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{=0A= document.writeln('

3D"Click

');=0A= }=0A= =0A= function get_text_link(url, linktext)=0A= {=0A= document.writeln('' + = linktext + '');=0A= }=0A= =0A= function get_htmlftext_link(a, b) {=0A= document.writeln("" + = b + ", ");=0A= }=0A= =0A= ------=_NextPart_000_000C_01CA3D3E.0EADFA70 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://download.interscience.wiley.com/freeflow/js/personalisation.js /*=0A= * Project : Interscience Redesign=0A= * File : personalisation.js=0A= * Creation Date : 25th March 2003=0A= * Author : Jonathan Sugar=0A= *=0A= * Various functions to deliver personalisation functionality.=0A= */=0A= =0A= // Pre-declare userName so it's available when we need to test if the = page=0A= // assembler ran OK=0A= var userName =3D null;=0A= var shcrtCookie =3D null;=0A= =0A= // Browser detect globals:=0A= var uaStr =3D navigator.userAgent.toLowerCase();=0A= var uaVer =3D parseInt(navigator.appVersion);=0A= // for detecting netscape 4=0A= var netscape =3D (uaStr.indexOf("mozilla") > -1);=0A= var netscape4 =3D (netscape && uaVer < 5);=0A= =0A= // Factory Methods=0A= =0A= // Simple wraper object to encapsulate the data for a co-branding item.=0A= function coBrandingItem(image, imageAltText, href)=0A= {=0A= //alert("Making new coBrandingItem:\nImage: " + image + = "\nimageAltText" + imageAltText + "\nhref: " + href);=0A= this.image =3D image;=0A= this.imageAltText =3D imageAltText;=0A= this.href =3D href;=0A= }=0A= =0A= /*=0A= * Insert the 'Customer co-branding' section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= *=0A= * 1. Insert customer co branding. Loop through 'customerCoBranding' array=0A= * inserting XHTML where needed.=0A= *=0A= * NOTE: the array contains ONLY the resource location (the location of a = gif=0A= * on the file system), the required XHTML is then added where needed. = This way=0A= * we maintain the seperation between presentation and content.=0A= */=0A= function insertCustomerCoBranding(maxBranding)=0A= {=0A= if( customerCoBranding[0].image !=3D "none")=0A= {=0A= document.writeln('

');=0A= document.writeln('

');=0A= document.writeln('

' + imageAltText + '
' + imageAltText + = '
More Providers

');=0A= document.writeln('

');=0A= document.writeln('

3D""

');=0A= }=0A= }=0A= =0A= /*=0A= * Insert the 'My Area' section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= * Updated: Ben Marvell, April 2006=0A= *=0A= * My profile link absolute as this file isnt only run from w3 anymore.=0A= */=0A= function insertMyArea(userdata)=0A= {=0A= // just in case the page assembler isn't running.=0A= =0A= =0A= if (userName !=3D null)=0A= {=0A= document.writeln("

" + = userName.substring(0,33) + "..., access your
" + userName + = ", access your

My Profile"); =0A= } =0A= document.writeln(getLoginOutLink());=0A= //CR 3652 getAthenStatus() as added=0A= document.writeln(getAthenStatus());=0A= document.writeln("

");=0A= } else {=0A= if(typeof(environment)!=3D'undefined' && = environment=3D=3D'PRODUCTION') {=0A= document.writeln("

Log Out");=0A= document.writeln("

"); =0A= }=0A= }=0A= if (menuloadedflag){=0A= wisMenu();=0A= }=0A= }=0A= =0A= /*=0A= * Insert the Customer Admin 'Login/Logout' link=0A= * -----------------------------=0A= *=0A= * Author: Bobby Jack, December 2004.=0A= *=0A= */=0A= function insertAdminLink(page)=0A= {=0A= val =3D getCookie("AID");=0A= =0A= if (val && val !=3D "-1")=0A= {=0A= document.writeln('

Logout');=0A= }=0A= else=0A= {=0A= document.writeln('

');=0A= }=0A= }=0A= =0A= /*=0A= * Insert the username section.=0A= * -----------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= *=0A= * 1. Insert the username.=0A= */=0A= function insertUserName()=0A= {=0A= document.write(userName);=0A= }=0A= =0A= /*=0A= * Return the appropriate link to the login/logout cgi=0A= * ---------------------------------------------------=0A= *=0A= * Author: Jonathan Sugar, March 2003.=0A= * Updated: Ben Marvell, April 2006=0A= * Had to change the links absolute as this file isnt only run from w3 = anymore.=0A= */=0A= function getLoginOutLink()=0A= {=0A= if(userName !=3D "")=0A= {=0A= return "

";=0A= }else=0A= {=0A= return "

";=0A= }=0A= }=0A= =0A= /*=0A= * Return the appropriate link to the shopping cart content page=0A= * ---------------------------------------------------=0A= *=0A= * Author: Zhiming & Warrell June 2007=0A= */=0A= function getShCrtLink(logged)=0A= {=0A= if(logged =3D=3D true )=0A= return "

";=0A= else=0A= return "

";=0A= }=0A= =0A= function toggleShCrtLink()=0A= {=0A= shcrtCookie =3D readCookie("SHCRT");=0A= if(shcrtCookie !=3D null)=0A= {=0A= if(document.all)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= document.getElementById("shcrt_id").style.display =3D "block";=0A= }=0A= }=0A= else=0A= {=0A= if(document.all)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= document.getElementById("shcrt_id").style.display =3D "none";=0A= }=0A= }=0A= }=0A= =0A= =0A= =0A= function readCookie(name)=0A= {=0A= var nameEQ =3D name + "=3D";=0A= var ca =3D document.cookie.split(';');=0A= for(var i=3D0; iYou are logged in = to Athens';=0A= }=0A= else=0A= {=0A= return "

";=0A= }=0A= =0A= } ------=_NextPart_000_000C_01CA3D3E.0EADFA70 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://download.interscience.wiley.com/freeflow/js/site.js /************************************************************************= ***************************=0A= FILE: site.js=0A= AUTHOR: Jonathan Sugar, Chris Neatby-Smith and Digital Pulp. July = 2003.=0A= =0A= DESCRIPTION: Javascript relating to site wide functionality across =0A= InterScience.=0A= =0A= INCLUDES FUNCTIONS=0A= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= Search Functions=0A= ----------------=0A= =0A= selectAndSubmit() - for deciding where to submit a query based on the = state of the form.=0A= processallContentSearch() - for validating form input for an all = content search.=0A= processTitleSearch() - for validating form input for a Title search.=0A= checkCitationSearch() - for validating the citation search form.=0A= selectAll() - check all the checkboxes in a form.=0A= do_popup() - stasa Acronym Finder window popup function.=0A= =0A= =0A= Other Functions=0A= ---------------=0A= =0A= fixInlineLists() - fix netscape 4.x bug where

