You can now ensure that vehicles are always state of the art.
By allowing the innovative technology to simply and reliably optimize the vehicle without requiring a visit to the repair shop.
The data transfer is shielded by a complex security architecture with end-to-end encryption.
For maximum security against attackers, a unique encryption is made for each consumer;
Found a bug? No Problem!
Developers can work remotely and roll their codes directly to the
vehicles OTA.

Go to StatisticsUnique custom updates for each and every consumer, with each ECU having its own custom security stack.
Your car will be online. Anytime, anywhere.
Updates occur while the system is running, without interrupting the user.
All downloads are optimized.
Only the new changed will be downloaded.
Cars can go back to a previous firmware version if problems found.


---
layout: post
title: ''
date: '2009-06-28T10:27:18-07:00'
tags:
tumblr_url: http://alexhwilliams.tumblr.com/post/131784686/teamseesmic-co-learners-in-the-school-of-social
---
TeamSeesmic - co-learners in the school of social media
Loic LeMeur moves Seesmic toward becoming a community about social media.


- Login to LiveJournal with the following form:
- The browser will prompt you to save a file to your local machine.
This file will be the contents of your posts/comments from
LiveJournal in a Blogger export file.
- Login to your Blogger
or create a new user.
- Once logged in, click on the Create a Blog link from the
user dashboard, and then click on the Import Blog Tool
- Follow the instructions and upload your Blogger export file when
prompted.
- After completing the import wizard, you should have a set of
imported posts from LiveJournal that you can now publish to
Blogger. Have fun!
|


