Review for 10.1186/1471-230X-2-23 (2002)

Reviewer's□report 
□Expression□of□cytokine□mRNA□and□secretion□of□tumor□necrosis□factor-a□by□gallbladder□ Title: 
epithelial□cells:□Response□to□bacterial□lipopolysaccharides 
Authors: 
Christopher□E□Savard□( cesavard@u.washington.edu )
Thane□A□Blinman□( blinman@email.chop.edu )
Ho-Soon□Choi□( choihs@hanyang.ac.kr )
Sung-Koo□Lee□( sklee@amc.seoul.kr )
Stephen□J□Pandol□( Stephen.Pandol@med.va.gov )
Sum□P□Lee□( splee@u.washington.edu )
□1□ □10□Sep□2002 Version: Date: 
□Dr□KA□Papadakis Reviewer: 
□A□paper□whose□findings□are□important□to□those□with□closely□related□research□ Level□of□interest: 
interests 
□Unable□to□decide□on□acceptance□or□rejection□until□the□authors□have□ Advice□on□publication: 
responded□to□the□compulsory□revisions 
Savard,□et□al.□describes□the□effect□of□LPS□stimulation□on□cytokine□and□chemokine□mRNA□expression□ 
in□an□in□vitro□model□system□of□biliary□epithelial□cells.□In□addition□they□analyzed□TNF□protein□production 
in□response□to□LPS.□The□work□presented□is□interesting□and□novel,□and□the□manuscript□is□well□written.□ 
However,□I□have□several□comments□on□some□of□the□experiments,□the□presentation□of□the□data□and□ 
the□conclusions□they□support.□ 
Comments□to□the□authors□ 
1.□Addition□of□a□molecular□weight□marker□in□Figure□1□would□enable□the□reader□to□better□appreciate□the 
size□of□each□band.□Did□the□authors□perform□time□course□experiments□to□determine□the□optimal□ 
duration□of□exposure□of□MGBE□to□LPS□in□the□mRNA□expression□of□the□different□cytokines□and□ chemokines□analyzed?□The□absence□of□IL-10□and□KC□mRNA□induction□in□response□to□LPS□in□the□ 
RT-PCR□experiment□shown□does□not□necessarily□mean□that□the□mRNAs□are□absent.□Did□the□authors□ 
test□a□positive□control□where□the□IL-10□and□KC□primers,□shown□in□Table□1,□yielded□the□expected□ 
amplification□product?□Such□a□control□could□be□LPS□stimulated□mouse□macrophages,□which□respond□ 
to□LPS□by□producing□both□IL-10□and□KC.□ 
2.□In□Figure□1,□did□the□authors□try□to□optimize□the□PCR□reaction□for□RANTES□to□eliminate□unexpected□ 
bands□(contamination□by□genomic□DNA,□nonspecific□annealing□of□primers)?□ 
3.□In□Figure□2,□the□upregulation□of□TNF□mRNA□is□not□terribly□convincing.□How□many□cycles□were□used□ 
for□each□PCR□reaction?□How□many□cycles□for□the□ARP□gene?□These□conditions□should□be□stated□in□ 
the□Figure□legend.□Ideally□real-time□PCR□should□be□used□to□quantify□the□amount□of□RNA□induced□by□ 
LPS.□ 4.□It□would□be□preferable□that□the□authors□don't□use□the□term□"cytokine"□to□encompass□both□cytokines□ and□chemokines.□RANTES,□MIP-2,□MCP-1,□and□KC□should□be□separately□referred□as□"chemokines"□ 
throughout□the□manuscript.□In□addition□these□chemokines□should□also□be□named□(in□parentheses)□ 
according□to□the□recent□nomenclature□that□was□developed□for□this□group□of□chemotactic□proteins□ (Zlotnik,□A□and□O.□Yoshie,□Chemokines:□A□new□classification□system□and□their□role□in□immunity.□ Immunity,□2000;12:121-7)□ 
5.□The□conclusion□in□the□abstract□should□support□the□data□presented□in□the□manuscript.□The□term□ 
"produce"□in□the□first□page□in□the□Conclusion□section□of□the□abstract□imply□that□cytokines□are□ 
produced□at□the□protein□level□but□this□was□shown□only□for□TNF.□It□could□be□rephrased□as□"can□express 
mRNA□for□cytokines□and□chemokines".□Similarly□in□the□second□line□"can□alter□their□cytokine□mRNA□ 
pattern".□ 
Competing□interests: 
None□declared.