tags with images = display both an image and=0A= the default bullet. =0A= =0A= = *************************************************************************= **************************/=0A= =0A= var http =3D false;=0A= if(navigator.appName =3D=3D "Microsoft Internet Explorer") {=0A= http =3D new ActiveXObject("Microsoft.XMLHTTP");=0A= } else {=0A= http =3D new XMLHttpRequest();=0A= } =0A= =0A= // Global Variables=0A= var debug =3D false;=0A= var first =3D true;=0A= =0A= // Browser detect globals:=0A= var uaStr =3D navigator.userAgent.toLowerCase();=0A= var uaVer =3D parseInt(navigator.appVersion);=0A= // for detecting netscape 4=0A= var netscape =3D (uaStr.indexOf("mozilla") > -1);=0A= var netscape4 =3D (netscape && uaVer < 5);=0A= =0A= /*=0A= * Used to switch search mode between APS and TitleFinder on quicksearch = boxes=0A= * = -------------------------------------------------------------------------= --=0A= *=0A= * 1. Decides which search is required based on the value of the radio = buttons=0A= * in 'document.formSelector.whichForm'.=0A= *=0A= * 2. Calls individual functions to process the forms and submit them.=0A= *=0A= */=0A= function selectAndSubmit()=0A= {=0A= var formSelection;=0A= =0A= // find out which form we need to process.=0A= var selector =3D document.formSelector.whichForm;=0A= for (var i =3D 0; i < selector.length; i++){=0A= if (selector[i].checked){=0A= formSelection =3D selector[i].value;=0A= }=0A= }=0A= =0A= // Call the relevent function to process the required form.=0A= if(formSelection =3D=3D "allContentSearch"){=0A= processallContentSearch(document.allContentSearchForm, = document.formSelector)=0A= }=0A= else if(formSelection =3D=3D "searchTitles"){=0A= processTitleSearch(document.searchTitles, document.formSelector)=0A= }=0A= else{=0A= return false;=0A= }=0A= =0A= //@@@DM It was anoying me this error =0A= return false;=0A= }=0A= =0A= /*=0A= *Process the ADS search form=0A= *=0A= */=0A= function trim(str)=0A= {=0A= return str.replace(/(^\s*)|(\s*$)/g,"");=0A= }=0A= =0A= function ADSquery(t) {=0A= var keyword =3D trim(t.getElementsByTagName("input")[0].value);=0A= if (keyword!=3D"") {=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= t.target=3D"popup";=0A= t.qsearch.value=3Dkeyword;=0A= return true;=0A= = //processADSSearch(document.ADSSearchForm,document.ADSQueryForm,keyword);=0A= } else {=0A= alert("Please input a keyword for ADS searching before clicking go");=0A= return false;=0A= }=0A= }=0A= /*=0A= function processADSSearch(hiddenForm ,queryForm , str) {=0A= var terms =3D str;=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= formToSubmit.qsearch.value =3D terms;=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= formToSubmit.action =3D = "http://adsabs.harvard.edu/cgi-bin/nph-basic_connect"=0A= formToSubmit.target =3D "popup";=0A= formToSubmit.submit();=0A= }=0A= */=0A= =0A= /*=0A= * Process the allContentSearch form=0A= * ---------------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= function processallContentSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.WISsearch1.value =3D formToProcess.terms.value;=0A= formToSubmit.submit(); =0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= /*=0A= * Process the Title Search form=0A= * -----------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= =0A= function processTitleSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.query.value =3D formToProcess.terms.value;=0A= formToSubmit.submit();=0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= function doCitationSearch()=0A= {=0A= var form =3D document.citsearchform;=0A= if(checkCitationSearch())=0A= {=0A= form.action =3D "/search/allsearch";=0A= form.submit();=0A= }=0A= }=0A= =0A= // Preprocesses the arguments sent to CitationSearch and checks validity = of user inputs.=0A= function checkCitationSearch()=0A= {=0A= var supplementFound =3D 0; //increment if found;=0A= var hyphenFound =3D 0; //increment if found;=0A= var fm =3D document.citsearchform;=0A= var errmsg =3D "Please use the valid digits '0' to '9'";=0A= // remove all spaces=0A= fm.volume.value =3D fm.volume.value.replace(/ /g, "");=0A= fm.issue.value =3D fm.issue.value.replace(/ /g, "");=0A= fm.pages.value =3D fm.pages.value.replace(/ /g, "");=0A= =0A= if (fm.issn.value.length > 0 && fm.volume.value.length > 0 && = fm.pages.value.length > 0)=0A= {=0A= // check page range=0A= var pageRange =3D fm.pages.value;=0A= var volume =3D fm.volume.value;=0A= var issue =3D fm.issue.value;=0A= var continueFlag =3D 0;=0A= //check the volume=0A= // if (isNaN(volume))=0A= // {=0A= // // volume failed checking=0A= // alert("Volume is invalid. "+errmsg+".");=0A= // return false;=0A= // }=0A= //check the issue=0A= if ((issue.length !=3D 0) && isNaN(issue))=0A= {=0A= // issue failed checking=0A= alert("Issue is invalid. "+errmsg+" or leave blank.");=0A= return false;=0A= }=0A= //check the page range=0A= if (isNaN(pageRange))=0A= {=0A= var isValid =3D false;=0A= // check for hyphen=0A= var hyphenIndex =3D pageRange.indexOf("-");=0A= if ((hyphenIndex !=3D -1) && (hyphenIndex =3D=3D = pageRange.lastIndexOf("-")))=0A= {=0A= // we have only one hyphen, so extract each number=0A= var firstNum =3D pageRange.substring(0, hyphenIndex);=0A= var secondNum =3D pageRange.substring(hyphenIndex + 1);=0A= // check for S=0A= var firstS =3D firstNum.indexOf("S");=0A= var secondS =3D secondNum.indexOf("S");=0A= if (firstS =3D=3D secondS)=0A= {=0A= if ((firstS =3D=3D -1) && isNum(firstNum) && isNum(secondNum) && = (firstNum.length > 0) && (secondNum.length > 0))=0A= isValid =3D true; // both inputs are digits=0A= else if (firstS =3D=3D 0)=0A= {=0A= var firstNumNoS =3D firstNum.substring(1);=0A= var secondNumNoS =3D secondNum.substring(1);=0A= if (isNum(firstNumNoS) && isNum(secondNumNoS) && = (firstNumNoS.length > 0) && (secondNumNoS.length > 0))=0A= isValid =3D true; // both inputs are Sdigits=0A= }=0A= =0A= }=0A= }=0A= if (isValid)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= alert("The page range is invalid. "+errmsg+" and a hyphen '-' if = required.");=0A= return false;=0A= }=0A= }=0A= return true;=0A= }=0A= else=0A= {=0A= alert("Volume and pages are required fields. Please make sure data has = been entered for both fields.");=0A= return false;=0A= }=0A= }=0A= =0A= // returns true if a is an Integer.=0A= function isNum(a)=0A= { return !(isNaN(a)); }=0A= =0A= function fixInlineLists() {=0A= if (!document.getElementById) return;=0A= var allLIs =3D document.getElementsByTagName('li');=0A= var bulletNode =3D document.createElement('img');=0A= bulletNode.src = =3D"http://download.interscience.wiley.com/images/icon.bullet.gif";=0A= =0A= for (var i=3D0; iAbstract | References';=0A= }=0A= //alert("URL: " + loc + "\nStart Index: " + startIndex + "\nEnd Index: = " + endIndex + "\nPage: " + page + "\nLink Text: " + links);=0A= return links;=0A= }=0A= =0A= =0A= function selectAll(fm)=0A= {=0A= //var fm =3D document.viewSelectedForm;=0A= =0A= for (var i =3D 0; i < fm.elements.length; i++)=0A= {=0A= if (fm.elements[i].type =3D=3D 'checkbox' /*&& fm.elements[i].name = =3D=3D 'ID'*/)=0A= {=0A= fm.elements[i].checked =3D true;=0A= }=0A= }=0A= }=0A= =0A= =0A= function do_popup(file)=0A= {=0A= var w =3D window.open(file, 'popWin', = 'width=3D350,height=3D500,scrollbars=3Dyes');=0A= =0A= }=0A= =0A= =0A= function mailTool(itemID, useurl, skin)=0A= {=0A= //var url =3D "/cgi-bin/mailform?item=3D"+itemID;=0A= var url =3D "/cgi-bin/mailform?item=3D" + itemID + "&skin=3D" + skin;=0A= var parameters =3D = 'status=3Dno,scrollbars=3Dno,resizable=3Dyes,width=3D400,height=3D475';=0A= =0A= if (useurl !=3D "")=0A= {=0A= url +=3D "&useurl=3D"+useurl;=0A= }=0A= =0A= if ((isNaN(parseInt(itemID))=3D=3Dtrue)||(arguments.length >=3D 4 && = arguments[3] =3D=3D 'cluster'))=0A= {=0A= url +=3D '&type=3Dcluster';=0A= }=0A= =0A= //check whether the last 2 arguments is used for urllink.=0A= // urllink is needed to open up mail form for mrw pages on tomcat = port=0A= if (arguments.length>=3D5 && = arguments[arguments.length-2]=3D=3D'urllink' && = arguments[arguments.length-1]!=3D'')=0A= {=0A= url =3D arguments[arguments.length-1] + url;=0A= }=0A= =0A= if (arguments.length>=3D5 && arguments[3]=3D=3D'currentlink' && = arguments[4]!=3D'')=0A= {=0A= url =3D url + '¤tLink=3D' + arguments[4];=0A= =0A= //processing specific cases=0A= if (arguments[4]=3D=3D'jcatalogjsphdr')=0A= {=0A= //get the issn and pass it the next stage for processing=0A= var currloc =3D location.href;=0A= var issnloc =3D currloc.indexOf("&issn=3D");=0A= if (issnloc !=3D -1)=0A= {=0A= url =3D url + '¤tISSN=3D' + = currloc.substring(issnloc+6);=0A= }=0A= }=0A= // issn is needed for cocoon rendered mrw to form the final url.=0A= else if ( (arguments[4]=3D=3D'cocoonmrwhome') || = (arguments[4]=3D=3D'cocoonmrwarticle') )=0A= {=0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'issn' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tISSN=3D' + arguments[6];=0A= }=0A= //getting the parameters of the currently shown mrw from the = url=0A= //the method used here is a little crude, it extracts the = values from the current url of the page the user is in =0A= //to pass it to the background program to reconstruct the = requred url=0A= if (arguments[4]=3D=3D'cocoonmrwhome')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= var mrwFileParam =3D mrwParamArr[6];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= mrwFileParam =3D = mrwFileParam.substring(0,seploc);=0A= }=0A= seploc =3D mrwFileParam.indexOf("?");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwUrlParam=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= =0A= }=0A= else if (arguments[4]=3D=3D'cocoonmrwarticle')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= url =3D url + '&mrwAid=3D' + mrwParamArr[7];=0A= url =3D url + '&mrwVersion=3D' + mrwParamArr[8];=0A= var mrwFileParam =3D mrwParamArr[9];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= }=0A= }=0A= }=0A= =0A= //checking if a html file exists in the url=0A= var tmpfname =3D location.href;=0A= var loc =3D tmpfname.indexOf("?");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(0,loc);=0A= }=0A= loc =3D tmpfname.indexOf(".html");=0A= if (loc !=3D -1)=0A= {=0A= loc =3D tmpfname.lastIndexOf("/");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(loc+1);=0A= url =3D url + '¤tFile=3D' + tmpfname;=0A= }=0A= }=0A= =0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'currentyear' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tYear=3D' + arguments[6];=0A= }=0A= =0A= eval("wileyMailTool =3D = window.open('"+url+"','MailTool','"+parameters+"')");=0A= =0A= // If object exists ensure has focus=0A= if (eval("wileyMailTool") && window.focus)=0A= eval("wileyMailTool").focus();=0A= }=0A= =0A= =0A= /*=0A= * 1. Find the links we need to alter.=0A= * 2. For each link apply the new styles we need.=0A= */=0A= function fixAbsRefRendering()=0A= { =0A= var table =3D document.getElementById('prerendered');=0A= var anchors =3D table.getElementsByTagName('a');=0A= for (var i =3D 1; i < anchors.length; i++)=0A= {=0A= if(anchors[i].href !=3D"") =0A= { =0A= applyAbsRefStyleCorrection(anchors[i]);=0A= =0A= }=0A= }=0A= }=0A= =0A= function applyAbsRefStyleCorrection(currentLink)=0A= {=0A= currentLink.style["color"] =3D "#369";=0A= currentLink.style["fontWeight"] =3D "normal";=0A= currentLink.style["marginLeft"] =3D "3px";=0A= currentLink.style["marginRight"] =3D "5px";=0A= }=0A= =0A= function populatePage(formName)=0A= {=0A= if (formName.Page.value =3D=3D '')=0A= {=0A= formName.Page.value =3D document.location;=0A= }=0A= }=0A= =0A= function equalizeColumnWidth(numFeatures)=0A= {=0A= var numberOfFeatures =3D numFeatures; =0A= // Percentage width of table to use for each column.=0A= var percentPerFeature =3D Math.floor(100 / numberOfFeatures); =0A= // Number of columns in total. Content cols + seperators. =0A= =0A= document.writeln("");=0A= for(var i =3D 0; i < numberOfFeatures; i++)=0A= { =0A= document.writeln(' 3D"blank"

');=0A= }=0A= document.writeln("");=0A= }=0A= =0A= function old_equalizeColumnWidth(numFeatures)=0A= {=0A= var numberOfFeatures =3D numFeatures; =0A= // Percentage width of table to use for each column.=0A= var percentPerFeature =3D Math.floor(100 / numberOfFeatures); =0A= // Number of columns in total. Content cols + seperators. =0A= =0A= if(debug)=0A= {=0A= alert("Number of feature boxes: " + numberOfFeatures + "\nPercent per = feature: " + percentPerFeature);=0A= }=0A= =0A= document.writeln("");=0A= for(var i =3D 0; i < numberOfFeatures; i++)=0A= { =0A= if (netscape4 =3D=3D false)=0A= {=0A= document.writeln(' 3D"blank"

'); =0A= }=0A= }=0A= document.writeln("");=0A= }=0A= =0A= function writeFBoxCell()=0A= {=0A= if(first)=0A= {=0A= document.write('');=0A= }else=0A= {=0A= document.write('');=0A= }=0A= numFeatures ++;=0A= }=0A= =0A= =0A= //=0A= // escape apostrophe characters within a string=0A= //=0A= function encode(str)=0A= {=0A= out =3D '';=0A= =0A= for (a =3D 0; a < str.length; a++)=0A= {=0A= c =3D str.charAt(a);=0A= =0A= if (c =3D=3D "'")=0A= {=0A= out +=3D '\\';=0A= }=0A= =0A= out +=3D c;=0A= }=0A= =0A= return out;=0A= }=0A= =0A= =0A= //=0A= //=0A= //=0A= function escapeEntities(str)=0A= {=0A= while (str.indexOf("'") > -1)=0A= {=0A= pos =3D str.indexOf("'");=0A= str =3D str.substr(0, pos) + '\\\'' + str.substr(pos + 6);=0A= }=0A= =0A= return str;=0A= }=0A= =0A= =0A= // new popup function less intrusive just uses the DOM to pickup links = with an exwin class.=0A= // currently implemented for Figure popups on D&T homepage.=0A= =0A= function exLinks()=0A= {=0A= if (document.getElementById)=0A= {=0A= var aObject =3D document.getElementsByTagName("a");=0A= for (var x=3D0; x" + = liels[j].innerHTML + "

You do not have = anything saved in this menu.