Documentation
Part V - Build FlickrView for production
In the previous parts, Developing a Dojo Mobile Application, Implementing FeedView and Implementing Settings View, we created the HTML, CSS, and JavaScript code required to power the FlickrView mobile application. This tutorial will focus on updating the code for deployment, leveraging the Dojo build system to keep the application compact for production, and a basic review of the entire Dojo Mobile-powered application.
- Difficulty: Intermediate
- Dojo Version: 1.9
- Series: FlickrView
Dojo Mobile and Builds
Creating a build for Dojo Mobile applications is extremely important because we want our mobile applications to be a small as possible. Let's walk through the steps to create a compact build of our Dojo Mobile application: FlickrView.
Dojo's Build System
Typical build scripts are found within the Dojo Toolkit's util/buildscripts
directory, so, for the rest of the tutorial, you will need a local copy of a Dojo
source distribution. If you don't have that already, visit
dojotoolkit.org/download.
Make sure you get a source distribution, that includes the
util
directory. Once you have that, copy the
dojo, dijit, dojox
and util
directories
into the js
directory of your sample application. Your file structure
should now be as follows.
Build Profile
Let's create a build profile for FlickrView. The build profile is a configuration file that says what the build will include, and what options are used. The Dojo build system is fully documented on the Creating Builds tutorial, we will just explain briefly what it contains here.
The profile starts with some options:
"basePath"
defines the origin for all the relative paths in this profile;"action": "release"
is the standard action for a build"releaseDir"
specifies the directory where the build results will be output"cssOptimize"
option is useful for a mobile build since it makes sure all the CSS files that make up each theme are concatenated into one single CSS file
var profile = { "basePath": "./", "action": "release", "releaseDir": "../release/js", "selectorEngine": "acme", "stripConsole": "normal", "copyTests": false, "cssOptimize": "comments.keepLines", "mini": true, "optimize": "closure", "layerOptimize": "closure", "localeList": "en-us",
Next are the layers definition:
In this tutorial, we define only one layer that contains all the modules needed by
the application. A layer will result in one .js
file being generated,
and in our case, we define it as the dojo/dojo layer. It will include only the
Dojo code that we actually use in our application, and the custom code that is
specific to our application.
Note that we could have split our applications into multiple layers, for example one
layer for the dojo code and one layer for the application code. But in the context
of a mobile application deployed on networks with high latencies, having multiple
requests could have a significant impact on the overall application launch time. For
the same reason, we specify the builder to include the localized bundle into the
layer (by means of the includeLocales
property).
layers: { "dojo/dojo": { customBase: true, includeLocales: ["en-us"], include: [ "dojox/mobile/parser", "dijit/registry", "dojox/mobile/compat", "dojox/mobile/ScrollableView", "dojox/mobile/ListItem", "dojox/mobile/FormLayout", "dojox/mobile/TextBox", "dojox/mobile/RadioButton", "dojox/mobile/Heading", "dojox/mobile/EdgeToEdgeList", "dojox/mobile/RoundRect", "dojox/mobile/Switch", "dojo/cldr/nls/de/gregorian", "dojo/cldr/nls/fr/gregorian", "dojo/cldr/nls/it/gregorian", "dojo/cldr/nls/ko/gregorian", "dojo/cldr/nls/pt/gregorian", "dojo/cldr/nls/es/gregorian", "dojo/cldr/nls/zh/gregorian", "dojo/cldr/nls/zh-hk/gregorian", "flickrview/FeedView", "flickrview/SettingsView" ] } },
Then we define a set of has features:
Features are a way to isolate pieces of code that can be conditionally included or excluded (see dojo/has).
For example, we set the "ie"
feature to
undefined
: by this, we state that we don't want the application to run
on old Internet Explorer (only on mobile browsers), so we accept that any
IE-specific code is excluded from the build.
staticHasFeatures: { "dom-addeventlistener": true, "dom-qsa": true, "json-stringify": true, "json-parse": true, "bug-for-in-skips-shadowed": false, "dom-matches-selector": true, "native-xhr": true, "array-extensible": true, "ie": undefined, "quirks": false, "dojo-sync-loader": false, "ie-event-behavior": 0 }, packages: [ { name: "dojo", location: "dojo" }, { name: "dijit", location: "dijit" }, { name: "dojox", location: "dojox" }, { name: "flickrview", location: "flickrview" } ] };
This build profile is included as flickrview/js/flickrview-app.profile.js
in the downloadable archive available at the end of this article.
Running the Build
Let's switch to the command line and build our layers based on the build profile above:
Unix
cd js/util/buildscripts ./build.sh profile=../../flickrview-app.profile.js
Windows
cd js\util\buildscripts .\build.bat profile=..\..\flickrview-app.profile.js
The build process is a mix of JavaScript and Java (for code
minification/optimization) tasks. By default, the unix build.sh
script
uses node.js as the default runtime if available, or falls back
to Rhino otherwise (this choice is motivated by the fact that
node.js usually much more faster than Rhino).
The Windows build.bat
script, on the other hand, only leverages
Rhino. But that does not mean you can’t build with
node.js on windows! Here is the command line to do it manually:
.\node ..\..\dojo\dojo.js load=build --profile ../../flickrview-app.profile.js
After the build is completed, navigate to the release/js/flickrview/
directory to view the result.
Implementing the Build
To implement our newly created build files, update flickrview.html
First, update the path to Dojo, so that we now use the dojo.js
file
that was created by the build instead of the original dojo.js
.
This corresponds to the dojo/dojo
layer in our profile, and the
resulting file is in release/js/dojo/dojo.js
, so we update the path
of the Dojo script tag accordingly. We don't need to do other modifications since
the layer contains all the modules the application needs.
<script type="text/javascript" src="./release/js/dojo/dojo.js"></script> <script> require([...], function(...){ // existing require calls // ... existing code }); // ... </script>
Finally, we can also simplify the dojoConfig
section like this :
<script type="text/javascript"> dojoConfig = { async: true, parseOnLoad: false, mblHideAddressBar: true, extraLocale: ["en-us", "fr-fr", "de-de", "it-it", "ko-kr", "pt-br", "es-us", "zh-hk"] }; </script>
A build version of the HTML file, flickrview-build.html
, is included in
the downloadable archive. You can launch this file once you have run the build.
Review
FlickrView is complete! Our simple mobile application has been templated (HTML), styled (CSS), coded (JavaScript), and built for production!
Let's review what we learned in the process of create FlickrView:
- The basic widgets included within Dojo Mobile
- The format which to declaratively code dojox/mobile widgets with HTML and programmatically create widgets with JavaScript
- How to use dojo/request/script to retrieve JSON-formatted data from Flickr
- How to extend dojox/mobile base widgets
- Strategies behind following good JavaScript practices but also keeping code compact and minimized in dependencies
- The special build process needed for dojox/mobile applications
In this tutorial, we saw that Dojo Mobile is an outstanding mobile application framework complete with themes and widgets to match mobile device controls. Dojo Mobile is also easy to learn, extend, and dynamically populate with content. FlickrView, a basic three-view application, was very easy to create, thanks to Dojo Mobile!
Download The Source
Resources & references
- Dojo Mobile Reference Guide
- The Dojo Toolkit API
- Creating Builds (Tutorial)
- Get Dojo! (Downloads)


