Reviewer's report
 Melatonin reduces cytokine induced expression of MAdCAM-1: Implications for IBD therapy. Title:
Authors:
Makotoh Sasaki MD (msasak@lsuhsc.edu )
Paul Jordan MD (pjorda1@lsuhsc.edu )
Takashi Joh MD (jalexa@lsuhsc.edu )
Makotoh Itoh MD (jalexa@lsuhsc.edu )
Michael Jenkins (michael.jenkins@virgin.net )
J. Steven Alexander PhD (jalexa@lsuhsc.edu )
 1  27 Mar 2002 Version: Date:
 Wayne Patton Reviewer:
 A paper whose findings are important to those with closely related research interests Level of interest:
 Unable to decide on acceptance or rejection until the authors have responded Advice on publication:
to the compulsory revisions
Comments: 
Overview: This manuscript demonstrates that melatonin inhibits TNF-alpha-induced MAdCAM-1 
expression. The reviewer recommends publishing the manuscript after the authors make the cited 
compulsory revisions. This reviewer would classify the article as a paper whose findings are important to
those with closely related research interests. The quality of written English in the manuscript is 
acceptable. 
Compulsory revisions: 
1. Inflammatory bowel disease should be spelled out and not abbreviated "IBD" in the title. 
2. "Cytokine" should be replaced with "tumor necrosis factor alpha" in the title, as this is the only 
cytokine investigated. Alternatively, other cytokines should be included in the study. 
3. In Materials and Methods, the detection reagent used to visualize the MAdCAM-1 by immunoblotting should be stated. Was detection performed by chemiluminescence using ECL reagent from Amersham 
Life Sciences or by colorimetric methods using diaminobenzidine? 
4. In the same section, it should be stated that SVEC4-10 is an SV-40 transformed mouse lymphoid endothelial cell line. 5. How was protein load normalized lane-to-lane on the Western blot? For example, was a solution 
protein assay performed prior to electrophoresis? Were blots stained for total protein prior to 
immunoblotting to verify equal loading? Does cell number and hence protein content change during the 24 hour incubation period with TNF-alpha? 6. Results: 1ng/ml TNF-alpha is shown to increase MAdCAM-1 expression 5-fold. Information 
concerning the dose-response effect of this cytokine should be included. Is the maximal response 
achieved with 1ng/ml cytokine? 7. Results: Is the induction of MAdCAM-1 specific? That is, if another cell surface adhesion molecule, such as occludin were assayed by Western blotting, would its expression increase to a similar extent? 
8. Results: Is the induction of MAdCAM-1 specific to TNF-alpha? Do other cytokines, such as 
interleukin-1 also induce MAdCAM-1 expression? This might be addressed in the Discussion section 
rather than the Results section. 
9. Discussion: How does the potency of the melatonin effect compare to the other agents 
(dexamethasone, IL-10) tested by the investigators in previous studies? 10. Discussion: Can a circulating blood level of 1 mM melatonin realistically be achieved in vivo? How does this dose compare with pharmacologically relevant levels of melatonin used in sleep therapy? 
Competing interests:
The reviewer has no financial competing interests with regard to the paper. From 1987-1989, the 
reviewer did serve in the same academic laboratory as the senior author of the paper. This laboratory 
investigated cytoskeletal regulation of endothelial cell inflammation.
Reviewer's report
 Melatonin reduces cytokine induced expression of MAdCAM-1: Implications for IBD therapy. Title:
Authors:
Makotoh Sasaki MD (msasak@lsuhsc.edu )
Paul Jordan MD (pjorda1@lsuhsc.edu )
Takashi Joh MD (jalexa@lsuhsc.edu )
Makotoh Itoh MD (jalexa@lsuhsc.edu )
Michael Jenkins (michael.jenkins@virgin.net )
J. Steven Alexander PhD (jalexa@lsuhsc.edu )
 1  27 Mar 2002 Version: Date:
 Wayne Patton Reviewer:
 A paper whose findings are important to those with closely related research interests Level of interest:
 Unable to decide on acceptance or rejection until the authors have responded Advice on publication:
to the compulsory revisions
Comments: 
Overview: This manuscript demonstrates that melatonin inhibits TNF-alpha-induced MAdCAM-1 
expression. The reviewer recommends publishing the manuscript after the authors make the cited 
compulsory revisions. This reviewer would classify the article as a paper whose findings are important to
those with closely related research interests. The quality of written English in the manuscript is 
acceptable. 
Compulsory revisions: 
1. Inflammatory bowel disease should be spelled out and not abbreviated "IBD" in the title. 
2. "Cytokine" should be replaced with "tumor necrosis factor alpha" in the title, as this is the only 
cytokine investigated. Alternatively, other cytokines should be included in the study. 
3. In Materials and Methods, the detection reagent used to visualize the MAdCAM-1 by immunoblotting should be stated. Was detection performed by chemiluminescence using ECL reagent from Amersham 
Life Sciences or by colorimetric methods using diaminobenzidine? 