";=0A= else=0A= tmpinnerhtml +=3D "

" = + liels[j].innerHTML + "

"; =0A= } =0A= } else {=0A= //setup var to see if more is required=0A= var more =3D (subnodeslength < lilength) ? true : = false; =0A= var flag =3D true;=0A= for (var j=3D0; j" + liels[j].innerHTML + = "";=0A= } else if (j > lilength-3){=0A= if (j =3D=3D lilength-2){=0A= var url =3D liels[j].innerHTML.split('"')[1];=0A= tmpinnerhtml +=3D "

" + liels[j].innerHTML + "

";=0A= } else {=0A= tmpinnerhtml +=3D "

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Brain Pathology

ISSN: 1750-3639 (Online)

ISSN: 1015-6305 (Print)

Volume 19, Issue 4, 2009.

Pages: 694=96712

Top of Page
Abstract
INTRODUCTION
PRINCIPLES OF DC THERAPY IN GLIOMA
DESIGN OF THERAPEUTIC STUDIES
RESULTS
CONCLUSIONS
ACKNOWLEDGMENTS
References

Tables: 1, 2

List of=20 Images

Abstract=20 Only

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/************************************************************************= ***************************=0A= FILE: site.js=0A= AUTHOR: Jonathan Sugar, Chris Neatby-Smith and Digital Pulp. July = 2003.=0A= =0A= DESCRIPTION: Javascript relating to site wide functionality across =0A= InterScience.=0A= =0A= INCLUDES FUNCTIONS=0A= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= Search Functions=0A= ----------------=0A= =0A= selectAndSubmit() - for deciding where to submit a query based on the = state of the form.=0A= processallContentSearch() - for validating form input for an all = content search.=0A= processTitleSearch() - for validating form input for a Title search.=0A= checkCitationSearch() - for validating the citation search form.=0A= selectAll() - check all the checkboxes in a form.=0A= do_popup() - stasa Acronym Finder window popup function.=0A= =0A= =0A= Other Functions=0A= ---------------=0A= =0A= fixInlineLists() - fix netscape 4.x bug where

tags with images = display both an image and=0A= the default bullet. =0A= =0A= = *************************************************************************= **************************/=0A= =0A= var http =3D false;=0A= if(navigator.appName =3D=3D "Microsoft Internet Explorer") {=0A= http =3D new ActiveXObject("Microsoft.XMLHTTP");=0A= } else {=0A= http =3D new XMLHttpRequest();=0A= } =0A= =0A= // Global Variables=0A= var debug =3D false;=0A= var first =3D true;=0A= =0A= // Browser detect globals:=0A= var uaStr =3D navigator.userAgent.toLowerCase();=0A= var uaVer =3D parseInt(navigator.appVersion);=0A= // for detecting netscape 4=0A= var netscape =3D (uaStr.indexOf("mozilla") > -1);=0A= var netscape4 =3D (netscape && uaVer < 5);=0A= =0A= /*=0A= * Used to switch search mode between APS and TitleFinder on quicksearch = boxes=0A= * = -------------------------------------------------------------------------= --=0A= *=0A= * 1. Decides which search is required based on the value of the radio = buttons=0A= * in 'document.formSelector.whichForm'.=0A= *=0A= * 2. Calls individual functions to process the forms and submit them.=0A= *=0A= */=0A= function selectAndSubmit()=0A= {=0A= var formSelection;=0A= =0A= // find out which form we need to process.=0A= var selector =3D document.formSelector.whichForm;=0A= for (var i =3D 0; i < selector.length; i++){=0A= if (selector[i].checked){=0A= formSelection =3D selector[i].value;=0A= }=0A= }=0A= =0A= // Call the relevent function to process the required form.=0A= if(formSelection =3D=3D "allContentSearch"){=0A= processallContentSearch(document.allContentSearchForm, = document.formSelector)=0A= }=0A= else if(formSelection =3D=3D "searchTitles"){=0A= processTitleSearch(document.searchTitles, document.formSelector)=0A= }=0A= else{=0A= return false;=0A= }=0A= =0A= //@@@DM It was anoying me this error =0A= return false;=0A= }=0A= =0A= /*=0A= *Process the ADS search form=0A= *=0A= */=0A= function trim(str)=0A= {=0A= return str.replace(/(^\s*)|(\s*$)/g,"");=0A= }=0A= =0A= function ADSquery(t) {=0A= var keyword =3D trim(t.getElementsByTagName("input")[0].value);=0A= if (keyword!=3D"") {=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= t.target=3D"popup";=0A= t.qsearch.value=3Dkeyword;=0A= return true;=0A= = //processADSSearch(document.ADSSearchForm,document.ADSQueryForm,keyword);=0A= } else {=0A= alert("Please input a keyword for ADS searching before clicking go");=0A= return false;=0A= }=0A= }=0A= /*=0A= function processADSSearch(hiddenForm ,queryForm , str) {=0A= var terms =3D str;=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= formToSubmit.qsearch.value =3D terms;=0A= = window.open("","popup","toolbar=3Dyes,location=3Dyes,status=3Dyes,menubar= =3Dno,scrollbars=3Dyes,resizable=3Dyes,width=3D800,height=3D500");=0A= formToSubmit.action =3D = "http://adsabs.harvard.edu/cgi-bin/nph-basic_connect"=0A= formToSubmit.target =3D "popup";=0A= formToSubmit.submit();=0A= }=0A= */=0A= =0A= /*=0A= * Process the allContentSearch form=0A= * ---------------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= function processallContentSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.WISsearch1.value =3D formToProcess.terms.value;=0A= formToSubmit.submit(); =0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= /*=0A= * Process the Title Search form=0A= * -----------------------------=0A= * 1. Perform form validation, checking for empty fields.=0A= * Give the user a relevent message if required.=0A= *=0A= * 2. Process/Assign the appropriate form elements and submit the form.=0A= */=0A= =0A= function processTitleSearch(hiddenForm, queryForm)=0A= {=0A= var formToSubmit =3D hiddenForm;=0A= var formToProcess =3D queryForm;=0A= =0A= // Do some basic form validation=0A= if(formToProcess.terms.value !=3D "")=0A= {=0A= formToSubmit.query.value =3D formToProcess.terms.value;=0A= formToSubmit.submit();=0A= }=0A= else=0A= {=0A= alert("You must enter a search term in the text box before you click = go.");=0A= }=0A= }=0A= =0A= function doCitationSearch()=0A= {=0A= var form =3D document.citsearchform;=0A= if(checkCitationSearch())=0A= {=0A= form.action =3D "/search/allsearch";=0A= form.submit();=0A= }=0A= }=0A= =0A= // Preprocesses the arguments sent to CitationSearch and checks validity = of user inputs.=0A= function checkCitationSearch()=0A= {=0A= var supplementFound =3D 0; //increment if found;=0A= var hyphenFound =3D 0; //increment if found;=0A= var fm =3D document.citsearchform;=0A= var errmsg =3D "Please use the valid digits '0' to '9'";=0A= // remove all spaces=0A= fm.volume.value =3D fm.volume.value.replace(/ /g, "");=0A= fm.issue.value =3D fm.issue.value.replace(/ /g, "");=0A= fm.pages.value =3D fm.pages.value.replace(/ /g, "");=0A= =0A= if (fm.issn.value.length > 0 && fm.volume.value.length > 0 && = fm.pages.value.length > 0)=0A= {=0A= // check page range=0A= var pageRange =3D fm.pages.value;=0A= var volume =3D fm.volume.value;=0A= var issue =3D fm.issue.value;=0A= var continueFlag =3D 0;=0A= //check the volume=0A= // if (isNaN(volume))=0A= // {=0A= // // volume failed checking=0A= // alert("Volume is invalid. "+errmsg+".");=0A= // return false;=0A= // }=0A= //check the issue=0A= if ((issue.length !=3D 0) && isNaN(issue))=0A= {=0A= // issue failed checking=0A= alert("Issue is invalid. "+errmsg+" or leave blank.");=0A= return false;=0A= }=0A= //check the page range=0A= if (isNaN(pageRange))=0A= {=0A= var isValid =3D false;=0A= // check for hyphen=0A= var hyphenIndex =3D pageRange.indexOf("-");=0A= if ((hyphenIndex !