title=Registration date=2018-11-10 type=page status=published ~~~~~~ Registration is entirely free :)


$xpath-form.startTag()
#if (!$xpath-form.fields.xpathExpression.valid)
$xpath-form.fields.xpathExpression.error
#end
XPath expression:
${xpath-form.fields.xpathExpression}
XPath value:
${xpath-form.fields.xpathValue}
${xpath-form.fields.check}${xpath-form.fields.clear}
${xpath-form.fields.request}
$xpath-form.endTag()
#if($isXpathChecked)
Result:
$xpathQueryResult
#end


Let's make a label.
Build
OS
None
Windows 7
Windows 8
Windows 8.1
Windows 10
Linux
macOS
OS X 10.11
Warranty
Sold As Seen
3 Months
6 Months
12 Months
WiFi
No
Yes
Price
£
Print
Label
Summary
Test Results


Compares specified registry subkeys or entries.
For examples of how to use this command, see Examples.
Syntax
reg compare <KeyName1> <KeyName2> [{/v ValueName | /ve}] [{/oa | /od | /os | on}] [/s]
Parameters
Parameter Description
<KeyName1> Specifies the full path of the first subkey to be compared. To specify a remote computer, include the computer name (in the format \\ComputerName\) as part of the KeyName. Omitting \\ComputerName\ causes the operation to default to the local computer. The KeyName must include a valid root key. Valid root keys for the local computer are: HKLM, HKCU, HKCR, HKU, and HKCC. If a remote computer is specified, valid root keys are: HKLM and HKU.
<KeyName2> Specifies the full path of the second subkey to be compared. To specify a remote computer, include the computer name (in the format \\ComputerName\) as part of the KeyName. Omitting \\ComputerName\ causes the operation to default to the local computer. Specifying only the computer name in KeyName2 causes the operation to use the path to the subkey specified in KeyName1. The KeyName must include a valid root key. Valid root keys for the local computer are: HKLM, HKCU, HKCR, HKU, and HKCC. If a remote computer is specified, valid root keys are: HKLM and HKU.
/v <ValueName> Specifies the value name to compare under the subkey.
/ve Specifies that only entries that have a value name of null should be compared.
[{/oa | /od | /os | on}] Specifies how to display the results of the compare operation. The default is /od. See the specific options below.
/oa Specifies that all differences and matches are displayed. By default, only the differences are listed.
/od Specifies that only differences are displayed. This is the default behavior.
/os Specifies that only matches are displayed. By default, only the differences are listed.
/on Specifies that nothing is displayed. By default, only the differences are listed.
/s Compares all subkeys and entries recursively.
/? Displays help for reg compare at the command prompt.
Remarks
The following table lists the return values for reg compare.
Value Description
0 The comparison is successful and the result is identical.
1 The comparison failed.
2 The comparison was successful and differences were found.
The following table lists the symbols displayed in the results.
Symbol Description
= KeyName1 data is equal to KeyName2 data.
< KeyName1 data is less than KeyName2 data.
> KeyName1 data is greater than KeyName2 data.
Examples
To compare all values under the key MyApp with all values under the key SaveMyApp, type:
REG COMPARE HKLM\Software\MyCo\MyApp HKLM\Software\MyCo\SaveMyApp
To compare the value for the Version under the key MyCo and the value for the Version under the key MyCo1, type:
REG COMPARE HKLM\Software\MyCo HKLM\Software\MyCo1 /v Version
To compare all subkeys and values under HKLM\Software\MyCo on the computer named ZODIAC with all subkeys and values under HKLM\Software\MyCo on the local computer, type:
REG COMPARE \\ZODIAC\HKLM\Software\MyCo \\. /s