4. In the same section, it should be stated that SVEC4-10 is an SV-40 transformed mouse lymphoid endothelial cell line. 5. How was protein load normalized lane-to-lane on the Western blot? For example, was a solution 
protein assay performed prior to electrophoresis? Were blots stained for total protein prior to 
immunoblotting to verify equal loading? Does cell number and hence protein content change during the 24 hour incubation period with TNF-alpha? 6. Results: 1ng/ml TNF-alpha is shown to increase MAdCAM-1 expression 5-fold. Information 
concerning the dose-response effect of this cytokine should be included. Is the maximal response 
achieved with 1ng/ml cytokine? 7. Results: Is the induction of MAdCAM-1 specific? That is, if another cell surface adhesion molecule, such as occludin were assayed by Western blotting, would its expression increase to a similar extent? 
8. Results: Is the induction of MAdCAM-1 specific to TNF-alpha? Do other cytokines, such as 
interleukin-1 also induce MAdCAM-1 expression? This might be addressed in the Discussion section 
rather than the Results section. 
9. Discussion: How does the potency of the melatonin effect compare to the other agents 
(dexamethasone, IL-10) tested by the investigators in previous studies? 10. Discussion: Can a circulating blood level of 1 mM melatonin realistically be achieved in vivo? How does this dose compare with pharmacologically relevant levels of melatonin used in sleep therapy? 
Competing interests:
The reviewer has no financial competing interests with regard to the paper. From 1987-1989, the 
reviewer did serve in the same academic laboratory as the senior author of the paper. This laboratory 
investigated cytoskeletal regulation of endothelial cell inflammation.
Please review this paper.
user
Reviewer's report
 Melatonin reduces cytokine induced expression of MAdCAM-1: Implications for IBD therapy. Title:
Authors:
Makotoh Sasaki MD (msasak@lsuhsc.edu )
Paul Jordan MD (pjorda1@lsuhsc.edu )
Takashi Joh MD (jalexa@lsuhsc.edu )
Makotoh Itoh MD (jalexa@lsuhsc.edu )
Michael Jenkins (michael.jenkins@virgin.net )
J. Steven Alexander PhD (jalexa@lsuhsc.edu )
 1  27 Mar 2002 Version: Date:
 Wayne Patton Reviewer:
 A paper whose findings are important to those with closely related research interests Level of interest:
 Unable to decide on acceptance or rejection until the authors have responded Advice on publication:
to the compulsory revisions
Comments: 
Overview: This manuscript demonstrates that melatonin inhibits TNF-alpha-induced MAdCAM-1 
expression. The reviewer recommends publishing the manuscript after the authors make the cited 
compulsory revisions. This reviewer would classify the article as a paper whose findings are important to
those with closely related research interests. The quality of written English in the manuscript is 
acceptable. 
Compulsory revisions: 
1. Inflammatory bowel disease should be spelled out and not abbreviated "IBD" in the title. 
2. "Cytokine" should be replaced with "tumor necrosis factor alpha" in the title, as this is the only 
cytokine investigated. Alternatively, other cytokines should be included in the study. 
3. In Materials and Methods, the detection reagent used to visualize the MAdCAM-1 by immunoblotting should be stated. Was detection performed by chemiluminescence using ECL reagent from Amersham 
Life Sciences or by colorimetric methods using diaminobenzidine? 
4. In the same section, it should be stated that SVEC4-10 is an SV-40 transformed mouse lymphoid endothelial cell line. 5. How was protein load normalized lane-to-lane on the Western blot? For example, was a solution 
protein assay performed prior to electrophoresis? Were blots stained for total protein prior to 
immunoblotting to verify equal loading? Does cell number and hence protein content change during the 24 hour incubation period with TNF-alpha? 6. Results: 1ng/ml TNF-alpha is shown to increase MAdCAM-1 expression 5-fold. Information 
concerning the dose-response effect of this cytokine should be included. Is the maximal response 
achieved with 1ng/ml cytokine? 7. Results: Is the induction of MAdCAM-1 specific? That is, if another cell surface adhesion molecule, such as occludin were assayed by Western blotting, would its expression increase to a similar extent? 
8. Results: Is the induction of MAdCAM-1 specific to TNF-alpha? Do other cytokines, such as 
interleukin-1 also induce MAdCAM-1 expression? This might be addressed in the Discussion section 
rather than the Results section. 
9. Discussion: How does the potency of the melatonin effect compare to the other agents 
(dexamethasone, IL-10) tested by the investigators in previous studies? 10. Discussion: Can a circulating blood level of 1 mM melatonin realistically be achieved in vivo? How does this dose compare with pharmacologically relevant levels of melatonin used in sleep therapy? 
Competing interests:
The reviewer has no financial competing interests with regard to the paper. From 1987-1989, the 
reviewer did serve in the same academic laboratory as the senior author of the paper. This laboratory 
investigated cytoskeletal regulation of endothelial cell inflammation.
assistant