=3D -1) && (hyphenIndex =3D=3D = pageRange.lastIndexOf("-")))=0A= {=0A= // we have only one hyphen, so extract each number=0A= var firstNum =3D pageRange.substring(0, hyphenIndex);=0A= var secondNum =3D pageRange.substring(hyphenIndex + 1);=0A= // check for S=0A= var firstS =3D firstNum.indexOf("S");=0A= var secondS =3D secondNum.indexOf("S");=0A= if (firstS =3D=3D secondS)=0A= {=0A= if ((firstS =3D=3D -1) && isNum(firstNum) && isNum(secondNum) && = (firstNum.length > 0) && (secondNum.length > 0))=0A= isValid =3D true; // both inputs are digits=0A= else if (firstS =3D=3D 0)=0A= {=0A= var firstNumNoS =3D firstNum.substring(1);=0A= var secondNumNoS =3D secondNum.substring(1);=0A= if (isNum(firstNumNoS) && isNum(secondNumNoS) && = (firstNumNoS.length > 0) && (secondNumNoS.length > 0))=0A= isValid =3D true; // both inputs are Sdigits=0A= }=0A= =0A= }=0A= }=0A= if (isValid)=0A= {=0A= return true;=0A= }=0A= else=0A= {=0A= alert("The page range is invalid. "+errmsg+" and a hyphen '-' if = required.");=0A= return false;=0A= }=0A= }=0A= return true;=0A= }=0A= else=0A= {=0A= alert("Volume and pages are required fields. Please make sure data has = been entered for both fields.");=0A= return false;=0A= }=0A= }=0A= =0A= // returns true if a is an Integer.=0A= function isNum(a)=0A= { return !(isNaN(a)); }=0A= =0A= function fixInlineLists() {=0A= if (!document.getElementById) return;=0A= var allLIs =3D document.getElementsByTagName('li');=0A= var bulletNode =3D document.createElement('img');=0A= bulletNode.src = =3D"http://download.interscience.wiley.com/images/icon.bullet.gif";=0A= =0A= for (var i=3D0; iAbstract | References';=0A= }=0A= //alert("URL: " + loc + "\nStart Index: " + startIndex + "\nEnd Index: = " + endIndex + "\nPage: " + page + "\nLink Text: " + links);=0A= return links;=0A= }=0A= =0A= =0A= function selectAll(fm)=0A= {=0A= //var fm =3D document.viewSelectedForm;=0A= =0A= for (var i =3D 0; i < fm.elements.length; i++)=0A= {=0A= if (fm.elements[i].type =3D=3D 'checkbox' /*&& fm.elements[i].name = =3D=3D 'ID'*/)=0A= {=0A= fm.elements[i].checked =3D true;=0A= }=0A= }=0A= }=0A= =0A= =0A= function do_popup(file)=0A= {=0A= var w =3D window.open(file, 'popWin', = 'width=3D350,height=3D500,scrollbars=3Dyes');=0A= =0A= }=0A= =0A= =0A= function mailTool(itemID, useurl, skin)=0A= {=0A= //var url =3D "/cgi-bin/mailform?item=3D"+itemID;=0A= var url =3D "/cgi-bin/mailform?item=3D" + itemID + "&skin=3D" + skin;=0A= var parameters =3D = 'status=3Dno,scrollbars=3Dno,resizable=3Dyes,width=3D400,height=3D475';=0A= =0A= if (useurl !=3D "")=0A= {=0A= url +=3D "&useurl=3D"+useurl;=0A= }=0A= =0A= if ((isNaN(parseInt(itemID))=3D=3Dtrue)||(arguments.length >=3D 4 && = arguments[3] =3D=3D 'cluster'))=0A= {=0A= url +=3D '&type=3Dcluster';=0A= }=0A= =0A= //check whether the last 2 arguments is used for urllink.=0A= // urllink is needed to open up mail form for mrw pages on tomcat = port=0A= if (arguments.length>=3D5 && = arguments[arguments.length-2]=3D=3D'urllink' && = arguments[arguments.length-1]!=3D'')=0A= {=0A= url =3D arguments[arguments.length-1] + url;=0A= }=0A= =0A= if (arguments.length>=3D5 && arguments[3]=3D=3D'currentlink' && = arguments[4]!=3D'')=0A= {=0A= url =3D url + '¤tLink=3D' + arguments[4];=0A= =0A= //processing specific cases=0A= if (arguments[4]=3D=3D'jcatalogjsphdr')=0A= {=0A= //get the issn and pass it the next stage for processing=0A= var currloc =3D location.href;=0A= var issnloc =3D currloc.indexOf("&issn=3D");=0A= if (issnloc !=3D -1)=0A= {=0A= url =3D url + '¤tISSN=3D' + = currloc.substring(issnloc+6);=0A= }=0A= }=0A= // issn is needed for cocoon rendered mrw to form the final url.=0A= else if ( (arguments[4]=3D=3D'cocoonmrwhome') || = (arguments[4]=3D=3D'cocoonmrwarticle') )=0A= {=0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'issn' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tISSN=3D' + arguments[6];=0A= }=0A= //getting the parameters of the currently shown mrw from the = url=0A= //the method used here is a little crude, it extracts the = values from the current url of the page the user is in =0A= //to pass it to the background program to reconstruct the = requred url=0A= if (arguments[4]=3D=3D'cocoonmrwhome')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= var mrwFileParam =3D mrwParamArr[6];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= mrwFileParam =3D = mrwFileParam.substring(0,seploc);=0A= }=0A= seploc =3D mrwFileParam.indexOf("?");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwUrlParam=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= =0A= }=0A= else if (arguments[4]=3D=3D'cocoonmrwarticle')=0A= {=0A= var currloc =3D location.href;=0A= var mrwParamArr =3D currloc.split("/");=0A= if (mrwParamArr.length >=3D 7)=0A= {=0A= url =3D url + '&mrwPath=3D' + mrwParamArr[5];=0A= url =3D url + '&mrwAid=3D' + mrwParamArr[7];=0A= url =3D url + '&mrwVersion=3D' + mrwParamArr[8];=0A= var mrwFileParam =3D mrwParamArr[9];=0A= var seploc =3D mrwFileParam.indexOf("#");=0A= if (seploc !=3D -1)=0A= {=0A= url =3D url + '&mrwSection=3D' + = mrwFileParam.substring(seploc+1);=0A= url =3D url + '&mrwDocType=3D' + = mrwFileParam.substring(0,seploc);=0A= }=0A= else=0A= {=0A= url =3D url + '&mrwDocType=3D' + mrwFileParam;=0A= }=0A= }=0A= }=0A= }=0A= }=0A= =0A= //checking if a html file exists in the url=0A= var tmpfname =3D location.href;=0A= var loc =3D tmpfname.indexOf("?");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(0,loc);=0A= }=0A= loc =3D tmpfname.indexOf(".html");=0A= if (loc !=3D -1)=0A= {=0A= loc =3D tmpfname.lastIndexOf("/");=0A= if (loc !=3D -1)=0A= {=0A= tmpfname =3D tmpfname.substring(loc+1);=0A= url =3D url + '¤tFile=3D' + tmpfname;=0A= }=0A= }=0A= =0A= if (arguments.length>=3D7 && arguments[5]=3D=3D'currentyear' && = arguments[6]!=3D'')=0A= {=0A= url =3D url + '¤tYear=3D' + arguments[6];=0A= }=0A= =0A= eval("wileyMailTool =3D = window.open('"+url+"','MailTool','"+parameters+"')");=0A= =0A= // If object exists ensure has focus=0A= if (eval("wileyMailTool") && window.focus)=0A= eval("wileyMailTool").focus();=0A= }=0A= =0A= =0A= /*=0A= * 1. Find the links we need to alter.=0A= * 2. For each link apply the new styles we need.=0A= */=0A= function fixAbsRefRendering()=0A= { =0A= var table =3D document.getElementById('prerendered');=0A= var anchors =3D table.getElementsByTagName('a');=0A= for (var i =3D 1; i < anchors.length; i++)=0A= {=0A= if(anchors[i].href !=3D"") =0A= { =0A= applyAbsRefStyleCorrection(anchors[i]);=0A= =0A= }=0A= }=0A= }=0A= =0A= function applyAbsRefStyleCorrection(currentLink)=0A= {=0A= currentLink.style["color"] =3D "#369";=0A= currentLink.style["fontWeight"] =3D "normal";=0A= currentLink.style["marginLeft"] =3D "3px";=0A= currentLink.style["marginRight"] =3D "5px";=0A= }=0A= =0A= function populatePage(formName)=0A= {=0A= if (formName.Page.value =3D=3D '')=0A= {=0A= formName.Page.value =3D document.location;=0A= }=0A= }=0A= =0A= function equalizeColumnWidth(numFeatures)=0A= {=0A= var numberOfFeatures =3D numFeatures; =0A= // Percentage width of table to use for each column.=0A= var percentPerFeature =3D Math.floor(100 / numberOfFeatures); =0A= // Number of columns in total. 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return hwnd; } =0A= function pagetitle() {return 'Brain Pathology Vol. 19, 4 Pages: = 694-712'; } =0A= function dochead(hwnd, spec) {var title =3D pagetitle(); = hwnd.document.open(); = hwnd.document.write(''+spec+title+''); } =0A= function doctail(hwnd) {hwnd.document.write(''); } =0A= function pagehead(hwnd) {hwnd.document.write('