Book-A-Meal
Sign Up
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Reset Password
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"A pleasant free game downloader" according to
the
review at DIYGamer.com. It's an app store
for free Windows games. It's a garden, where indie
games grow! The YouTube video at the right gives an
impression of Indiegame Garden.
Download (version
Beta-6)
Options:
Easy - run the ClickOnce web installer directly by
clicking this link (works in Internet Explorer only!)
Download
the installer here and run it. The installer downloads
the software and should also install any required Microsoft
libraries (such as XNA 4.0).
For Portable App fans like me, download
the ZIP file here; unzip somewhere for example the
Desktop; and run IndiegameGarden.exe.
System requirements
Windows XP or Windows 7 PC with somewhat modern graphics
card [XNA "Reach" profile, for the insiders.]
Fast Internet connection (for the automatic game downloads)
Play
Enable your internet connection. Run Indiegame Garden, wait a
moment (first time may be longer), and check the panel that
appears on the top left which keys to use. Have fun! Future
updates will automatically show in the form of an update icon top
left in your garden.


Get total downloads
{{ packageName }}
was downloaded
{{ totalDownloads.toLocaleString() }}
times.
Package
{{ packageName }}
is not found.


Deployed by {{ user }}, cheers!
{% if pr_merges %}
We just deployed the following PRs:
{% for pr in pr_merges %}
-
#{{ pr.number }} {{ pr.title }}
opened by @{{ pr.opened_by }}
+{{ pr.additions }}
−{{ pr.deletions }}
{% for label in pr.labels %}
{{ label.name }}
{% endfor %}
{% endfor %}
{% for label, prs in prs_by_label.items %}
{{ label }}
{% endfor %}
{% else %}
No PRs were merged since last deploy.
{% endif %}
Here's the complete diff on github: {{ compare_url }}


This is the 5th Edition of technoVIT, an international level technical festival of VIT Chennai. It gives us immense pride and pleasure in presenting technoVIT'19. This technical extravaganza will be filled with numerous activities such as workshops and technical events organised by the various schools of the University with the primary objective being, “nurture professional skills and impart fine qualities to the students”. It also has gaming events to thrill and create an environment with fun and frolic. Additionally, technical exhibitions from various organisations are being hosted. We, at VIT Chennai promise the audience an atmosphere of immense joy leaving them the thirst for more.
Get Set Go!


{{template "header.html" .Header}}
{{template "navbar.html" .}}Are you looking for a speaker for a tech event, or want to speak at a tech event?
There are currently {{.Details.Talks}} talks from {{.Details.Speakers}} speakers, who can speak at {{.Details.Locations}} locations and {{.Details.Groups}} groups wanting speakers at {{.Details.Places}} places.
Passwords are set to show by default, we do this because the likelihood of being shoulder surfed is pretty small and entering a complex password is made a lot harder on small screens when the password is masked. There is an Article by Bruce Schneier detailing it in more depth.
The only cookie set is when you login, so we recognise you and don't ask for a username/password every time you go to a new page. Viewing without being logged in doesn't involve any cookies, we aren't interested in tracking you.


public class TimeManager
extends java.util.Observable
| Modifier and Type | Field and Description |
|---|---|
int |
incomputeNextDelay1
This is for Test ONLY
|
int |
incomputeNextDelay2
This is for Test ONLY
|
| Modifier and Type | Method and Description |
|---|---|
java.time.ZonedDateTime |
getDateTime()
This method is used to get the ZonedDateTime object stored in this object.
|
java.lang.String |
getTime()
This method is used to get the time (yyyy/MM/dd/HH/mm) stored in the object.
|
static TimeManager |
getTimer()
This method is used to get the instance of this singleton class.
|
void |
setTime(java.lang.String t)
This is for Test ONLY
|
void |
setZonedDateTime(java.time.ZonedDateTime z)
This is for Test ONLY
|
void |
testNotify()
This is for Test ONLY
|
void |
timing()
This method starts the timer at 0 minute in the next hour and notify the observers every hour.
|
public int incomputeNextDelay1
public int incomputeNextDelay2
public void setZonedDateTime(java.time.ZonedDateTime z)
public void setTime(java.lang.String t)
public void testNotify()
public static TimeManager getTimer()
public java.lang.String getTime()
public java.time.ZonedDateTime getDateTime()
public void timing()


