From: Subject: Can neural stem cells be used to track down and destroy migratory brain tumor cells while also providing a means of repairing tumor-associated damage? -- Noble 97 (23): 12393 -- Proceedings of the National Academy of Sciences Date: Mon, 14 Jan 2008 09:43:08 +0100 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01C85691.DECA84C0" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3198 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01C85691.DECA84C0 Content-Type: text/html; charset="Windows-1252" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/cgi/content/full/97/23/12393 Can neural stem cells be used to track down and = destroy migratory brain tumor cells while also providing a means of = repairing tumor-associated damage? -- Noble 97 (23): 12393 -- = Proceedings of the National Academy of Sciences
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PNAS | November 7, 2000 | vol. 97 | no. 23 |=20 12393-12395

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Commentary
Can neural stem = cells be=20 used to track down and destroy migratory brain tumor cells while also = providing=20 a means of repairing tumor-associated damage?

Mark = Noble*=20

Center for Cancer Biology, University of Rochester Medical Center,=20 601 Elmwood Avenue, Box 633, Rochester, NY 14642=20


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Successful eradication of central nervous system (CNS) tumors is an=20 exceptionally complex problem, in part because of the = devastating=20 effects that injury to the brain or spinal cord can have on = normal=20 function. Such injury is associated with brain tumors for = multiple=20 reasons. In part, it is well established that both = reperfusion and=20 compressive forces, which are unavoidable consequences of = tumor=20 growth, are injurious to the CNS. It is likely that other = mechanisms=20 also contribute to tumor-related damage, such as abnormal ion = fluxes=20 and triggering of glutamate release from damaged cells, = possibly even=20 from the tumor cells themselves. Moreover, it is clear that=20 therapeutic regimes used to treat CNS tumors may themselves = cause=20 injury. Such a conclusion is based on the increasing = recognition that=20 treatment with chemotherapeutic agents and/or radiation often = is=20 associated with significant cognitive impairment (1-3). = The=20 physiological basis for this cognitive impairment is not = wholly=20 known, although a number of studies have demonstrated that = radiation=20 kills oligodendrocytes, stem cells of the subventricular = zone, and=20 precursor cells of the dentate gyrus of the hippocampus in = rodent=20 models (1, 4-7). =

Taking the above considerations as a starting point, the obvious = conclusion=20 emerges that a useful advance in brain tumor therapy would be = to=20 develop therapeutic approaches that would both kill tumor = cells and=20 repair injury to the damaged CNS. Repairing CNS damage = requires the=20 recruitment of endogenous stem cells (or lineage-restricted = precursor=20 cells) or the transplantation of cells with the capacity to = carry out=20 repair, thus making of particular interest attempts to use = neural=20 stem cells as therapeutic delivery vehicles.

Recently, Benedetti et al. (8) = reported that=20 neural stem cells genetically modified to produce IL-4 could promote = tumor=20 regression and prolonged survival in mice that have been = injected=20 intracranially with the GL261 mouse glioma cell line. These=20 intriguing results did, however, leave unanswered a number of = questions of relevance to construction of clinical trials (9). For = example, it is necessary to determine whether any particular = CNS stem=20 cell or lineage-restricted precursor cell offers advantages = as a=20 therapeutic delivery vehicle and whether the therapeutic = agent that=20 these cells are modified to produce will damage normal CNS = tissue.=20 In addition, it is problematic that many of the models used = to=20 study gliomas in mice and rats do not reproduce all of the=20 important characteristics of malignant gliomas in the human,=20 including variability of phenotypes within individual tumors = and the=20 expression of radioresistance and chemoresistance. In = particular, as=20 the GL261 glioma cell line does not exhibit the migratory=20 characteristics in vivo that are such an important = feature of=20 human CNS neural tumors, the studies of Benedetti et = al. (8) did = not shed=20 light on the value of neural stem cells as delivery vehicles = when=20 confronted with a tumor in which cells have disseminated = large=20 distances from the original tumor mass.

This issue of PNAS provides an important new contribution from Aboody = and=20 colleagues (10) = that=20 suggests that neural stem cells might prove an effective = therapeutic=20 vehicle even when a migratory tumor cell population is the = target for=20 treatment. In these studies, transplanted neural stem cells = were=20 shown to have the ability to migrate toward an intracranial = tumor=20 cell mass. This ability of stem cells to migrate toward a = tumor mass=20 was seen when stem cells were injected at intracranial sites=20 distant from the tumor, and even after somatic injection into = the=20 tail vein. Moreover, some neural stem cells migrated so as to = be=20 juxtaposed with tumor cells that had themselves become = distributed=20 away from the primary tumor mass. Neural stem cells = engineered to=20 produce cytosine deaminase, which converts 5-fluorocytosine = to the=20 oncolytic drug 5-fluorouracil, were able to kill tumor cells = in=20 vitro and cause objective reductions in tumor mass in=20 vivo. Thus, it appears that the extensive migratory = capacity of=20 neural stem cells is retained in the tumor environment and = may be=20 advantageously applied both to delivering these novel drug = delivery=20 vehicles to the primary tumor and at distances removed from = the=20 central tumor mass.