3D"About

Online ISSN: = 1096-9896 Print ISSN: 0022-3417
The Journal of = Pathology
Volume 214, Issue 3, 2008. Pages: 328-336

Copyright =C2=A9 2007 Pathological Society of Great = Britain and Ireland. Published by John Wiley & Sons, Ltd.

'); } =0A= function copyright(hwnd) {var title =3D pagetitle(); = hwnd.document.write(title.bold() + '
Copyright Journal compilation = © 2009 International Society of Neuropathology

'); } =0A= function imagelink(hwnd, iname) {if (iname.indexOf(' ') > -1) {var = images =3D iname.split(' ');for (var i =3D 0; i < images.length; ++i) = {hwnd.document.write('

');}} = else {hwnd.document.write('

');}}=0A= function otherlinks(hwnd, iname, size) { hwnd.document.write('View List of Images = '); hwnd.document.write('

'); } =0A= =0A= =0A= function caption_f1(hwnd) = {hwnd.document.write('Figure 1. Electroporation of = DCs with RNA-molecules is feasible without loss of cell function in = vitro. A. Immature DCs were left untouched (no EP) or = electroporated (EP) and immediately afterwards matured with = 1 µg/mL (Escherichia coli) lipopolysaccharide for 24 = hours. Mature DCs were analyzed by flow cytometry for surface marker = expression. EP significantly lowered the expression of CD80 and CD86 on = DCs (overall analysis of variance P < 0.001, = *P < 0.05). B. The capacity to stimulate = allogeneic cells was assessed in a mixed lymphocyte reaction in 96 well = format with total 2 × 10⁵ splenocytes from = naive BALB/C mice as responder (R) cells and DCs from C57BL/6 mice as = stimulator (S) cells. Unstimulated cells and phytohemagglutinin (PHA) = were used as negative and positive controls, respectively. Responder to = stimulator ratios (R : S) ranged from 5:1 to 50:1. After 4 = days of culture, thymidine incorporation was measured by pulsing = cultures for 18 hours with 1 µCi [³H] thymidine = per well. Mean counts per minute (cpm) are depicted. C. One = million DCs were electroporated with 1 µg mRNA encoding = enhanced green fluorescent protein (eGFP) and eGFP expression (grey = curve in FL1-Height histogram) was assessed by flow cytometry 24 hours = after electroporation. The filled curve represents nonelectroporated = DCs.'); }=0A= function display_f1(size) { var i =3D new Array(); i[0] =3D 'nf1'; i[1] = =3D 'nf1'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f1', size); imagelink(hwnd, i[size]); = caption_f1(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= function caption_f2(hwnd) = {hwnd.document.write('Figure 2. RNA-loaded DCs induce a = T cell-mediated antitumor immune response in vitro. T cells = pooled from spleen and lymph nodes of a naive C57BL/6 mouse were = stimulated ex vivo (two stimulation rounds with DC : T = cell ratio of 1:10 with addition of 20 U/mL of IL-2) with either = mock-loaded DCm (DCm-mock), DCm electroporated with total RNA extracted = from Lewis lung carcinoma cells (DCm-LLC-RNA) or DCm electroporated with = total RNA extracted from GL261 glioma cells (DCm-GL261-RNA). = Unstimulated T cells were used as the control. Stimulated T cells were = then used as effector cells and coincubated with GL261 tumor cells in an = effector to target ratio of 10:1. Tumor cell viability was assessed by = measuring the metabolization of = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan = (measured at 570 nm) by living cells after 2 days of co-culture. = Pooled results (n =3D 3) are depicted as optical density = values at 570 nm (OD₅₇₀) and compared to GL261 target = cells without effector cells. Overall analysis of variance = P < 0.001; **P < 0.01, = ***P < 0.001.'); }=0A= function display_f2(size) { var i =3D new Array(); i[0] =3D 'nf2'; i[1] = =3D 'nf2'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f2', size); imagelink(hwnd, i[size]); = caption_f2(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= function caption_f3(hwnd) = {hwnd.document.write('Figure 3. Immunotherapy in the = GL261 experimental orthotopic glioma mouse model.A. Overview = of the prophylactic DC vaccination model. Adult C57BL/6 mice are = orthotopically challenged with 5 × 10⁵ = syngeneic GL261 malignant glioma cells injected 1.5 mm posterior = and 1 mm lateral from the bregma at a depth of 3 mm under = stereotactic guidance. Read-out consists of monitoring general health = parameters (weight and neurological symptoms), overall survival = analysis, in vivo bioluminescence (BLI) requiring GL261 cells = expressing luciferase in white mutant C57BL/6.Cg.Tyrc-2J/J mice, and = detailed immune monitoring both systemically (in blood, spleen and = tumor-draining cervical lymph nodes) and locally within the brain. = Immunotherapy in our preventive treatment setting consists of either = active immunization, decreasing regulatory T cell (Treg)-mediated immune = suppression, or combined treatment. Active immunization is performed = through intraperitoneal (IP) vaccination on day 14 and day 7 before = tumor challenge with one million mature bone-marrow derived CD11c+ DCs = that were loaded ex vivo with tumor antigens (in the form of = total RNA). In some experiments, animals were preconditioned with a = single intraperitoneal injection (250 µg) of the depleting = anti-CD25 monoclonal antibody (clone PC61). This in vivo = depletion of CD25-expressing cells was performed 1 week before the first = vaccination with DC (or 3 weeks before tumor challenge if no DC = vaccination was administered). B. Pooled survival data = represented as Kaplan–Meier graph. Overall log rank = P < 0.001. Survival curves of animals vaccinated = with DCm-GL261-RNA (green curve, n =3D 27), DCm-GL261-L (DC = loaded with GL261 lysate) (red curve, n =3D 19), = DCm-splenocyte-RNA (blue curve, n =3D 5) and untreated animals = (black curve, n =3D 13) are depicted.'); }=0A= function display_f3(size) { var i =3D new Array(); i[0] =3D 'nf3'; i[1] = =3D 'nf3'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f3', size); imagelink(hwnd, i[size]); = caption_f3(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= function caption_f4(hwnd) = {hwnd.document.write('Figure 4. HGG-IMMUNO-2003 = protocol: DC vaccination for adults with relapsed GBM. Patients with = relapsed GBM were reoperated (removal of as much tumor tissue as = feasible) and received subsequent immunotherapy. Patients were divided = among four consecutive cohorts.A. The age (expressed in years = on the y-axis) for the four cohorts is shown, and median age is = indicated. B. The number of patients with total resection or less = than total resection of relapsed GBM is shown for each cohort = (ND =3D not documented). C. Progression-free survival = (PFS) for the four cohorts of patients. D. Overall survival (OS) = for the four cohorts of patients.'); }=0A= function display_f4(size) { var i =3D new Array(); i[0] =3D 'nf4'; i[1] = =3D 'nf4'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f4', size); imagelink(hwnd, i[size]); = caption_f4(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= function caption_f5(hwnd) = {hwnd.document.write('Figure 5. MRI findings during and = after DC vaccination for relapsed GBM. Transverse T1-weighted MR images = after contrast administration, obtained 1 day after debulking surgery = for relapsed GBM (early postoperative period), 6 weeks after the first = vaccine (6 w after V1), 6 months after the first vaccine (6 m = after V1), 9 months after the first vaccine (9 m after V1) and = after long-term follow-up (LT follow-up), 3 to 6 years after the first = vaccine according to the case (3–6 years after V1). Case 1 is a = patient with a resection in the left frontal lobe. MR imaging showed = discrete contrast enhancement around the resection cavity 6 weeks after = administration of the first vaccine. No contrast enhancement was = documented in follow-up imaging. Case 2 is a patient after surgery in = the right frontal lobe. Transient contrast enhancement was seen at 6 and = 9 months after the start of the vaccination therapy. Follow-up MR scans = showed subsequent resolution of the contrast enhancement. Case 3 shows = follow-up MR images in a patient with surgery in the left frontal lobe. = Over consecutive time points, the resection cavity collapsed and a = comparable area of contrast enhancement has remained stable for 3 years. = Case 4 is a patient with a resection in the right frontoparietal region. = Subsequent MRI shows an increasing region of contrast enhancement, = suggestive of tumor relapse/progression.'); }=0A= function display_f5(size) { var i =3D new Array(); i[0] =3D 'nf5'; i[1] = =3D 'nf5'; var hwnd =3D newfigwin(); dochead(hwnd, ''); copyright(hwnd); = otherlinks(hwnd, 'f5', size); imagelink(hwnd, i[size]); = caption_f5(hwnd); doctail(hwnd); hwnd.document.close(); hwnd.focus(); }=0A= =0A= =0A= function imagelist() {var hwnd =3D newfigwin(); dochead(hwnd, 'List of = Images - '); copyright(hwnd); =0A= hwnd.document.write('');=0A= =0A= =0A= hwnd.document.write('');=0A= hwnd.document.write('');=0A= hwnd.document.write('');=0A= hwnd.document.write('');=0A= hwnd.document.write('');hwnd.document.write('

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Brain Pathology

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Volume 19, Issue 4, Pages 694-712

Published Online: 4 Sep 2009

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MINI-SYMPOSIUM: Immunotherapy of = Gliomas

Dendritic Cell Therapy of High-Grade=20 Gliomas

Stefaan Van = Gool=20 ¹ ; Wim Maes=20 ² ; Hilko Ardon=20 ³ ; Tina Verschuere=20 ² ; Sofie Van = Cauter=20 ⁴ ; Steven De = Vleeschouwer=20 ³

¹ Laboratory of = Experimental Immunology, and Department of Child & Woman, = Catholic=20 University of Leuven, Leuven, Belgium.
² Laboratory of = Experimental Immunology, Catholic University of Leuven, Leuven,=20 Belgium.
³ Laboratory=20 of Experimental Immunology, and Department of Neurosciences, = Catholic=20 University of Leuven, Leuven, Belgium.
⁴ Department of = Radiology, Catholic University of Leuven, Leuven, Belgium.=20

Correspondence to Stefaan = Van Gool, MD,=20 PhD, Pediatric Neuro-oncology, University Hospital Leuven, = Catholic=20 University of Leuven, Herestraat 49, 3000 Leuven, Belgium (E-mail: = stefaan.vangool@uz.kuleuve= n.be)=20

Stefaan Van Gool is senior clinical investigator at the = Fund for=20 Scientific Research Flanders (FWO-V). Wim Maes is supported by the = Olivia=20 Hendrickx Research Fund. Hilko Ardon and Tina Verschuere are = research=20 fellows supported by the Institute for the Promotion of Innovation = by=20 Science and Technology in Flanders (IWT). Sofie Van Cauter is = research=20 fellow in the Department of Radiology. Steven De Vleeschouwer is = supported=20 by the Klinisch Onderzoeksfonds UZ Leuven.

Copyright Journal compilation =A9 2009 = International Society of Neuropathology

KEYWORDS

clinical trials =95 dendritic cells =95 = glioblastoma =95=20 high-grade glioma =95 immunotherapy =95 translational = medicine

ABSTRACT

3DAbstract=20

The prognosis of patients with malignant glioma is = poor in=20 spite of multimodal treatment approaches consisting of = neurosurgery,=20 radiochemotherapy and maintenance chemotherapy. Among innovative = treatment=20 strategies like targeted therapy, antiangiogenesis and gene = therapy=20 approaches, immunotherapy emerges as a meaningful and feasible = treatment=20 approach for inducing long-term survival in at least a = subpopulation of=20 these patients. Setting up immunotherapy for an inherent = immunosuppressive=20 tumor located in an immune-privileged environment requires = integration of=20 a lot of scientific input and knowledge of both tumor immunology = and=20 neuro-oncology. The field of immunotherapy is moving into the = direction of=20 active specific immunotherapy using autologous dendritic cells = (DCs) as=20 vehicle for immunization. In the translational research program of = the=20 authors, the whole cascade from bench to bed to bench of active = specific=20 immunotherapy for malignant glioma is covered, including proof of=20 principle experiments to demonstrate immunogenicity of = patient-derived=20 mature DCs loaded with autologous tumor lysate, preclinical in vivo experiments in a murine orthotopic glioma = model,=20 early phase I/II clinical trials for relapsing patients, a phase = II trial=20 for patients with newly diagnosed glioblastoma (GBM) for whom=20 immunotherapy is integrated in the current multimodal treatment, = and=20 laboratory analyses of patient samples. The strategies and results = of this=20 program are discussed in the light of the internationally = available=20 scientific literature in this fast-moving field of basic science = and=20 translational clinical research.

Received 15 June 2009; accepted 17 June=20 2009.

DIGITAL OBJECT IDENTIFIER (DOI)

10.1111/j.1750-3639.2009.00316.x About=20 DOI

=20

=20

=20

INTRODUCTION

High-grade gliomas (HGGs) consist of World Health=20 Organization (WHO) grade III and WHO grade IV neoplasms:=20 anaplastic/malignant gliomas respectively glioblastomas (GBMs). = While GBMs=20 are the most frequent and most malignant of these tumors, they = still=20 belong to the category of orphan diseases, with a yearly incidence = of=20 about three to four per 100 000 adults (42)=20 and two per million children (120).=20 The treatment for these patients consists primarily of surgery in = order to=20 debulk the tumoral mass for symptomatic relief and to obtain = tissue for=20 histological diagnosis, as well as radiochemotherapy to induce = optimal=20 local tumor control. In spite of improved surgical and = radiotherapeutic=20 techniques, and the addition of temozolomide to the multimodal = treatment=20 strategy, the prognosis of patients with GBM remains poor: the = median=20 overall survival (OS) is about 15 months, with 88% of patients = dead within=20 3 years (117,=20 118). Indeed, even after maximal treatment with surgery, = radiotherapy=20 and chemotherapy, relapse is universal and is believed to be due = to the=20 extensive spread of tumor cells into surrounding regions of the = brain (68,=20 21). At the time of relapse, the prognosis is particularly = poor, with=20 reports of 100% mortality within 1.5 years (13).=20 As such, there is obviously a need for more effective new = therapies. One=20 of the particular challenges with classical chemotherapeutic = strategies is=20 overcoming the blood-brain barrier. Therefore, much of today's = preclinical=20 research is focused on alternate approaches, such as more = selective=20 therapies, which specifically target intracellular signaling = pathways or=20 surface molecules (44,=20 84), antiangiogenesis strategies (46)=20 and especially immunotherapy. Acknowledging that different aspects = of=20 immunotherapy for malignant gliomas have been extensively reviewed = in the=20 last couple of years (149,=20 97, 66, 144, 94, 96, 28, 41, 27, 116, 2, 3, 133, 102, 17, 140, 38, = 79,=20 141), the aim of the present paper is to present our own = translational=20 research program efforts as a disease-specific paradigm for active = specific immunotherapy, rather than contributing one more = encyclopedic=20 review of dendritic cell (DC) therapy overall.

