gelatin tannate may act simply by binding to CT, thus preventing its activity in the intestine; the theory that the gelatin tannate may line the gut when given at 12 hrs (but NOT at 6 hrs) before toxin, makes no sense and avoids the possibility of simple pre-binding of the toxin, preventing it from reaching its binding site. The study design used is inappropriate and potentially misleading. The authors have failed to note in the text the need to test the gelatin tannate when given after CT fluid production has started.

Author Response

We apologize for the misunderstanding concerning the objective of the model used in our study, namely intestinal loops inoculated with cholera toxin. Indeed, herein we did not aim to evaluate the efficacy of the mucoprotectants tested against cholera-toxin-induced diarrhea. This was the reason we did not administer these compounds once the effect of the cholera toxin was established. Our objective was to evaluate whether their polymeric structure conferring film-forming properties were able to protect against fluid secretion, in our case by limiting access of the adverse secretagogue cholera toxin to the intestinal epithelium. We fully agree with your comment that possible trapping of cholera toxin by generation of the protective film and interplay with the intestinal mucus network could occur. This argument has been added to the Discussion section (lines 233-235, and 247 on Tracked Changes version). Further, to better clarify our purpose we have amended the title of our study to: “Effect of two mucoprotectants, gelatin tannate and xyloglucan plus gelatin, on cholera toxin-induced water secretion in rats” instead of “Effect of two mucoprotectants, gelatin tannate and xyloglucan plus gelatin, on cholera toxin-induced diarrhea in rats”.

We hope you will find these clarifications and amendments to your satisfaction.