JCB-10-0135.R2
Responses to the Reviewers’ and the Editorial Comments
August 30, 2010
Gary Stein
Executive Editor,
Journal of Cellular Biochemistry
betsy.bronstein@umassmed.edu
jcb@umassmed.edu
Manuscript #JCB-10-0135.R2
Title: Insufficient Peroxiredoxin-2 Expression in Uterine NK Cells Obtained from a Murine Model
of Abortion
Authors: Guangjie Yin, Cui Li, Bin Shan, Wenjing Wang, Hong Chen, Yanmin Zhong, Jingfang Di,
Qide Lin, Yi Lin
Dear Dr. Gary Stein,
We have revised the attached manuscript in keeping with the comments of the reviewers and the
editorial. The comments are presented below in bold, and our responses are italicized. Both a
marked version of our revised manuscript and a clean copy are submitted on-line.
We thank you and the reviewers for their time and the attention paid to our manuscript. We hope
our efforts have rendered the manuscript acceptable for publication in Journal of Cellular
Biochemistry.
Sincerely yours,
Yi Lin
Department of Obstetrics and Gynecology
Institute of Obstetrics and Gynecology
Renji Hospital
School of Medicine
Shanghai Jiaotong University
Shanghai 200001
China
Fax: +86 21 50390610
E-mail: yilinonline@gmail.com
Responses to the Comments
Reviewer: 1
Comments to the Author
In their revised manuscript, the authors have adequately addressed the initial responses of
this reviewer through additional data, clarification of methodology and discussion of
scientific limitations with regard to impact on interpretation of data.
Data presented in Figure 7 is exciting and lends credence to the authors’ line of investigation
and recognition of PRX-2 as a potentially important regulatory protein. The results
presented from the 51Cr-release assay demonstrating increased cytolytic activity of the
PRX-2 deficient uNK cells from –DBA/2J mice compared to uNK cells from –BALB/c mice
are particularly compelling and hint at elucidating a relevant, functional impact of uNK
cell-associated PRX-2 on feto-maternal tolerance. If the cytotoxicity of uNK cells derived
from both –BALB/c and –DBA/2J mice could be shown to increase with the
pre-treatment/presence of neutralizing antibody to PRX-2, this would further strengthen the
authors’ conclusions.
Thank you for your thoughtful comments. In our marked version, inserted or added words are
highlighted as blue-colored words, while deleted words are highlighted as red-colored words.
Now, Figure 7 is new Figure 5 in the revised manuscript. Additional 51Cr-releasing assay was
performed to investigate the cytotoxicity of uterine NK cells derived from both CBA/J×BALB/c
and CBA/J×DBA/2J mice after PRX-2-blockade by using its neutralizing antibody. The
cytotoxicity was increased after inhibition of PRX-2 and the results were discussed briefly in the
revised manuscript (In the marked revision, lines 187-190 in the Materials and Methods section;
lines 271-284 in the Results section; and lines 393-398 in the Discussion section; lines 584-601 in
Figure Legends; new Figure 5).
Reviewer: 2
Comments to the Author
1.
It cannot be unequivocally inferred from the data presented whether PRX-2
expression is in the uterine NK cells or infiltrating NK cells. It is highly recommended that
authors utilize double immunofluorescence staining or demonstrate a lack of infiltrating
peripheral NK cells.
Thank you for your thoughtful comments. In our marked version, inserted or added words are
highlighted as blue-colored words, while deleted words are highlighted as red-colored words.
Multi-vision immunofluorescence staining has been performed. In these experiments, uterine
NK cells were stained green with FITC-conjugated DBA-lectin, PRX-2-producing cells were
stained red with rabbit anti-mouse PRX-2 antibody and PE-conjugated anti-rabbit IgG. Nuclei
were stained using DAPI (blue). Uterine NK cell and PRX-2 co-localization was confirmed by
using these experiments (In the marked revision, the Materials and Methods section, lines 79-94;
Results section, lines 211-221; Discussion section, lines 313-319, 345-348; Figure legends, lines
542-553; new Fig. 1).
2.
If the authors choose to retain 2D & MS electrophoresis data, it would indeed be a
comprehensive experimental design. However, in that case differential protein expression
must be documented and select proteins identified by MS analysis. Subsequently, the
rationale for focusing on PRX-2 should be explained. Otherwise, western blot analysis
presented by authors is adequate demonstration of differential expression.
Following your thoughtful suggestions, 2-DE and MS data were deleted and Western blot analysis
was kept in the manuscript to demonstrate differential expression of PRX-2.
3.
2D gel analysis reveals additional proteins that are down-regulated in CBA / J x
DBA/2J as compared with CBA / J x BALB/c mice. Why have the authors excluded those
proteins as being responsible for or associated with embryo resorption?
Other down-regulated proteins may or may not be responsible for or associated with embryo
resorption. They may act independently or co-work with PRX-2. Limited by available resource in
our lab, no further experiments were performed to investigate the possible roles these proteins
may play in the modulation of embryo development. Since 2-DE and MS data have been deleted,
these parts were not mentioned in the revised manuscript.
4.
Lack of isotype control antibodies in neutralizing experiment (Fig 6) is troubling. The
observed effects could be the result of the antibody itself and not the neutralization. In
addition, there is no data in this experiment to support reduction of PRX-2 expression. A
western blot analysis is required.
Additional experiments were performed in which isotype control antibody was used and data were
compared with those in which neutralizing Ab was used to block PRX-2. In addition, Western blot
analysis was performed to confirm reduced expression of PRX-2 after PRX-2-blockade (lines 185,
187-190, 197-198, 259, 262, 265, 271-284, new Figures 4 and 5).
We wish to thank the referees for their useful comments. I hope that the revised manuscript is now
acceptable for publication.