The possibility that transplanted neural stem cells also might repair = damage=20 associated with the occurrence and/or treatment of a brain = tumor can=20 only be assessed at the moment in the context of other = efforts to use=20 stem cell transplantation in the repair of CNS injury. Of = particular=20 interest are recent studies on the ability of hypoxic injury = to alter=20 the behavior of endogenous stem cells of the CNS. In these = studies,=20 it was found that experimental induction of ischemic lesions = was=20 associated with increases in the division of stem cells of = the=20 subventricular zone and a preferential migration of newly = generated=20 cells into the region of injury. Moreover, this recruitment = of=20 endogenous cells with the capacity for division is associated = with=20 the generation of new neurons and oligodendrocytes that = become=20 incorporated into the CNS parenchyma in an apparently normal = manner=20 (Evan Snyder, personal communication). Whether similar = behavior will=20 be seen with neural stem cells transplanted into = tumor-bearing brain=20 is not yet known, but numerous studies showing that = transplanted CNS=20 stem cells and lineage-restricted precursor cells will = readily=20 generate new neurons, oligodendrocytes, and astrocytes in = vivo=20 gives one reason to be hopeful in this regard.

The studies of Aboody and colleagues bring us closer to the point = where=20 clinical trials will be initiated on the use of neural stem = cells to=20 treat CNS tumors, thus making it essential to consider what=20 additional preclinical evidence is required to reach this = point and=20 how such clinical trials themselves might be structured. A = number of=20 relevant questions have been raised previously, including the = need to=20 examine tumor cell killing in animal models that mimic the = human=20 condition more closely than is generally the case (9).= In=20 addition, a particularly critical issue derives from the fact = that=20 this is a treatment that might confer two wholly different = kinds of=20 benefits, one on survival and one on repair of CNS damage.=20 Structuring clinical trials to gain useful insights into = these=20 distinct possibilities is particularly challenging, = particularly=20 if3D---as has generally been the case3D---obvious therapeutic success is not achieved in the = first attempts.

Among the questions that need to be assessed in preclinical studies, = some of=20 the most important are those related to determining in more = detail=20 whether stem cells can really preferentially migrate toward = tumor=20 cells present in small numbers, and perhaps even distributed = as=20 individual cells. The information provided in the studies of = Aboody=20 et al. (10) is = insufficient to distinguish unambiguously between the = hypothesis that=20 stem cells are able to "track down" individual tumor cells = and the=20 possibility that the instances in which these cells are = colocalized=20 instead represent utilization of identical migratory = substrates by=20 tumor cells and stem cells. Both of these influences may be = relevant,=20 as stem cells might be expected to migrate toward a source of = growth=20 factor production (as occurs in a tumor mass), but it also is = possible that tumor cells migrate along identical substrate = pathways=20 to those used by normal migratory CNS cells. If migration = along=20 identical substrate pathways is the predominant reason = transplanted=20 stem cells and tumor cells are juxtaposed, this would = introduce a=20 strong element of chance in the occurrence of this event. If = so,=20 the great majority of disseminated tumor cells might be = expected=20 to escape killing. As tumor cells that have migrated away = from=20 the central tumor mass play a major role in brain tumor=20 recurrences, it is essential to determine with greater = accuracy the=20 extent to which neural stem cells might be guided to small = numbers=20 of tumor cells. Precise quantitative experiments in which = small=20 tumors are established and then labeled neural stem cells are = transplanted into and/or around the tumor bed, followed by=20 determination of the frequency with which migratory tumor = cells and=20 migratory neural stem cells are in contact with each other = are=20 required to address these questions.

Closely correlated with the question of whether transplanted stem = cells can=20 successfully home to the location of dispersed tumor cells = are the=20 issues of division and differentiation of the stem cells, and = the=20 number of tumor cells that can be killed by a single stem = cell. These=20 are finely graded balances that are closely interrelated. If = the stem=20 cells continue to divide after transplantation, then it is = possible=20 that they will themselves create an inappropriate cell mass. = If, in=20 contrast, they do not divide (as appears to be the case in = the=20 present studies), then as tumor cells continue their own = division=20 they eventually will become too numerous for the = therapeutically=20 modified stem cells to kill directly. Thus, if the mode of = tumor cell=20 killing requires close proximity to the transplanted stem = cells, as=20 might be expected in the studies of Aboody et al., = then the=20 action of the stem cells would be expected to only be = temporarily=20 effective. Moreover, if the transplanted stem cells = differentiate=20 into neurons or oligodendrocytes, then their migratory = capacity will=20 be compromised. It is possible, for these reasons, that = killing of=20 tumor cells and repair of CNS damage might require = transplantation of=20 two different stem cell populations, only one of which has = been=20 modified to kill the tumor cells. Still further = considerations of=20 importance are whether the therapeutic agent produced by the=20 transplanted stem cells causes injury to normal brain cells, = how to=20 engineer the stem cells to cease producing the therapeutic = protein=20 when it is no longer necessary to do so, and whether the use = of=20 nonautologous stem cells eventually will trigger an immune = reaction=20 against the cells they produce (as discussed previously, ref. = 9). =