=20

=20

PRINCIPLES OF DC THERAPY IN = GLIOMA

=20

=20

Active specific immunotherapy

Immunotherapy of HGGs covers a broad field = comprising=20 several approaches. These include passive immunotherapy with = monoclonal=20 antibodies (mAb) (132),=20 passive immune stimulation with cytokines (restorative) (86,=20 11, 148, 145, 39), treatment with stimulated T cells = (adoptive) (113,=20 106, 100) and active specific immunotherapy. In the latter = approach,=20 DCs are most commonly used to stimulate the immune system, given = their=20 role as the most powerful antigen-presenting cells (APCs) known to = date.=20 DCs are a subset of white blood cells, critical to most aspects of = adaptive immunity due to their central role as specialized APCs in = the=20 initiation phase of T cell responses (83,=20 54). Typically, DCs reside as immature cells in almost every = organ and=20 tissue at the interface of potential pathogen entry sites. In this = state,=20 they continuously sample antigens. This sampling, however, results = in=20 effective antigen presentation only when DCs are also triggered by = danger=20 signals derived from pathogens, tissue damage or signs of = inflammation. As=20 danger-triggered DCs start to mature and get activated, they = up-regulate=20 chemokine receptors, which guide them to draining lymph nodes. = There, the=20 mature DCs are capable of inducing primary T cell responses due to = their=20 high levels of major histocompatibility complex (MHC), adhesion = and=20 costimulatory molecule expression. Only mature DCs show the = typical=20 dendritic morphology with multiple cytoplasmic extensions (7).=20 Unlike other APCs like macrophages, monocytes and B cells, DCs are = able to=20 present and cross-present the antigenic peptides in the context of = both=20 MHC Class II and Class I molecules, respectively (110,=20 109). In this way, they can prime not only CD4+ T helper = cells, but=20 also CD8+ cytotoxic T cells (23).=20 Both effector cell types are believed to be necessary to induce an = effective cell-mediated immune response (34,=20 72, 121). Direct recognition of tumor cells by unprimed T = cells, in=20 contrast, induces T cell anergy rather than immunity as the = costimulatory=20 molecules are missing on the tumor cells (127,=20 126).

DCs are not only sentinels in the adaptive immune = response,=20 but have also been shown to be strong activators of natural killer = (NK)=20 cells and NK T cells (31),=20 thus linking the innate and adaptive immune responses. In this = way, both=20 tumor cells with and without expression of MHC Class I molecules = can=20 theoretically be killed (8).=20 All these particular characteristics make DCs the perfect = adjuvants in=20 active specific immunotherapeutic strategies, in which one aims to = induce=20 a specific immune response in vivo(6,=20 136, 119, 45, 59). Gliomas have been shown to express several=20 tumor-associated antigens (TAAs), including epidermal growth = factor=20 receptor isoform III (85),=20 glycoprotein 240 (69),=20 tenascin (128),=20 survivin (139),=20 squamous cell carcinoma-associated reactive antigen for cytotoxic = T cells=20 1 (57),=20 the alpha-2 chain of interleukin 13 receptor (IL-13Rα2 chain) = (93),=20 melanoma-associated antigens like tyrosinase, tyrosinase-related = protein 1=20 and 2, gp100, melanoma antigen (MAGE)-1 and MAGE-3 (18,=20 115, 75, 76) and Immunoselected Melanoma-2 (AIM-2) (77),=20 all extensively reviewed by Dunn et al(35).=20 Moreover, GBM patients exhibit circulating tumor-specific CD8+ T = cells (122).=20 Until now, however, a universally expressed glioma TAA with a = critical=20 downstream cell survival-related function has not been identified. = Therefore, just targeting the known TAAs using individual peptides = would=20 inherently lead to immune escape because of the positive clonal = selection=20 of antigen-loss variants (48).=20 In other words, those tumor cell clones that do not express the = particular=20 targeted TAA (anymore) will escape immune rejection and thus have = an=20 important proliferation advantage as compared with the cell clones = that=20 still express the targeted TAA. That heterogeneity in TAA = expression in=20 gliomas represents the main reason why most investigators favor = the use of=20 whole-tumor cell antigens as a source of TAAs to load the DCs. For = this,=20 acid-eluted membrane peptides (73,=20 147, 74), apoptotic bodies after gamma-irradiation (130),=20 tumor lysates (142,=20 143, 146, 135, 134, 25, 111, 30) and fusion of DC with tumor = cells (64,=20 65) have all been used to load DCs. Of note, Khan et al(63)=20 isolated proteins out of paraffin-embedded tissue to load the DCs. = To our=20 knowledge, no tumoral RNA has been used to load DCs for = administration to=20 patients thus far. In a comparative analysis, the loading with = apoptotic=20 bodies or total tumoral RNA seemed to be superior to loading with = lysate=20 or fusion of HGG cells with DCs (95).=20 However, loading with apoptotic bodies of HGGs has also been shown = to=20 increase the risk of inducing tolerogenic DCs via the = cyclooxygenase-2=20 (COX2) pathway (4).=20 Loading DCs with specific antigens that play key roles in tumor = biology=20 has also been attempted (138,=20 19, 20). Because glioma cancer stem cells might represent the=20 therapy-resistant fraction of HGGs, cancer stem cell-redirected=20 immunotherapy has been explored (99,=20 98). In our translational research program, we developed=20 patient-derived autologous mature DCs loaded with lysate of the = autologous=20 HGG (DCm-HGG-L) for in vitro investigation = and for=20 clinical applications. In our murine model, we used DCm loaded = with lysate=20 of the GL261 murine glioma tumor cells or with total RNA of GL261 = cells=20 (DCm-GL261-RNA). The GL261 glioma cell line is derived from=20 Methylcholantrene-induced glioma tumor cells in C57BL/6J = mice.

=20

=20

Experimental rodent models for = malignant=20 glioma

=20

=20

Hurdles associated with current = animal=20 models

Over the last 4 decades, hundreds of reports of = heterotopic=20 and orthotopic rat and mouse GBM models have been published. It is = extremely important to be aware of some fundamental differences = between=20 those models, for example, between models exploiting spontaneous = tumor=20 formation in genetically engineered mice versus=20 engrafted tumor models that are established by implantation of = primary=20 tumor cells or tumor cell lines. Although spontaneous tumor models = are=20 mimicking the clinical situation of gliomagenesis much better than = the=20 engrafted models, the main drawbacks are the poor reproducibility, = low=20 tumor penetrance, prolonged latency for tumor formation and the = need for=20 advanced in vivo imaging techniques. On the = other=20 hand, because engrafted models lack the stepwise genetic changes = occurring=20 during tumor progression, many of them are well circumscribed, = lack the=20 human counterpart of microvascular proliferation and rarely = recapitulate=20 the tumor-of-origin phenotype. Nevertheless, based on greater=20 reproducibility, engrafted models are better suited for evaluating = preclinical therapies such as DC immunotherapy, as long as the = models are=20 studied in immunocompetent animals (16,=20 43, 22).

=20

=20

Data from the literature

Below, we briefly summarize the most relevant data = on the=20 murine GL261 glioma model, as this model is also used in our = translational=20 research program. Ni et al underscored = the=20 immunogenicity of the GL261 tumor cells by treating mice with = intracranial=20 glioma with tumor extract-pulsed cloned DCs. Cured animals showed=20 increased delayed-type hypersensitivity (DTH) responses to GL261 = cells,=20 with long-term tumor protection (89).=20 Aoki et al showed that pulsing DCs = with a=20 complex of tumor extract with cationic liposomes induces an = antitumor=20 immune response against intracerebral glioma in which CD8+ T cells = are=20 involved (5).=20 Protective immunity against intracranial glioma growth obtained = through=20 immunization with either lysate- or RNA-loaded DCs was reported by = Insug=20 et al, while adding recombinant IL-12 = to the=20 vaccine regimen further improved its efficacy (58).=20 A survival benefit of combining vaccination with lysate-pulsed DCs = and=20 interferon (IFN)-β gene therapy was demonstrated by Saito = et al(112).=20 Similarly, the group of Okada et al = revealed=20 that the sequential intratumoral delivery of an IFN-α = encoding adenoviral=20 vector and DCs induced long-term survival and specific cytotoxic T = lymphocyte (CTL) activity (123).=20 Furthermore, the same group showed that intratumoral = administration of=20 DCs, genetically engineered to secrete IFN-α, enhances the = efficacy of=20 peripheral vaccines with cytokine gene-transduced tumor cells (70).=20 Kjaergaard et al observed complete = tumor=20 regression of established intracranial tumors with infiltration of = both=20 CD4+ and CD8+ T cells using vaccines created through electrofusion = of DCs=20 and irradiated tumor cells (67).=20 Survivin, a member of the apoptosis inhibition family of proteins, = has=20 been the GL261 TAA of choice for Ciesielski et al In particular, the authors exploited = the=20 xenogeneic differences between human and murine survivin sequences = to=20 develop a more immunogenic tumor vaccine (19,=20 20). The efficacy of systemic immunotherapy with DCs loaded = with GL261=20 antigens was confirmed by Pellegatta et al=20 Additionally, these authors introduced the concept of cancer stem = cells in=20 this model and reported that DC targeting of such stem cells = within the=20 GL261 tumor cell pool provides a higher level of protection = against GL261=20 glioma (99,=20 98). Recently, Grauer et al = illustrated the=20 pronounced impact of FoxP3+ regulatory T cells (Tregs) in this = model and=20 suggested that Treg elimination is a prerequisite for successful=20 eradication of established glioma using tumor lysate-pulsed DCs (50,=20 51).

=20

=20

The concept of DC treatment in the = orthotopic=20 GL261 mouse model

We implemented the concept of active glioma = immunotherapy=20 with RNA-loaded DCs in the GL261 murine model step-by-step. The = rationale=20 for loading DCs with total RNA from tumoral origin is to be able = to=20 provide DC immunotherapy for those glioma patients where = insufficient=20 tumor tissue is available to produce an optimal amount of = DCm-HGG-L, for=20 example, in patients with a small tumor mass of a relapsed tumor = located=20 in a previously irradiated area. Moreover, RNA molecules are = translated=20 into proteins by the DCs themselves and those proteins are = subsequently=20 processed in the endogenous pathway of antigen processing, = resulting in=20 optimal priming of CD8+ T cells. Researchers have dedicated a lot = of=20 effort to the improvement of RNA transfection methods. Three = techniques=20 have been investigated: passive transfection, lipid-mediated = transfection=20 and electroporation (47,=20 53). Using the latter technique, immature DCs are transfected = with=20 15 =B5g of total tumoral RNA per million DCs through = exponential decay=20 electroporation (300 V and 150 =B5F) with a GenePulser=20 electroporator (Bio-Rad, Nazareth, Belgium)(82,=20 81). By now, electroporation has proven to be the method of = choice for=20 the introduction of exogenous RNA molecules into DCs. It does not = require=20 additional reagents and is compatible with clinical use (37).=20 With this technology, improving the intrinsic characteristics of = DCs such=20 as cytokine, chemokine and costimulator expression through = transfection=20 with additional mRNA species becomes possible and seems very = promising (1,=20 12).

