The Role of New 3D Pathology and Lymphocyte Expression of Interstitial Inflammation in Pediatric-Onset Lupus Nephritis
Round 1
Reviewer 1 Report
The study carried out by Yung-Chieh Huang et al. shows that the analysis based on 3D pathology is useful in the diagnosis of Lupus Nephritis. In general, the study and the article presented are correct, well described, well presented, and with coherent and well-discussed results. However, there are some minor aspects that must be reviewed and corrected before its final publication:
- Please, review the expressions in Latin such as in situ, which must be written in italics.
- The official abbreviation for hours is h, not hr. It must be changed.
- The abbreviation DAPI (line 89) is not described.
- Line 89 needs a parenthesis.
- Figures 1A, B and C need a descriptive title.
- Table 1:
* Why is the p-value of the parameter Age of renal biopsy not presented?
* Why do some continuous variables express variability as -+ and others as interval? What does each of these two forms of expression mean? It must be explained in the footer of the figure.
* How have differences in continuous variables been statistically analyzed? It is not explained in the Statistics section, and should be included.
- Table 2: the meaning of the asterisks is not explained in the footer.
- Table 3: why are symbols used to explain the abbreviations? I think it is not necessary to use symbols that can confuse the reader.
- I think it would be appropriate to include a subsection on limitations of the study in the discussion of the paper.
Author Response
The study carried out by Yung-Chieh Huang et al. shows that the analysis based on 3D pathology is useful in the diagnosis of Lupus Nephritis. In general, the study and the article presented are correct, well described, well presented, and with coherent and well-discussed results. However, there are some minor aspects that must be reviewed and corrected before its final publication:
- Please, review the expressions in Latin such as in situ, which must be written in italics.
Answer: Thank you for remind. Now, the 5 in situ written in the original submission were corrected as “in situ”. In fact, this manuscript is sent for MDPI English editing service. All bugs in English writing will be corrected.
- The official abbreviation for hours is h, not hr. It must be changed.
Answer: Thank you for remind. We changed hr to h at
- The last 8th line, page 2
- Line 4,7,10 of the 2.5 of Material and methods, page 4.
- The abbreviation DAPI (line 89) is not described.
Answer: Thank you for remind. We added the whole name as “4′,6-Diamidino-2-phenylindole dihydrochloride” at line 89 before the DAPI
- Line 89 needs a parenthesis.
Answer: Yes, now line 89 is written as “proprietary clearing solution and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)”
- Figures 1A, B and C need a descriptive title.
Answer: Thank you for remind. We added the descriptive titles as below:
- Figure 1A 3D image for cell nuclear and membrane of renal tissue.
There is also a parenthesis below Figure 1A as “(Cell nuclei were stained as red and membrane as green.)”
- Figure 1B Cropped 2D image after “pseudo-needle” track procedure from 3D tissue image.
- Figure 1C Workflow of tissue processing, staining, and final output of the 3D image results.
- Table 1:
* Why is the p-value of the parameter Age of renal biopsy not presented?
Answer: Sorry, the p value is 0.317, which has added to Table 1.
* Why do some continuous variables express variability as -+ and others as interval? What does each of these two forms of expression mean? It must be explained in the footer of the figure.
Answer: Thank you for remind. Now, all continuous variables in Table 1 are expressed as mean±SD.
* How have differences in continuous variables been statistically analyzed? It is not explained in the Statistics section, and should be included.
Answer: Thank you for remind. Now we added the sentence “The Mann–Whitney U test to compare those continuous variables between the two groups.” in the 2nd line of “Statistics”.
- Table 2: the meaning of the asterisks is not explained in the footer.
Answer: Thank you for remind. The meaning of the asterisks are expressed as “*p<0.05; **p<0.01” in the footer.
- Table 3: why are symbols used to explain the abbreviations? I think it is not necessary to use symbols that can confuse the reader.
Answer: Thank you for the concern. Now we only use abbreviation symbols for TA: tubular atrophy and IF: interstitial fibrosis.
- I think it would be appropriate to include a subsection on limitations of the study in the discussion of the paper.