For the patient with a malignant glioma, concern over a number of the = above=20 questions is an unrealistic luxury. In these very needy = patients, it=20 is likely that trials will go ahead before all relevant = information=20 is obtained in experimental animals, and it is critical that = such=20 clinical trials are designed so that knowledge is gained even = in=20 failure. Thus, if one does not see prolonged regression of = tumor=20 mass, or enhancement of neurological function, in clinical = trials,=20 how will it be possible to determine the reasons for this = apparent=20 failure? How can one identify variables that might be = manipulated to=20 obtain more successful outcomes? Such questions can only be = answered=20 by transplanting cells that are permanently labeled and = making=20 certain that a thorough analysis will be conducted on the = brains of=20 patients who die at various time points after receiving stem = cell=20 transplants. Such a label could be intrinsic to the cell, as = would be=20 the case if neural stem cells from a male were transplanted = into the=20 CNS of a female patient (thus enabling recognition of = transplanted=20 cells with Y chromosome-specific probes), or could be = expressed=20 because of genetic modification of the transplanted cells. It = also=20 will be important to make certain that patients who seem = likely to=20 die receive injections of BrdUrd, so as to determine whether = the=20 transplanted stem cells continue to partake in DNA synthesis. = BrdUrd=20 administration to glioma patients has been used to label = tumor cells=20 before biopsy or surgery and also has been used as a = radiosensitizing=20 agent (as described, for example, in refs. 11-14).= BrdUrd=20 also is taken up by normal human brainstem cells or precursor = cells=20 that are engaged in DNA synthesis in situ in the CNS = of=20 patients with brain tumors (15). = Thus, it is=20 well established that BrdUrd can be used to label dividing = stem cells=20 in the human CNS. In case division of transplanted stem cells = continues for long periods after transplantation into the = human CNS,=20 the BrdUrd injection will need to be as close in time to the = point of=20 death as is possible in some patients to prevent dilution of = label to=20 undetectable levels. To allow for the possibility that cell = cycle=20 times are long, it would be advantageous if BrdUrd delivery = could be=20 continuous over a period of 8-24 h. Appropriate informed = consent=20 would need to be obtained for all such experiments, but their = importance warrants examination of this = possibility.

By combining the use of labeled stem cells with BrdUrd = administration, it=20 will be possible to obtain a great deal of information that = will not=20 be observable through standard neurological and radiological=20 examination. The ability to unambiguously recognize = transplanted=20 cells will allow determination of whether they form aberrant = growths,=20 whether they become incorporated into the subventricular zone = or=20 other germinal zones, whether a population of dividing cells = is=20 retained for long periods, and whether the transplanted cells = generate new neurons, oligodendrocytes, and astrocytes. It = also would=20 be advantageous to determine whether the tumors of patients = treated=20 with this approach have any molecular characteristics (e.g.,=20 expression of a truncated or amplified receptor for epidermal = growth=20 factor) that would allow tumor cells to be identified with=20 immunohistochemical markers. Although it will not be possible = in the human to quantify the apposition between stem cells = and=20 tumor cells with the accuracy that is possible in preclinical = models, it is nonetheless important to use any means = available=20 to at least estimate the extent to which this hoped-for = association=20 actually occurs.

As discussed, the problems that remain to be resolved in obtaining = benefit=20 from this novel therapeutic approach are formidable ones.=20 Nonetheless, the consistent failure, year after year, to=20 significantly increase the survival of patients with = malignant=20 brain tumors makes it clear that obtaining dramatic = improvements=20 in outcome in this devastating disease requires the = development=20 of fundamentally new treatment strategies. A number of such=20 treatments have been proposed, including vaccination, gene = therapy,=20 pharmacological blockade of specific receptors involved in = glioma=20 growth, and the application of angiogenesis inhibitors. All = of these=20 various treatments have been applied successfully in rodent = models,=20 but it is not yet clear whether any will prove of clinical=20 value. As, thus far, none of the treatments of brain tumors = that=20 have been successful in preclinical models have worked in = human=20 patients, the need for continued research in this arena = remains as=20 great as it has ever been.


    Footnotes

See companion article on page 12846.=20

* E-mail: mark_noble{at}urmc.rochester.edu .=20


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=20 CiteULike   =20 Complore   =20 Connotea   =20 Del.icio.us   <= IMG=20 alt=3D"Add to Digg" = src=3D"http://www.pnas.org/icons/shared/socbookmark/digg.gif"=20 border=3D0> Digg    What's this? =

Companion=20 article to this Commentary:=20

From the Cover: Neural stem cells display extensive = tropism for=20 pathology in adult brain: Evidence from intracranial gliomas=20
Karen S. Aboody, Alice Brown, Nikolai G. Rainov, Kate A. Bower, = Shaoxiong=20 Liu, Wendy Yang, Juan E. Small, Ulrich Herrlinger, Vaclav Ourednik, = Peter McL.=20 Black, Xandra O. Breakefield, and Evan Y. Snyder
PNAS 2000 97: = 12846-12851.=20 [Abstract]<= /A> [Full = Text]=20  =20