Initially, we focused on testing of our cellular = product=20 (murine DCs electroporated with total RNA from GL261 tumor cells,=20 DCm-GL261-RNA) in an experimental in vitro = design.=20 The procedure of electroporation caused only minor changes in the = DC=20 phenotype (Figure 1a),=20 and did not alter the stimulatory capacity of DCs in a mixed = lymphocyte=20 reaction (Figure 1b),=20 while the expression of enhanced green fluorescent protein at the = protein=20 level could be measured (Figure 1c).=20 In vitro, DCm-GL261-RNA were able to = stimulate T=20 cells to become specific effector T cells with cytotoxic activity = against=20 the GL261 target cells (Figure 2).=20 We noticed a nonspecific T cell cytotoxic activity when stimulated = with=20 mock-electroporated DCm or DCm electroporated with total RNA from = Lewis=20 lung cancer (DCm-LCC-RNA). Nevertheless, specificity of the = induced immune=20 response could be demonstrated in vivo(81).=20 The data obtained from these in vitro = stimulation=20 experiments confirmed that DCs loaded with tumor antigens in the = form of=20 RNA molecules are capable of inducing a T cell-mediated antitumor = immune=20 response. These results are consistent with published preclinical = data=20 from our group (82,=20 81) and others (10).

=20

= Figure 1. Electroporation of DCs with RNA-molecules is = feasible=20 without loss of cell function in vitro. A. Immature DCs were left untouched (no EP) = or=20 electroporated (EP) and immediately afterwards matured with=20 1 =B5g/mL (Escherichia coli)=20 lipopolysaccharide for 24 hours. Mature DCs were analyzed by = flow=20 cytometry for surface marker expression. EP significantly = lowered=20 the expression of CD80 and CD86 on DCs (overall analysis of = variance=20 P < 0.001, *P < 0.05). B. The=20 capacity to stimulate allogeneic cells was assessed in a = mixed=20 lymphocyte reaction in 96 well format with total=20 2 =D7 10⁵ splenocytes from = naive=20 BALB/C mice as responder (R) cells and DCs from C57BL/6 mice = as=20 stimulator (S) cells. Unstimulated cells and = phytohemagglutinin=20 (PHA) were used as negative and positive controls, = respectively.=20 Responder to stimulator ratios (R : S) ranged from = 5:1 to=20 50:1. After 4 days of culture, thymidine incorporation was = measured=20 by pulsing cultures for 18 hours with 1 =B5Ci=20 [³H] thymidine per well. Mean counts = per=20 minute (cpm) are depicted. C. One = million DCs=20 were electroporated with 1 =B5g mRNA encoding enhanced = green=20 fluorescent protein (eGFP) and eGFP expression (grey curve = in=20 FL1-Height histogram) was assessed by flow cytometry 24 = hours after=20 electroporation. The filled curve represents = nonelectroporated=20 DCs.

[Normal View=20 ]

=20

= Figure 2. RNA-loaded DCs = induce a T=20 cell-mediated antitumor immune response in=20 vitro. T cells pooled from spleen and lymph nodes of = a naive=20 C57BL/6 mouse were stimulated ex vivo = (two=20 stimulation rounds with DC : T cell ratio of 1:10 = with=20 addition of 20 U/mL of IL-2) with either mock-loaded = DCm=20 (DCm-mock), DCm electroporated with total RNA extracted from = Lewis=20 lung carcinoma cells (DCm-LLC-RNA) or DCm electroporated = with total=20 RNA extracted from GL261 glioma cells (DCm-GL261-RNA). = Unstimulated=20 T cells were used as the control. Stimulated T cells were = then used=20 as effector cells and coincubated with GL261 tumor cells in = an=20 effector to target ratio of 10:1. Tumor cell viability was = assessed=20 by measuring the metabolization of=20 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide = to=20 formazan (measured at 570 nm) by living cells after 2 = days of=20 co-culture. Pooled results (n =3D 3) are depicted = as optical=20 density values at 570 nm = (OD₅₇₀) and=20 compared to GL261 target cells without effector cells. = Overall=20 analysis of variance P < 0.001;=20 **P < 0.01, ***P < 0.001. =

[Normal=20 View ]

Since in vitro systems = merely partially=20 mimic the complex tumor microenvironment and cell interactions = in vivo, we proceeded to validate our findings in = the in vivo experimental mouse glioma model (82,=20 81). In this model, we hope to unravel new mechanisms = governing glioma=20 immunology that can eventually be exploited in translational = oncology=20 practice (Figure 3a).=20 We opted for prophylactic immunization because others reported the = GL261=20 model to be highly aggressive, necessitating additional = intervention for=20 curative settings (40,=20 78). Moreover, this model reflects the clinical therapeutic = setting,=20 at least locally in the brain, in which DC vaccination is given at = a stage=20 of minimal residual disease after (sub)total resection and not at = the time=20 of bulky disease. We adapted the model in order to monitor tumor = growth=20 consecutively with bioluminescence imaging (BLI) (82).=20 We have shown that there is a strong correlation of the BLI signal = with=20 ex vivo measured tumor volumes. Moreover,=20 implementing optical imaging in our experimental glioma model = resulted in=20 a more detailed characterization of GL261 glioma behavior in=20 vivo, demonstrating midline crossing of tumor cells, along = with the=20 observation of an initial tumor cell adaptation phase before = consistent=20 tumor progression.

=20

= Figure 3. Immunotherapy in the GL261 experimental orthotopic = glioma=20 mouse model. A. Overview of = the=20 prophylactic DC vaccination model. Adult C57BL/6 mice are=20 orthotopically challenged with=20 5 =D7 10⁵ syngeneic GL261 = malignant=20 glioma cells injected 1.5 mm posterior and 1 mm = lateral=20 from the bregma at a depth of 3 mm under stereotactic = guidance.=20 Read-out consists of monitoring general health parameters = (weight=20 and neurological symptoms), overall survival analysis, in vivo bioluminescence (BLI) requiring = GL261 cells=20 expressing luciferase in white mutant C57BL/6.Cg.Tyrc-2J/J = mice, and=20 detailed immune monitoring both systemically (in blood, = spleen and=20 tumor-draining cervical lymph nodes) and locally within the = brain.=20 Immunotherapy in our preventive treatment setting consists = of either=20 active immunization, decreasing regulatory T cell = (Treg)-mediated=20 immune suppression, or combined treatment. Active = immunization is=20 performed through intraperitoneal (IP) vaccination on day 14 = and day=20 7 before tumor challenge with one million mature bone-marrow = derived=20 CD11c+ DCs that were loaded ex vivo = with tumor=20 antigens (in the form of total RNA). In some experiments, = animals=20 were preconditioned with a single intraperitoneal injection=20 (250 =B5g) of the depleting anti-CD25 monoclonal = antibody (clone=20 PC61). This in vivo depletion of=20 CD25-expressing cells was performed 1 week before the first=20 vaccination with DC (or 3 weeks before tumor challenge if no = DC=20 vaccination was administered). B. = Pooled=20 survival data represented as Kaplan=96Meier graph. Overall = log rank=20 P < 0.001. Survival = curves of=20 animals vaccinated with DCm-GL261-RNA (green curve,=20 n =3D 27), DCm-GL261-L (DC loaded with GL261 = lysate) (red=20 curve, n =3D 19), DCm-splenocyte-RNA (blue curve,=20 n =3D 5) and untreated animals (black curve,=20 n =3D 13) are depicted.

[Normal=20 View ]

Vaccination with DCm-GL261-RNA prolonged survival = and could=20 cure nearly half of the treated animals subjected to subsequent = tumor=20 challenge (81).=20 As shown in Figure 3b,=20 we could not detect a difference in outcome in this model when the = mice=20 were treated with DCm-GL261-RNA vs. DCm-GL261-L (median survival = of 36 and=20 34 days, respectively, compared to 21 days for untreated mice). = Mapping of=20 weight loss and tumor-induced neurologic deficit clearly = paralleled the=20 survival data. Finally, histological analysis revealed that = DCm-GL261-RNA=20 vaccination resulted in infiltration of lymphocytes and = nonlymphoid cells,=20 such as macrophages, particularly at the interface of the tumor = mass with=20 the normal brain parenchyma (81).=20 These findings are in agreement with the data previously reported = by Insug=20 et al(58).

=20

=20

Which cells are players in = anti-GL261 mouse=20 glioma immunotherapy?

Why vaccination only results in tumor protection = for half of=20 the treated mice is still not completely understood. Longitudinal = imaging=20 of tumor-bearing mice with BLI revealed that within the animals = that=20 eventually succumbed, distinct patterns of nonresponding and = partially=20 responding mice can be discriminated (82).=20 We hypothesize that there is a very delicate balance between = antitumor=20 immune responses on the one hand (either endogenous or = induced/boosted by=20 the DC vaccine) and natural and tumor-induced immune suppression = on the=20 other. In responding mice, it is apparent that treatment with DCs = shifts=20 this balance away from tolerance and toward immunity, resulting in = protection against tumor growth. In contrast, in mice refractory = to DC=20 vaccination, DCs might actually induce or expand = immune-suppressive Tregs=20 and/or immune-suppressive and tumor-promoting myeloid-derived = suppressor=20 cells (MDSCs) (103).

Our data provided a rationale to investigate = in-depth the=20 mode of action of DC treatment and to implement strategies that = are aimed=20 at lowering the detrimental immune suppression within the tumor=20 microenvironment. In order to study the individual role of = separate cell=20 populations, we used depleting mAb to selectively remove the = targeted cell=20 population (Figure 3a).=20 When CD8+ T cells were removed, tumor-challenged mice had worse = outcomes=20 as compared with the control-challenged mice, suggesting that the=20 injection of GL261 cells as such induces a minor endogenous CD8+ T = cell=20 response in the immunocompetent hosts. When CD8-depleted mice were = subsequently treated with DCm-GL261-RNA, the survival was still = prolonged=20 as compared with CD8+-depleted or control-untreated animals, but = none of=20 the mice ultimately survived. These data suggest that besides a = major role=20 of CD8+ T cells in inducing the antitumoral immune effect leading = to=20 long-term survival, other immune cells stimulated via DC = vaccination play=20 a role as well. When Tregs were depleted via anti-CD25 mAb, all = mice=20 survived the subsequent tumor challenge, without or with DC = treatment.=20 Although the injection of anti-CD25 mAb only transiently depleted=20 circulating CD4+CD25+FoxP3+ Tregs, we found an increased amount = and=20 specific antitumoral activity of brain-infiltrating lymphocytes in = these=20 Treg-depleted animals, even stronger than in the animals that were = treated=20 with DCm-GL261-RNA. Whereas an infiltration of MDSCs was found in = the=20 control tumor-challenged mice, similar to published data from = others (49,=20 125), we found an increased infiltration of macrophages with = M1=20 phenotype in the Treg-depleted animals, releasing or unmasking an=20 endogenous tumor rejecting immunosurveillance. Finally, upon = rechallenge=20 of long-term surviving mice after Treg depletion and/or DC = vaccination,=20 only those mice that were treated with DC vaccination depicted a = prolonged=20 antitumor protective immune response. Upon rechallenge, the = CD25-depleted=20 surviving animals that were not vaccinated showed mortality rates = similar=20 to untreated mice. All together, our data suggest that there is a = complex=20 interaction between different cell populations: tumor cells, = effector T=20 cells, memory T cells, Tregs and macrophages with different = functional=20 phenotypes. Most likely all these cells interact with each other, = and the=20 net result of complex interactions determines the outcome of the = mice upon=20 vaccination.