Hello there! My name is Vanesa and I am a busy mother of two beautiful children. I have been struggling all my life with weight loss and weight management. I started my keto lifestyle in 2017, and it helped me reach a healthy weight. I would like to share my experience with everyone that needs a change in their life, but can't do a traditional diet. The Keto journey is not an magic solution, but it's a lifestyle that can help you reach your goals and maintain it in the long-term.
Content Magement Team
- Vanesa Nasi, Founder
- Matt Nasi, Editor
- Hannah Jeffcoat, Marketing


Welcome to Model My Watershed - Runoff Simulation. See how changes in the amount of rainfall, the surfaces on which the rain falls and the texture of the soil change where the water goes. To begin, decide how much rain will fall by using the slide bar on the upper right. Then select the land-cover type where the rain will fall. Finally, pick a soil type. The coarsest soils (sandy soils) are on the left and the finest soils (clay loam) are on the right. As the simulation runs, follow where the water flows. Also, look at the column in the center that shows how much water goes in each direction.
Precipitation for 24-hour storm event
Land Cover
Hydrologic Soil Group
Apply our Site Storm Model to your watershed. Info and help at http://wikiwatershed.org/model/.


|
||||||||||
| PREV PACKAGE NEXT PACKAGE | FRAMES NO FRAMES |
| Interface Summary | |
|---|---|
| AES | Marker interface. |
| Hmac | Marker interface. |
| PrfFixed | General interface for pseudorandom function with fixed input and output lengths. |
| PrfVaryingInputLength | General interface for pseudorandom function with varying input length. |
| PrfVaryingIOLength | General interface for pseudorandom function with varying input and output lengths. |
| PrpFixed | General interface for pseudorandom permutation with fixed input and output lengths. |
| PrpVaryingIOLength | General interface for pseudorandom permutation with varying input and output lengths. |
| PseudorandomFunction | General interface for pseudorandom function. |
| PseudorandomPermutation | General interface for pseudorandom permutations which is sub-interface of pseudorandon function. |
| TripleDES | Marker interface. |
| Class Summary | |
|---|---|
| IteratedPrfVarying | This class is one implementation of pseudorandom function with varying IO, based on any prf with varying input length. |
| LubyRackoffPrpFromPrfVarying | The class LubyRackoffPrpFromPrfVarying is one implementation that has a varying input and output length. |
| PrfVaryingFromPrfVaryingInput | This class implements some common functionality of varying input and output length prf classes. |
| PrpFromPrfFixed | This class implements some common functionality of PrpFixed by having an instance of prfFixed. |
| PrpFromPrfVarying | This class implements some common functionality of PrpVaryingIOLength by having an instance of prfVaryingIOLength. |
|
||||||||||
| PREV PACKAGE NEXT PACKAGE | FRAMES NO FRAMES |


OLC Redmine Automator
To facilitate the sharing of the CFIA's genomic data and tools to analyze this data, we make use of the Redmine platform. Our instance of Redmine can be found here (note that you must be on the CFIA network in order to access Redmine).
If you have not yet gotten started with Redmine or want to know how the Redmine automator works and what it can do, take a look at our Getting Started page for instructions on how to start using Redmine and a brief overview of what Redmine can do.
For any issues or general help with Redmine, see the Troubleshooting page.