Answer: Yes, it is necessary. We added a paragraph mentioning two limitations in this study. It is written as
“There are several limitations in this study. The number of 3D pathology is limited. The technique has never been applied to human renal tissue. We only checked limited representative specimen. The selection criteria included the class IV LN in ISN/RPS classification, various scores of II and chronicity and the adequate thickness of the renal sample. This study only provides comparative data between traditional 2D pathology and this de novo 3D pathology. We used the “pseudo-needle” track to check the depth adequate for immunofluorescence (IF) stain of interstitial lymphocyte markers. The 3D IF stains for syndecan-1 or CD38 is not available at present.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
Review for Huang et al
This study focuses on interstitial inflammation II in lupus nephritis end stage renal disease (ESRD). 3D renal pathology, CD3+ expression, and B cell antigen expression was examined in 48 SLE patients with high II score and low chronicity. The study demonstrates that while 2D and 3D pathology were well correlated. However, syndecan-1 (a plasma cell marker) did not correlate with interstitial inflammation and CD3+ cells were found in sections that were negative for the B cell markers CD19 and CD20. This is largely a descriptive study, nevertheless yields some interesting observations that may help better define predictors of disease progression
Comments:
--the flow of the paper is choppy and the main findings could be articulated more clearly
-abstract lupus doesn’t cause long-term glucocorticoid/immune-suppressants use,prolonged treatment of lupus with glucocorticoid/immune suppressants is associated with a variety of issues. Rephrase this sentence, including ESRD
-overall, abstract could be organized better to emphasize major finidngs
-should provide a description of the therapies that are discussed (e.g. belimumab)
Author Response
Reviewer’s comments:
--the flow of the paper is choppy and the main findings could be articulated more clearly
Answer: Thank for comments. We modified the order of results. The main findings can be more articulated. Please refer to the revised manuscript.
-abstract lupus doesn’t cause long-term glucocorticoid/immune-suppressants use,prolonged treatment of lupus with glucocorticoid/immune suppressants is associated with a variety of issues. Rephrase this sentence, including ESRD
Answer: Thank you for your comment. Now we modify the original sentences” It causes long-term glucocorticoid/immune-suppressants use, and even the end stage renal disease (ESRD) in pSLE. “ to “It is one of the major causes for long-term glucocorticoid/immune-suppressants use in pSLE. It is now well known that high chronicity, especially the tubulointerstitial components in renal biopsy predicts a poor renal outcome, including end stage renal disease (ESRD).”
-overall, abstract could be organized better to emphasize major finidngs
Answer: Thanks for remind.
Now we added “Background”, “Material and methods”, ”Results” and “Conclusion” in the abstract. It can be more organized.
-should provide a description of the therapies that are discussed (e.g. belimumab)
Answer: Thank you for advice. Now we add two sentences in front of the original first sentence of the 4th paragraph of “Discussion”. The sentences were written as “Belimumab, a recombinant human IgG1λ monoclonal antibody, inhibits B-lymphocyte stimulator. It has been approved for patients with active autoantibody-positive SLE.”
Author Response File: Author Response.docx
Reviewer 3 Report
Dear author/s,
I found this paper “IJMS-2166001” has been poorly and carelessly written. If authors have the novel or extraordinary finding and used NEW 3D pathology technique, they must explain/present the results satisfactory. Here are some of my apparent comments-
1. Introduction should be wealthier with clear objectives and gap in research in the present study.
2. Some basic information is missing; like determination of class III, IV and V patient categories etc.
3. Methods are inadequately described like microscope name and used associated software for scanning etc. Follow this paper for microscopy and imaging https://doi.org/10.1371/journal.ppat.1007338
4. Surprisingly, figure legends are missing in all the figures. It should be separate from your results and underneath the figure with captions.
5. Results section is also seeming like incompletely described, explain your findings well in organized and cohesive manner.
Overall, there is a lot of errors including spellings (Line # 91, 134 cambered slides) and English language throughout the manuscript “IJMS-2166001”. I have highlighted and superficially mentioned some of them, though there are essential requirement of major corrections and proofreading. I will urge to do the substantial amendment to make the article more conventional and reader friendly, so that someone can follow/cite your work in future.
All the best.
Author Response
Comments and Suggestions for Authors
Honorable reviewer: Thanks a lot for your comments and advices. We have answered your comments point by point as below:
I found this paper “IJMS-2166001” has been poorly and carelessly written. If authors have the novel or extraordinary finding and used NEW 3D pathology technique, they must explain/present the results satisfactory. Here are some of my apparent comments-
1. Introduction should be wealthier with clear objectives and gap in research in the present study.
Answer: Thank you for the important comment. We added some words in the 2nd paragraph to interpret the objectives of this study more clearly. Please refer to the last 7 lines of this paragraph. Now, we modified as
“Alsuwaida AO et al. [10] demonstrated no association of II with end stage renal disease (ESRD). Rijnink EC et al. [11] found the components of II, including interstitial infiltrate score, either lymphocyte or granulocyte dominance, had no impact on ESRD in lupus nephritis. One explanation is the various concurrent chronicity scores noted in these studies. Nevertheless, limited sampling from renal tissue may not represent the full scale of II score or chronicity, a more detailed study in renal pathology is warranted.”