=20

=20

Human DCm-HGG-L

In preparation for potential clinical use, we = studied the=20 loading of human DCs with tumor lysates in = vitro, as=20 well as the potency of DCm-HGG-L (26,=20 29). DCs were differentiated out of monocytes, obtained after = two=20 adherence cycles of peripheral blood mononuclear cells. = Differentiation=20 was induced during 6 days with medium change and addition of IL-4 = and=20 Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) at day = 1, with=20 30% medium and 100% cytokine refreshments at days 3 and 5. Loading = with=20 lysate was performed at day 6, after which, maturation stimuli = were added=20 to the cultures for one day: IL-1β, tumor necrosis factor = (TNF)-α and=20 prostaglandin E2 (PGE2). The phenotypic and functional = characteristics of=20 these so-called early mature DCs have been described (26,=20 29). After two cycles of stimulation, the first stimulation in = the=20 presence of IL-6 and IL-12 and the second in the presence of IL-4 = and=20 IL-7, DCm-HGG-L were able to induce T cell proliferation. Upon=20 stimulation, a shift towards Th1 cytokine production was observed. = Finally=20 and most importantly, DCm-HGG-L induced tumoricidal and = tumor-suppressive=20 activity by the T cells, which was antigen specific, MHC specific = and MHC=20 restricted, pointing to CD8+ cytotoxic T cell activity. These = cells were=20 prepared as cellular product to be injected intradermally in the = shoulder=20 region of patients.

It is important to note here that multiple steps = in the=20 production process, as well as the administration of the DC = vaccine, are=20 amenable to significant improvement. In fact, no comparative = clinical data=20 are currently available pertaining to the optimal cellular source = of DCs,=20 cytokine cocktail to induce DC differentiation, source of tumor = material,=20 maturation cocktail, amount of cells to be injected, volume in = which the=20 cells should be suspended, route of injection, site of injection, = schedule=20 for vaccination, method for boosting the immune response, and = combinations=20 with other antitumoral and immunomodulatory treatment strategies. = Still,=20 progress has been made over the last few years, and currently, = most groups=20 use DCs that were matured ex vivo, = injecting these DC=20 vaccines intradermally or directly into lymph = nodes.

=20

=20

DESIGN OF THERAPEUTIC STUDIES

=20

=20

Selection of patients

Upon literature search, three case reports and = thirteen=20 phase I, phase I/II or phase II trials have been published on DC=20 vaccination of glioma patients since 2000 (Table 1).=20 The median patient number in these reports was only 12, ranging = from 1 to=20 56. The inclusion criteria, immunotherapeutic designs and = interpretations=20 varied significantly among and even within the reports, being = based=20 entirely on single-center approaches and hypotheses. Hence, no = firm=20 conclusions can be derived regarding the optimal immunotherapeutic = strategy, nor would it make sense to perform a meta-analysis on = these=20 data. Already in our first reports (25,=20 111), we stressed the importance of a minimal residual disease = setting=20 for adjuvant postoperative DC vaccination in HGG patients. In the = largest=20 series published to date (30),=20 we confirmed the importance of a gross total resection prior to DC = vaccination for GBM patients in a multivariate analysis. General = awareness=20 and agreement on this important issue is mounting and even = culminating in=20 the assumption that therapeutic tumor vaccinations should be = directed to=20 early disease states. Most of the reports describe patients = treated with=20 immunotherapy at a stage of minimal residual disease, after = gross-total or=20 near-total resection. Interestingly, patients have been under = maintenance=20 corticosteroid treatment just prior and/or during immunotherapy in = some=20 trials (142,=20 146, 143), which one would expect to have a negative effect on = the=20 generation of an effective immune response.

=20

Table 1. Overview of=20 reported clinical trials. Abbreviations: = CTL =3D cytotoxic T=20 lymphocyte; DC =3D dendritic cell; = DTH =3D delayed-type=20 hypersensitivity; GBM =3D glioblastoma multiforme;=20 IFNγ =3D interferon-γ; = IL-4 =3D interleukin-4;=20 JAM =3D just another method; MHC =3D major=20 histocompatibility complex; mo =3D month; = NK =3D natural=20 killer; no. =3D number; OS =3D overall = survival;=20 pat =3D patient; PBMC =3D peripheral blood = mononuclear=20 cell; PFS =3D progression-free survival;=20 qPCR =3D quantitative polymerase chain reaction;=20 rhIL-12 =3D recombinant human interleukin-12;=20 RTE =3D naive recent thymic emigrant T cell; = TTP =3D time=20 to progression; TTS =3D time to survival;=20 w =3D week.

Author	Year	No. of patients (type = of=20 trial)	Antigen source	Administration	Treatment schedule	Immune response	Clinical = response

Liau et al(73)	2000	1 (case report)	Allogeneic MHC class = I-matched GBM=20 peptides	Intradermal	3 vaccinations	T-cell proliferation	None
Yu et al(147)	2001	9 (phase I)	Tumor-specific MHC-I = associated=20 peptides	Subcutaneous	3 vaccinations at 2-week=20 intervals	Systemic CTL cytotoxicity = against=20 tumor (n =3D 4) (JAM assay)	Prolonged survival = compared to=20 control group
Kikuchi et al(64)	2001	8 (phase I)	DC fusion with autologous = glioma=20 cells	Intradermal	3=967 vaccinations at = 3-week=20 intervals	Increase in NK cells in = PMBCs=20 (n =3D 4); increased IFNγ in = supernatant=20 (n =3D 6)	Mixed response = (n =3D 1);=20 steroids during vaccination (n =3D 5); 2 = minor=20 responses
Yamanaka et al(142)	2003	10 (phase I-II)	Autologous tumor = lysate	Intradermal and/or = intratumor=20 (Ommaya)	1=9610 vaccinations at = 3-week=20 intervals	Increase in NK cells in = PBMCs=20 (n =3D 5); positive DTH reaction to tumor = lysate=20 (n =3D 3); increased T-cell mediated = antitumor=20 activity (n =3D 2); Elispot IFNγ	Minor response=20 (n =3D 2)
Wheeler et al(135)	2003	17 (phase I)	Autologous = tumor lysate	Not = reported	3 vaccinations at 2- week=20 intervals	Not reported = (study on=20 CD8+ RTE)	Not = reported

17 (phase II)	3 vaccinations at 2-week = intervals;=20 fourth vaccination 6 weeks after third=20 (n =3D 10)
Yu et al(146)	2004	14 (phase I-II)	Autologous tumor = lysate	Subcutaneous	3 vaccinations at 2-week=20 intervals	IFNγ release in PBMCs = (n =3D 6); expansion of CD8+ antigen = specific T cell=20 clones (n =3D 4); systemic T cell = cytotoxicity against=20 tumor (n =3D 1)	Significant increase in = median=20 survival
De Vleeschouwer et al(25)	2004	1 (case report)	Autologous tumor = lysate	Intradermal	6 vaccinations; w1, w3 and = further=20 every 4 weeks	Positive DTH reaction to = tumor=20 lysate	Long-lasting tumor-free = survival=20 (>7 years after vaccination)
Rutkowski et al(111)	2004	12 (phase I)	Autologous tumor = lysate	Intradermal	2=967 vaccinations; w1, w3 = and further=20 every 4 weeks	Positive DTH reaction to = tumor=20 lysate (n =3D 6)	Partial response = (n =3D 1);=20 tumor-free survival (n =3D 2; 5 years after=20 vaccination)
Kikuchi et al(65)	2004	15 (phase I-II)	DC fusion with autologous = glioma=20 cells	Intradermal	Vaccination + rhIL-12	Positive DTH reaction to = tumor=20 lysate (n =3D 15); increased cytotoxic = activity=20 (n =3D 2); increased intracellular IFNγ = in CD8+ T=20 cells (n =3D 1)	Partial response = (n =3D 4);=20 mixed response (n =3D 1)
Liau et al(74)	2005	12 (multicohort = dose-escalation=20 study)	Acid-eluted tumor = associated=20 peptides (autologous)	Intradermal	≤3 vaccinations	CTL response = (n =3D 6)	Median TTP = 19.9 mos=97OS 18 to=20 >58 mos=97median OS 35.8 mos
Yamanaka et al(143)	2005	24 (phase I-II)	Autologous tumor = lysate	Intradermal or=20 intradermal + intratumor (Ommaya)	1=9610 vaccinations at = 3-week=20 intervals	Positive DTH reaction to = tumor=20 lysate (n =3D 8); positive IFNγ Elispot = (n =3D 7)	Partial response = (n =3D 1);=20 minor response (n =3D 3); significantly = increased=20 median survival
Okada et al(92)	2007	2 (A) / 5 (B)	IL-4 transfected=20 fibroblasts + apoptotic glioma cells (A) /=20 autologous tumor lysate (B)	Intradermal	2 vaccinations with 2-week = interval	A: increased T cell = reactivity; B:=20 no response	A: clinical and = radiological=20 response in both patients; B: no response
Wheeler et al(134)	2008	34 (phase II)	Autologous tumor = lysate	Subcutaneous	3 vaccinations at 2-week = intervals;=20 fourth vaccination 6 weeks after third	Post-vaccine = antigen-directed IFNγ=20 response in PBMCs (qPCR-based assay) = (n =3D 17);=20 DTH-test resulted in cutaneous GBM in 1 pat (DTH was=20 subsequently discontinued)	Significant positive = correlation=20 between post-vaccine response magnitude on one hand = and TTS=20 and TTP spanning chemotherapy on the other = hand
Walker et al(130)	2008	13 (phase I)	Single-cell suspension of = autologous=20 tumor cells	Intradermal	Priming phase consisting = of 6=20 vaccinations at 2-week intervals; further vaccinations = at=20 6-week intervals	Increased T cell = infiltration in=20 post-vaccination tumor specimens compared to = pre-vaccination=20 specimens (n =3D 3)	9-month survival (9/13); = 12-month=20