Implements interfaces:
This file is part of Terminalor.
For the full copyright and license information, please view the LICENSE file that was distributed with this source code.
Terminalor DocParser Template Abstract class responsible for rendering all tags without induvidual renderers.
Located in /DocParser/Template/Abstract.php (line 16)
| Class | Description |
|---|---|
| Terminalor_DocParser_Template_Standard | This file is part of Terminalor. |
Build commands help for each command using given variables, each tag is parsed separetly using specified or default parser
Get all registred renderers
Get variables available to the template
Render given tag for specified command if tag has an induvidual renderer it will be parsed using it, otherwise default renderer _renderUnspecifiedTag() will be used
Set variables available for given command
Wrapper for setVar() method, allow to specify array of variables for given command
Check if given tag has a renderer
Find longest element in given array
Render tag using default renderer, this method is used for all tags withoud specified renderer
Documentation generated on Tue, 07 Feb 2012 14:40:53 +1100 by phpDocumentor 1.4.4


--- layout: full-width title: Help using the service breadcrumb: home --- Jump to: Using the library service Borrowing, charges & fines eBooks, eAudio & devices WiFi, computers & printing Privacy & legal Using the library service Finding and reserving titles Borrowing, renewing and returning titles Joining the library Updating your library account Suggesting a title Opening times and holidays Lost and replacement cards Borrowing, charges & fines Borrowing periods & overdue charges for physical titles eLibrary borrowing periods & allowances If we don't stock a title: borrowing from other library services Lost or damaged items eBooks, eAudio books, streaming, downloads & devices Using our eLibrary services Borrowing eReaders and iPads Borrowing BBC micro:bits Wifi, computers and printing in libraries Printing, copying, scanning & faxing Using library computers Using wifi in libraries What we filter on public PCs and our WiFi service Privacy & legal Privacy and cookies Library byelaws Trouble finding the information you need? Contact us<EMAIL_ADDRESS>01473 351249 @suffolklibrary Facebook
Jump to:
Trouble finding the information you need? Contact us:


Photographs, Landscape and Memory
“Landscape,” British historian Simon Schama writes, “is the work of the mind. Its scenery is built up as much from the strata of memory as from layers of rock.”
Living in an old quarter of Lisbon for much of the previous year, I often reflected on this relationship between landscape and memory. The castle I saw on my daily walk to the metro had been seen from the same vantage point by the eyes of Vasco de Gama, Lord Byron and Mussolini. In the earth beneath the castle, visitors can gaze down archeological shafts through the collapsed roofs of the partially excavated Roman city of Olisipo. Deeper still, the bricks, bones, and amphora of the Carthaginians who first built the city, and below that, Neolithic remnants: shards of antler and flint. All testaments to the forgotten lives of individual humans. They had seen the same periwinkle blue estuary of the River Tagus, walked the same granite-strewn hills.
But as the famously cryptic Greek philosopher Heraclitus put it, “We both step and do not step in the same rivers. We are and are not.” Landscapes offer an impression of permanence, but they are as much in flux as the contents of our own memories. Regenerated each spring, continually reshaped by earthquakes, exposure, sedimentation, and human activity, a landscape is both constant and constantly changing – just as our own bodies recreate themselves, sloughing off molecules and generating new ones at such a rapid clip that it is estimated that we replace most of our cells every decade or so.
When we at The Appendix started thinking about the theme of “Illusions” for our second issue, we decided to construe the term as broadly as possible. We wanted it to encompass magic tricks, deceit, forgery and lies, but also deeper illusions: illusions of permanence; illusions created by our own memories. For the cover of The Appendix Issue Two, we settled on a photograph-within-a-photograph depicting the same spot of ground in 2012 and circa 1960. The hand in the photo is mine as an adult; the photograph I’m holding shows my father and his sisters as children. Photographs are famously susceptible to trickery and hoaxes. Photography is also, as Susan Sontag noted, a process of metamorphosis that turns a moment in time into a frozen object, transforming “an event or person into something that can be possessed.”
In other words, photographs, like landscapes, create an illusion of permanence. The images below are others in the series that gave rise to our Issue Two cover. I took them with an iPhone camera in the summer of 2012 while visiting my deceased grandmother’s old house in Scituate, a largely Irish-American seacoast town south of Boston. While going through her family photo albums, I realized that many had been taken in locations that I still recognized decades later. But in contrasting these old photos with their present-day correlates, it was the differences between the two scenes that struck me. The juxtaposition reminded me of the novelist L.P. Hartley’s oft-quoted observation that “the past is a foreign country.”
Photographs are passports.