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left } DIV.ac_region #hw_ac_activation_form .hw_ac_important { COLOR: #a70716 } DIV.ac_region #hw_ac_activation_form TABLE { =09 } DIV.ac_region #hw_ac_activation_form FIELDSET { PADDING-RIGHT: 0px; PADDING-LEFT: 0px; PADDING-BOTTOM: 0px; MARGIN: = 0px; BORDER-TOP-STYLE: none; PADDING-TOP: 0px; BORDER-RIGHT-STYLE: none; = BORDER-LEFT-STYLE: none; BORDER-BOTTOM-STYLE: none } DIV.ac_region #hw_ac_activation_form FIELDSET LEGEND { DISPLAY: none } ------=_NextPart_000_0000_01C85691.DECA84C0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/javascript/ajax/xmlhttprequest.js /*=0A= =0A= Cross-Browser XMLHttpRequest v1.2=0A= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= Emulate Gecko 'XMLHttpRequest()' functionality in IE and Opera. Opera = requires=0A= the Sun Java Runtime Environment .=0A= =0A= by Andrew Gregory=0A= http://www.scss.com.au/family/andrew/webdesign/xmlhttprequest/=0A= =0A= This work is licensed under the Creative Commons Attribution License. To = view a=0A= copy of this license, visit = http://creativecommons.org/licenses/by-sa/2.5/ or=0A= send a letter to Creative Commons, 559 Nathan Abbott Way, Stanford, = California=0A= 94305, USA.=0A= =0A= Attribution: Leave my name and web address in this script intact.=0A= =0A= Not Supported in Opera=0A= ----------------------=0A= * user/password authentication=0A= * responseXML data member=0A= =0A= Not Fully Supported in Opera=0A= ----------------------------=0A= * async requests=0A= * abort()=0A= * getAllResponseHeaders(), getAllResponseHeader(header)=0A= =0A= */=0A= // IE support=0A= if (window.ActiveXObject && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= var msxmls =3D new Array(=0A= 'Msxml2.XMLHTTP.5.0',=0A= 'Msxml2.XMLHTTP.4.0',=0A= 'Msxml2.XMLHTTP.3.0',=0A= 'Msxml2.XMLHTTP',=0A= 'Microsoft.XMLHTTP');=0A= for (var i =3D 0; i < msxmls.length; i++) {=0A= try {=0A= return new ActiveXObject(msxmls[i]);=0A= } catch (e) {=0A= }=0A= }=0A= return null;=0A= };=0A= }=0A= // Gecko support=0A= /* ;-) */=0A= // Opera support=0A= if (window.opera && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= this.readyState =3D 0; // = 0=3Duninitialized,1=3Dloading,2=3Dloaded,3=3Dinteractive,4=3Dcomplete=0A= this.status =3D 0; // HTTP status codes=0A= this.statusText =3D '';=0A= this._headers =3D [];=0A= this._aborted =3D false;=0A= this._async =3D true;=0A= this._defaultCharset =3D 'ISO-8859-1';=0A= this._getCharset =3D function() {=0A= var charset =3D _defaultCharset;=0A= var contentType =3D = this.getResponseHeader('Content-type').toUpperCase();=0A= val =3D contentType.indexOf('CHARSET=3D');=0A= if (val !=3D -1) {=0A= charset =3D contentType.substring(val);=0A= }=0A= val =3D charset.indexOf(';');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= val =3D charset.indexOf(',');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= return charset;=0A= };=0A= this.abort =3D function() {=0A= this._aborted =3D true;=0A= };=0A= this.getAllResponseHeaders =3D function() {=0A= return this.getAllResponseHeader('*');=0A= };=0A= this.getAllResponseHeader =3D function(header) {=0A= var ret =3D '';=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= if (header =3D=3D '*' || this._headers[i].h =3D=3D header) {=0A= ret +=3D this._headers[i].h + ': ' + this._headers[i].v + '\n';=0A= }=0A= }=0A= return ret;=0A= };=0A= this.getResponseHeader =3D function(header) {=0A= var ret =3D getAllResponseHeader(header);=0A= var i =3D ret.indexOf('\n');=0A= if (i !=3D -1) {=0A= ret =3D ret.substring(0, i);=0A= }=0A= return ret;=0A= };=0A= this.setRequestHeader =3D function(header, value) {=0A= this._headers[this._headers.length] =3D {h:header, v:value};=0A= };=0A= this.open =3D function(method, url, async, user, password) {=0A= this.method =3D method;=0A= this.url =3D url;=0A= this._async =3D true;=0A= this._aborted =3D false;=0A= this._headers =3D [];=0A= if (arguments.length >=3D 3) {=0A= this._async =3D async;=0A= }=0A= if (arguments.length > 3) {=0A= opera.postError('XMLHttpRequest.open() - user/password not = supported');=0A= }=0A= this.readyState =3D 1;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= };=0A= this.send =3D function(data) {=0A= if (!navigator.javaEnabled()) {=0A= alert("XMLHttpRequest.send() - Java must be installed and = enabled.");=0A= return;=0A= }=0A= if (this._async) {=0A= setTimeout(this._sendasync, 0, this, data);=0A= // this is not really asynchronous and won't execute until the = current=0A= // execution context ends=0A= } else {=0A= this._sendsync(data);=0A= }=0A= }=0A= this._sendasync =3D function(req, data) {=0A= if (!req._aborted) {=0A= req._sendsync(data);=0A= }=0A= };=0A= this._sendsync =3D function(data) {=0A= this.readyState =3D 2;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= // open connection=0A= var url =3D new java.net.URL(new = java.net.URL(window.location.href), this.url);=0A= var conn =3D url.openConnection();=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= conn.setRequestProperty(this._headers[i].h, this._headers[i].v);=0A= }=0A= this._headers =3D [];=0A= if (this.method =3D=3D 'POST') {=0A= // POST data=0A= conn.setDoOutput(true);=0A= var wr =3D new = java.io.OutputStreamWriter(conn.getOutputStream(), this._getCharset());=0A= wr.write(data);=0A= wr.flush();=0A= wr.close();=0A= }=0A= // read response headers=0A= // NOTE: the getHeaderField() methods always return nulls for me :(=0A= var gotContentEncoding =3D false;=0A= var gotContentLength =3D false;=0A= var gotContentType =3D false;=0A= var gotDate =3D false;=0A= var gotExpiration =3D false;=0A= var gotLastModified =3D false;=0A= for (var i =3D 0; ; i++) {=0A= var hdrName =3D conn.getHeaderFieldKey(i);=0A= var hdrValue =3D conn.getHeaderField(i);=0A= if (hdrName =3D=3D null && hdrValue =3D=3D null) {=0A= break;=0A= }=0A= if (hdrName !=3D null) {=0A= this._headers[this._headers.length] =3D {h:hdrName, = v:hdrValue};=0A= switch (hdrName.toLowerCase()) {=0A= case 'content-encoding': gotContentEncoding =3D true; break;=0A= case 'content-length' : gotContentLength =3D true; break;=0A= case 'content-type' : gotContentType =3D true; break;=0A= case 'date' : gotDate =3D true; break;=0A= case 'expires' : gotExpiration =3D true; break;=0A= case 'last-modified' : gotLastModified =3D true; break;=0A= }=0A= }=0A= }=0A= // try to fill in any missing header information=0A= var val;=0A= val =3D conn.getContentEncoding();=0A= if (val !=3D null && !gotContentEncoding) = this._headers[this._headers.length] =3D {h:'Content-encoding', v:val};=0A= val =3D conn.getContentLength();=0A= if (val !=3D -1 && !gotContentLength) = this._headers[this._headers.length] =3D {h:'Content-length', v:val};=0A= val =3D conn.getContentType();=0A= if (val !=3D null && !gotContentType) = this._headers[this._headers.length] =3D {h:'Content-type', v:val};=0A= val =3D conn.getDate();=0A= if (val !=3D 0 && !gotDate) this._headers[this._headers.length] = =3D {h:'Date', v:(new Date(val)).toUTCString()};=0A= val =3D conn.getExpiration();=0A= if (val !=3D 0 && !gotExpiration) = this._headers[this._headers.length] =3D {h:'Expires', v:(new = Date(val)).toUTCString()};=0A= val =3D conn.getLastModified();=0A= if (val !=3D 0 && !gotLastModified) = this._headers[this._headers.length] =3D {h:'Last-modified', v:(new = Date(val)).toUTCString()};=0A= // read response data=0A= var reqdata =3D '';=0A= var stream =3D conn.getInputStream();=0A= if (stream) {=0A= var reader =3D new java.io.BufferedReader(new = java.io.InputStreamReader(stream, this._getCharset()));=0A= var line;=0A= while ((line =3D reader.readLine()) !=3D null) {=0A= if (this.readyState =3D=3D 2) {=0A= this.readyState =3D 3;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= }=0A= reqdata +=3D line + '\n';=0A= }=0A= reader.close();=0A= this.status =3D 200;=0A= this.statusText =3D 'OK';=0A= this.responseText =3D reqdata;=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onload) {=0A= this.onload();=0A= }=0A= } else {=0A= // error=0A= this.status =3D 404;=0A= this.statusText =3D 'Not Found';=0A= this.responseText =3D '';=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onerror) {=0A= this.onerror();=0A= }=0A= }=0A= };=0A= };=0A= }=0A= // ActiveXObject emulation=0A= if (!window.ActiveXObject && window.XMLHttpRequest) {=0A= window.ActiveXObject =3D function(type) {=0A= switch (type.toLowerCase()) {=0A= case 'microsoft.xmlhttp':=0A= case 'msxml2.xmlhttp':=0A= case 'msxml2.xmlhttp.3.0':=0A= case 'msxml2.xmlhttp.4.0':=0A= case 'msxml2.xmlhttp.5.0':=0A= return new XMLHttpRequest();=0A= }=0A= return null;=0A= };=0A= }=0A= ------=_NextPart_000_0000_01C85691.DECA84C0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/javascript/ajax/utility.js /************************************************************************= *****=0A= * javascript/ajax/utility.js=0A= *=0A= * Utility functions for working with XMLHttpRequest data.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Copy XML nodes into an HTMLElement. This effectively=0A= * clones XML markup which uses XHTML naming conventions=0A= * into an HTML DOM.