2. Some basic information is missing; like determination of class III, IV and V patient categories etc.
Answer: Thank you for the concerns.
a. We added the period of patient recruitment in the first sentence of “2.1 patients” as “From January 2006 to December 2016, we recruited 48 patients….”.
b. There is “Interpretation of renal pathology” mentioned as 2.4 in “Materials and
methods”. The renal biopsies from these patients were interpreted by ISN/RPS Classification. So we did not write it in 2.1”Patients”.
c. Their basic profile and related data at biopsy and renal pathology were shown as Table 1.
3. Methods are inadequately described like microscope name and used associated software for scanning etc. Follow this paper for microscopy and imaging https://doi.org/10.1371/journal.ppat.1007338
Answer: Thank you for providing the link. We found an article titled as “Klebsiella pneumoniae infection of murine neutrophils impairs their efferocytic clearance by modulating cell death machinery” through the link.
a. We searched the word “software” and found it is Image J software, rather than “ImageJ software”. Now we corrected the writing in this aspect.
b. The microscope and its software for immunofluorescence scan is now described as “Each slide was carefully mounted with cover slips for 2D image acquisition using FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with FV31S-SW software (Olympus, Tokyo, Japan).” as the last sentence of 2.5 Immunofluorescence stain in “Materials and Methods”.
The FV300 confocal laser scanning microscope can read immunofluorescence stain, both thin and thick slides are applicable.
4. Surprisingly, figure legends are missing in all the figures. It should be separate from your results and underneath the figure with captions.
Answer: Thank you for remind.
Figure 1A 3D image for cell nuclear and membrane of renal tissue.
There is also a parenthesis below Figure 1A as “(Cell nuclei were stained as red and membrane as green.)”
Figure 1B Cropped 2D image after “pseudo-needle” track procedure from 3D tissue image.
Figure 1C Workflow of tissue processing, staining, and final output of the 3D image results.
5. Results section is also seeming like incompletely described, explain your findings well in organized and cohesive manner.
Overall, there is a lot of errors including spellings (Line # 91, 134 cambered slides) and English language throughout the manuscript “IJMS-2166001”. I have highlighted and superficially mentioned some of them, though there are essential requirement of major corrections and proofreading. I will urge to do the substantial amendment to make the article more conventional and reader friendly, so that someone can follow/cite your work in future.
Answer: Thank you for your concern.
a. We have applied MDPI English editing service.
b. We tried to describe our results more clearly. There are many modifications in “Results”. We hope it can be more organized and cohesive.
b-1. We added sentences and Table 2 after Table1.
Among these 48 pSLE patients with LN, we selected 5 samples of LN class IV with various II scores for 3D pathology. The control was different obtained from a patient with minimal change nephrotic syndrome. Table 2 shows the scores from their original interpretation and average scores from different depth of the 3D pathology.
Table 2 Comparison of 2D and 3D average scores of interstitial inflammation, chronicity and items in chronicity in control and pSLE patients with lupus nephritis class IV
|
Patient |
Interstitial inflammation
|
Chronicity
|
Tubular atrophy |
Interstitial fibrosis |
Global/ segmental sclerosis |
Fibrous crescent |
|
|
Control |
*0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
|
|
1 |
0/0.75 |
2/0.75 |
1/0.75 |
1/0 |
0/0 |
0/0 |
|
|
2 |
0/0.1 |
3/1.4 |
1/0.9 |
1/0.4 |
1/0.1 |
0/0 |
|
|
3 |
1/1.64 |
3/4.45 |
1/2.45 |
1/1.82 |
1/0.18 |
0/0 |
|
|
4 |
3/2.133 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
|
|
5 |
3/2.333 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
|
*scores from original pathology interpretation/ average scores from different depth in 3D pathology
b-2 The original Table 2 now becomes Table 3. It is removed to area immediately after Table 2.
b-3 We re-arranged the order in “Results”. Now Figure 2 A, B, C and interpretation for Figure 2 are removed to area after Table 3.
Author Response File: Author Response.docx