MyGet - Hosted Package Management Server for NuGet, symbols, Npm, Bower, Maven and VSIX
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{{ content }}


Returns the arccosine of each element of current array.
Returns the hyperbolic arccosine of each element of current array.
Adds x
multiplied by alpha
to the current array.
Accelerated with BLAS ?axpy
.
Determines the angle between the current vector and x
.
Returns the arcsine of each element of current array.
Returns the hyperbolic arcsine of each element of current array.
Returns the arctangent of each element of current array.
Returns the hyperbolic arctangent of each element of current array.
Augments x
with current matrix.
Perform binary operation f
on x
in the current array.
Returns the cube root of each element of current array.
Returns smallest integer greater than or equal to of each element of current array.
Asserts if indices i, j, ..., n
are within the bounds of current array
Combines the current vector with x
Makes a copy of the class and underlying data
Returns the cosine of each element of current array.
Returns the hyperbolic cosine of each element of current array.
Computes the cross product of the current vector and the vector x
This operation can only be calculated for vectors with three components.
Otherwise it throws an exception.
The method returns a new (result) vector.
Gets the determinant of current matrix using LU factorization.
Gets the diagonal of current matrix.
Performs dot multiplication with x
and current array
Accelerated with BLAS ?dot
.
Gets eigenvalues and eigenvectors of the current matrix using the Jacobi method.
Accelerated with LAPACK ?geev
.
Checks if current array and x
are equal.
Asserts if current array and x
have the same shape
Asserts if current array and x
have the same length
Returns e^x of each element of current array, where x is the argument, and e is Euler's constant (2.718…), the base of the natural logarithm.
Returns subtracting 1 from exp(x) of each element of current array.
Fills the current array with a scalar value
Returns the largest integer less than or equal to a number of each element of current array.
Equivalent to TypedArray.prototype.forEach
.
Returns the nearest single precision float representation of each element of current array.
Gauss-Jordan elimination (i.e. returns the reduced row echelon form) of current matrix.
Gets the element at i, j, ..., n
from current vector.
Determines the inverse of current matrix using Gaussian elimination.
Accelerated with LAPACK ?getri
.
Returns the natural logarithm (log_e, also ln) of each element of current array.
Returns the natural logarithm (log_e, also ln) of 1 + x for each element of current array.
Returns the base 2 logarithm of each element of current array.
Returns the base 10 logarithm of each element of current array.
Performs full LU decomposition on current matrix.
Accelerated with LAPACK ?getrf
.
Performs LU factorization on current matrix.
Accelerated with LAPACK ?getrf
.
Equivalent to TypedArray.prototype.map
.
Gets the maximum value (smallest) element of current array.
Gets the arithmetic mean of current array.
Gets the minimum value (smallest) element of current array.
Multiplies current matrix with x
.
Accelerated with BLAS ?gemm
.
Calculates the norm of current array (also called L2 norm or Euclidean length).
Accelerated with BLAS ?nrm2
.
Normalizes current vector.
Returns each element of current array to the exponent power, that is, element^exponent.
Product of all elements of current array
Hadamard product of current matrix and x
Projects the current vector onto x
using the projection formula (y * (x * y / y * y))
.
Pushes a new value
into current vector.
Finds the rank of current matrix using gaussian elimination.
Gets the element-wise reciprocal of current array.
Equivalent to TypedArray.prototype.reduce
.
Reshapes current array
Returns the value of each element of current array rounded to the nearest integer.
Adds a multiple of one row multiplied by scalar
to another inside current matrix.
Multiplies all elements of current array with a specified scalar
.
Accelerated with BLAS ?scal
.
Sets the element at i, j, ..., n
to value
.
Returns the sign of each element of current array, indicating whether it is positive, negative or zero.
Returns the sine of each element of current array.