=0A= */=0A= function copy_xml_to_html(src, dst) {=0A= if (src.nodeType =3D=3D 1) { /* Node.ELEMENT_NODE */=0A= var e =3D document.createElement(src.nodeName);=0A= for (var i =3D 0; i < src.childNodes.length; i++) {=0A= copy_xml_to_html(src.childNodes[i], e);=0A= }=0A= for (var i =3D 0; i < src.attributes.length; i++) {=0A= var n =3D src.attributes[i].name;=0A= var v =3D unescape_xml_string(src.attributes[i].value); =0A= e.setAttribute(n, v);=0A= if (n =3D=3D "class") {=0A= e.className =3D v;=0A= }=0A= else if (n =3D=3D "style") {=0A= set_css_style(v, e, "");=0A= }=0A= }=0A= dst.appendChild(e);=0A= }=0A= else if (src.nodeType =3D=3D 3) { /* Node.TEXT_NODE */=0A= dst.appendChild(document.createTextNode(src.nodeValue));=0A= }=0A= }=0A= =0A= /* =0A= * It is unclear that this is the right thing to be calling=0A= * from copy_xml_to_html, but it appears that Safari decides=0A= * to convert & to the NCR #, and then encodes that=0A= * NCR to &%26%2338;. So, I'm going to treat the DOM Attr=0A= * value as a plain string, and run our XML string input=0A= * through the decoding routine below.=0A= */=0A= function unescape_xml_string(s) {=0A= return s.replace(/'/g, "'")=0A= .replace(/'/g, "'")=0A= .replace(/"/g, "\"")=0A= .replace(/"/g, "\"")=0A= .replace(/>/g, ">")=0A= .replace(/>/g, ">")=0A= .replace(/</g, "<")=0A= .replace(/</g, "<")=0A= .replace(/&/g, "&")=0A= .replace(/&/g, "&");=0A= }=0A= =0A= /*=0A= * Parse set of CSS rules and apply them to an element.=0A= * This is quite horrifying, but I'm unable to determine=0A= * how else to handle this with IE 6. FireFox and other=0A= * sane browsers let you simply set the style attribute=0A= * or use e.style.setProperty(rule, value, priority),=0A= * IE 6 appears to have neither of these capabilities..=0A= */=0A= function set_css_style(css, e, priority) {=0A= var rules =3D css.split(";");=0A= for (var i =3D 0; i < rules.length; i++) {=0A= var nvpair =3D rules[i].split(":");=0A= if (nvpair.length =3D=3D 2) {=0A= try {=0A= var name =3D nvpair[0]; /* style attribute */=0A= var value =3D nvpair[1]; /* attribute value */=0A= =0A= /*=0A= * For each possible style attribute, set the=0A= * appropriate style property in the element.=0A= */=0A= if (name =3D=3D "background") {=0A= e.style.background =3D value;=0A= }=0A= else if (name =3D=3D "background-attachment") {=0A= e.style.backgroundAttachment =3D value;=0A= }=0A= else if (name =3D=3D "background-color") {=0A= e.style.backgroundColor =3D value;=0A= }=0A= else if (name =3D=3D "background-image") {=0A= e.style.backgroundImage =3D value;=0A= }=0A= else if (name =3D=3D "background-position") {=0A= e.style.backgroundPosition =3D value;=0A= }=0A= else if (name =3D=3D "background-position-x") {=0A= e.style.backgroundPositionX =3D value;=0A= }=0A= else if (name =3D=3D "background-position-y") {=0A= e.style.backgroundPositionY =3D value;=0A= }=0A= else if (name =3D=3D "background-repeat") {=0A= e.style.backgroundRepeat =3D value;=0A= }=0A= else if (name =3D=3D "behavior") {=0A= e.style.behavior =3D value;=0A= }=0A= else if (name =3D=3D "border") {=0A= e.style.border =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom") {=0A= e.style.borderBottom =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-color") {=0A= e.style.borderBottomColor =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-style") {=0A= e.style.borderBottomStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-width") {=0A= e.style.borderBottomWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-collapse") {=0A= e.style.borderCollapse =3D value;=0A= }=0A= else if (name =3D=3D "border-color") {=0A= e.style.borderColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left") {=0A= e.style.borderLeft =3D value;=0A= }=0A= else if (name =3D=3D "border-left-color") {=0A= e.style.borderLeftColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left-style") {=0A= e.style.borderLeftStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-left-width") {=0A= e.style.borderLeftWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-right") {=0A= e.style.borderRight =3D value;=0A= }=0A= else if (name =3D=3D "border-right-color") {=0A= e.style.borderRightColor =3D value;=0A= }=0A= else if (name =3D=3D "border-right-style") {=0A= e.style.borderRightStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-right-width") {=0A= e.style.borderRightWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-style") {=0A= e.style.borderStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top") {=0A= e.style.borderTop =3D value;=0A= }=0A= else if (name =3D=3D "border-top-color") {=0A= e.style.borderTopColor =3D value;=0A= }=0A= else if (name =3D=3D "border-top-style") {=0A= e.style.borderTopStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top-width") {=0A= e.style.borderTopWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-width") {=0A= e.style.borderWidth =3D value;=0A= }=0A= else if (name =3D=3D "bottom") {=0A= e.style.bottom =3D value;=0A= }=0A= else if (name =3D=3D "clear") {=0A= e.style.clear =3D value;=0A= }=0A= else if (name =3D=3D "clip") {=0A= e.style.clip =3D value;=0A= }=0A= else if (name =3D=3D "color") {=0A= e.style.color =3D value;=0A= }=0A= else if (name =3D=3D "cssText") {=0A= e.style.Sets =3D value;=0A= }=0A= else if (name =3D=3D "cursor") {=0A= e.style.cursor =3D value;=0A= }=0A= else if (name =3D=3D "direction") {=0A= e.style.direction =3D value;=0A= }=0A= else if (name =3D=3D "display") {=0A= e.style.display =3D value;=0A= }=0A= else if (name =3D=3D "font") {=0A= e.style.font =3D value;=0A= }=0A= else if (name =3D=3D "font-family") {=0A= e.style.fontFamily =3D value;=0A= }=0A= else if (name =3D=3D "font-size") {=0A= e.style.fontSize =3D value;=0A= }=0A= else if (name =3D=3D "font-style") {=0A= e.style.fontStyle =3D value;=0A= }=0A= else if (name =3D=3D "font-variant") {=0A= e.style.fontVariant =3D value;=0A= }=0A= else if (name =3D=3D "font-weight") {=0A= e.style.fontWeight =3D value;=0A= }=0A= else if (name =3D=3D "height") {=0A= e.style.height =3D value;=0A= }=0A= else if (name =3D=3D "ime-mode") {=0A= e.style.imeMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-flow") {=0A= e.style.layoutFlow =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid") {=0A= e.style.layoutGrid =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-char") {=0A= e.style.layoutGridChar =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-line") {=0A= e.style.layoutGridLine =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-mode") {=0A= e.style.layoutGridMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-type") {=0A= e.style.layoutGridType =3D value;=0A= }=0A= else if (name =3D=3D "left") {=0A= e.style.left =3D value;=0A= }=0A= else if (name =3D=3D "letter-spacing") {=0A= e.style.letterSpacing =3D value;=0A= }=0A= else if (name =3D=3D "line-break") {=0A= e.style.lineBreak =3D value;=0A= }=0A= else if (name =3D=3D "line-height") {=0A= e.style.lineHeight =3D value;=0A= }=0A= else if (name =3D=3D "list-style") {=0A= e.style.listStyle =3D value;=0A= }=0A= else if (name =3D=3D "list-style-image") {=0A= e.style.listStyleImage =3D value;=0A= }=0A= else if (name =3D=3D "list-style-position") {=0A= e.style.listStylePosition =3D value;=0A= }=0A= else if (name =3D=3D "list-style-type") {=0A= e.style.listStyleType =3D value;=0A= }=0A= else if (name =3D=3D "margin") {=0A= e.style.margin =3D value;=0A= }=0A= else if (name =3D=3D "margin-bottom") {=0A= e.style.marginBottom =3D value;=0A= }=0A= else if (name =3D=3D "margin-left") {=0A= e.style.marginLeft =3D value;=0A= }=0A= else if (name =3D=3D "margin-right") {=0A= e.style.marginRight =3D value;=0A= }=0A= else if (name =3D=3D "margin-top") {=0A= e.style.marginTop =3D value;=0A= }=0A= else if (name =3D=3D "min-height") {=0A= e.style.minHeight =3D value;=0A= }=0A= else if (name =3D=3D "overflow") {=0A= e.style.overflow =3D value;=0A= }=0A= else if (name =3D=3D "overflow-x") {=0A= e.style.overflowX =3D value;=0A= }=0A= else if (name =3D=3D "overflow-y") {=0A= e.style.overflowY =3D value;=0A= }=0A= else if (name =3D=3D "padding") {=0A= e.style.padding =3D value;=0A= }=0A= else if (name =3D=3D "padding-bottom") {=0A= e.style.paddingBottom =3D value;=0A= }=0A= else if (name =3D=3D "padding-left") {=0A= e.style.paddingLeft =3D value;=0A= }=0A= else if (name =3D=3D "padding-right") {=0A= e.style.paddingRight =3D value;=0A= }=0A= else if (name =3D=3D "padding-top") {=0A= e.style.paddingTop =3D value;=0A= }=0A= else if (name =3D=3D "page-break-after") {=0A= e.style.pageBreakAfter =3D value;=0A= }=0A= else if (name =3D=3D "page-break-before") {=0A= e.style.pageBreakBefore =3D value;=0A= }=0A= else if (name =3D=3D "pixelBottom") {=0A= e.style.pixelBottom =3D value;=0A= }=0A= else if (name =3D=3D "pixelHeight") {=0A= e.style.pixelHeight =3D value;=0A= }=0A= else if (name =3D=3D "pixelLeft") {=0A= e.style.pixelLeft =3D value;=0A= }=0A= else if (name =3D=3D "pixelRight") {=0A= e.style.pixelRight =3D value;=0A= }=0A= else if (name =3D=3D "pixelTop") {=0A= e.style.pixelTop =3D value;=0A= }=0A= else if (name =3D=3D "pixelWidth") {=0A= e.style.pixelWidth =3D value;=0A= }=0A= else if (name =3D=3D "posBottom") {=0A= e.style.posBottom =3D value;=0A= }=0A= else if (name =3D=3D "posHeight") {=0A= e.style.posHeight =3D value;=0A= }=0A= else if (name =3D=3D "position") {=0A= e.style.position =3D value;=0A= }=0A= else if (name =3D=3D "posLeft") {=0A= e.style.posLeft =3D value;=0A= }=0A= else if (name =3D=3D "posRight") {=0A= e.style.posRight =3D value;=0A= }=0A= else if (name =3D=3D "posTop") {=0A= e.style.posTop =3D value;=0A= }=0A= else if (name =3D=3D "posWidth") {=0A= e.style.posWidth =3D value;=0A= }=0A= else if (name =3D=3D "right") {=0A= e.style.right =3D value;=0A= }=0A= else if (name =3D=3D "ruby-align") {=0A= e.style.rubyAlign =3D value;=0A= }=0A= else if (name =3D=3D "ruby-overhang") {=0A= e.style.rubyOverhang =3D value;=0A= }=0A= else if (name =3D=3D "ruby-position") {=0A= e.style.rubyPosition =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-3dlight-color") {=0A= e.style.scrollbar3dLightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-arrow-color") {=0A= e.style.scrollbarArrowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-base-color") {=0A= e.style.scrollbarBaseColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-darkshadow-color") {=0A= e.style.scrollbarDarkShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-face-color") {=0A= e.style.scrollbarFaceColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-highlight-color") {=0A= e.style.scrollbarHighlightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-shadow-color") {=0A= e.style.scrollbarShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-track-color") {=0A= e.style.scrollbarTrackColor =3D value;=0A= }=0A= else if (name =3D=3D "float") {=0A= e.style.styleFloat =3D value;=0A= }=0A= else if (name =3D=3D "table-layout") {=0A= e.style.tableLayout =3D value;=0A= }=0A= else if (name =3D=3D "text-align") {=0A= e.style.textAlign =3D value;=0A= }=0A= else if (name =3D=3D "text-align-last") {=0A= e.style.textAlignLast =3D value;=0A= }=0A= else if (name =3D=3D "text-autospace") {=0A= e.style.textAutospace =3D value;=0A= }=0A= else if (name =3D=3D "text-decoration") {=0A= e.style.textDecoration =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationBlink") {=0A= e.style.textDecorationBlink =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationLineThrough") {=0A= e.style.textDecorationLineThrough =3D value;=0A= }=0A= else if (name =3D=3D 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"vertical-align") {=0A= e.style.verticalAlign =3D value;=0A= }=0A= else if (name =3D=3D "visibility") {=0A= e.style.visibility =3D value;=0A= }=0A= else if (name =3D=3D "white-space") {=0A= e.style.whiteSpace =3D value;=0A= }=0A= else if (name =3D=3D "width") {=0A= e.style.width =3D value;=0A= }=0A= else if (name =3D=3D "word-break") {=0A= e.style.wordBreak =3D value;=0A= }=0A= else if (name =3D=3D "word-spacing") {=0A= e.style.wordSpacing =3D value;=0A= }=0A= else if (name =3D=3D "word-wrap") {=0A= e.style.wordWrap =3D value;=0A= }=0A= else if (name =3D=3D "writing-mode") {=0A= e.style.writingMode =3D value;=0A= }=0A= else if (name =3D=3D "z-index") {=0A= e.style.zIndex =3D value;=0A= }=0A= else if (name =3D=3D "zoom") {=0A= e.style.zoom =3D value;=0A= }=0A= }=0A= catch (e) {=0A= /* ignore error on attempt to set e.style.[property] */=0A= }=0A= }=0A= }=0A= }=0A= ------=_NextPart_000_0000_01C85691.DECA84C0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.pnas.org/javascript/entrez/callback.js /************************************************************************= *****=0A= * javascript/entrez/callback.js=0A= *=0A= * Entrez Linking callback to populate content box.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Execute callback to fill content box with Entrez Linking information.=0A= */=0A= function entrez_callback(pmid, callback_url) {=0A= /*=0A= * MSIE 5.5 and below have issues with the JavaScript=0A= * used for Entrez Linking. 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i < src.childNodes.length; i++) {=0A= copy_xml_to_html(src.childNodes[i], dst);=0A= }=0A= var tbl =3D tr.parentNode;=0A= tbl.replaceChild(dst, tr);=0A= }=0A= }=0A= req.open('GET', callback_url, true);=0A= req.send(null);=0A= }=0A= ------=_NextPart_000_0000_01C85691.DECA84C0 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://www.google-analytics.com/urchin.js //-- Google Analytics Urchin Module=0A= //-- Copyright 2007 Google, All Rights Reserved.=0A= =0A= //-- Urchin On Demand Settings ONLY=0A= var _uacct=3D""; // set up the Urchin Account=0A= var _userv=3D1; // service mode (0=3Dlocal,1=3Dremote,2=3Dboth)=0A= =0A= //-- UTM User Settings=0A= var _ufsc=3D1; // set client info flag (1=3Don|0=3Doff)=0A= var _udn=3D"auto"; // (auto|none|domain) set the domain name for cookies=0A= var _uhash=3D"on"; // (on|off) unique domain hash for cookies=0A= var _utimeout=3D"1800"; // set the inactive session timeout in seconds=0A= var _ugifpath=3D"/__utm.gif"; 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