Returns the hyperbolic sine of each element of current array.
Slices the current array in the corresponding dimension
Solves the equation AX = B (where A is current matrix and B is x
).
Accelerated with LAPACK ?gesv
.
Returns the positive square root of each element of current array.
Asserts if current matrix is square.
Subtracts x
from the current array.
Accelerated with BLAS ?axpy
.
Sum of array elements
Swaps two rows i
and j
in current matrix
Returns the tangent of each element of current array.
Returns the hyperbolic tangent of each element of current array.
Converts current vector into a JavaScript array.
Converts current vector into a readable formatted string.
Gets the trace of the matrix (the sum of all diagonal elements).
Transposes current matrix (mirror across the diagonal).
Returns the integer part of each element of current array, removing any fractional digits.
Returns the absolute value of each element of x
.
Returns the arccosine of each element of x
.
Returns the hyperbolic arccosine of each element of x
.
Adds y
multiplied by alpha
to x
.
Accelerated with BLAS ?axpy
.
Determines the angle between the x
and y
Constructor alias
Returns the arcsine of each element of x
.
Returns the hyperbolic arcsine of each element of x
.
Returns the arctangent of each element of x
.
Returns the hyperbolic arctangent of each element of x
.
Augments x
and y
.
Perform binary operation f
on y
in x
.
Returns the cube root of each element of x
.
Returns smallest integer greater than or equal to of each element of x
.
Asserts if indices i, j, ..., n
are within the bounds of x
Combines the vector x
with y
Makes a copy of x
Returns the cosine of each element of x
.
Returns the hyperbolic cosine of each element of x
.
Computes the cross product of the x
and the vector y
This operation can only calculated for vectors with three components.
Otherwise it throws an exception.
The method returns a new (result) vector.
Gets the determinant of x
.
Gets the diagonal of x
.
Performs dot multiplication with x
and y
.
Accelerated with BLAS ?dot
.
Gets eigenvalues and eigenvectors of x
using the Jacobi method.
Accelerated with LAPACK ?geev
.
Checks if x
and y
are equal.
Asserts if x
and y
have the same shape
Asserts if x
and y
have the same length
Returns e^x of each element of x
, where x is the argument,
and e is Euler's constant (2.718…), the base of the natural logarithm.
Returns subtracting 1 from exp(x) of each element of x
.
Creates an identity matrix of size n
and type type
.
Fills x
with a scalar value
Returns the largest integer less than or equal to a number of each element of x
.
Equivalent to TypedArray.prototype.forEach
.
Returns the nearest single precision float representation of each element of x
.
Gauss-Jordan elimination (i.e. returns the reduced row echelon form) of x
.
Gets the element at i, j, ..., n
from x
Determines the inverse of x
.
Accelerated with LAPACK ?getri
.
Returns the natural logarithm (log_e, also ln) of each element of x
.
Returns the natural logarithm (log_e, also ln) of 1 + x for each element of x
.
Returns the base 2 logarithm of each element of x
.
Returns the base 10 logarithm of each element of x
.
Performs full LU decomposition on x
.
Accelerated with LAPACK ?getrf
.
Performs LU factorization on x
.
Accelerated with LAPACK ?getrf
.
Creates a magic square matrix of size
Equivalent to TypedArray.prototype.map
.
Creates a matrix of r
rows and c
columns.
Gets the maximum value (largest) element of x
.
Accelerated with BLAS i?amax
.
Gets the arithmetic mean of x
.
Gets the minimum value (smallest) element of x
.
Multiplies two matrices x
and y
of matching dimensions.
Accelerated with BLAS ?gemm
.
Calculates the norm of current array (also called L2 norm or Euclidean length).
Accelerated with BLAS ?nrm2
.
Normalizes x
.
Creates an array containing ones (1
) of shape shape
Returns each element of x
to the exponent power, that is, element^exponent.
Product of all elements of x
.
Hadamard product of x
and y
Projects the y
onto x
using the projection formula (y * (x * y / y * y))
.
Pushes a new value
into x
.
Creates a vector containing random samples from a uniform distribution over [0, 1)
of shape shape
Creates an array containing a range (can be either ascending or descending)
of numbers specified by the arguments provided (e.g. NDArray.range(0, .5, 2)
gives an array containing all numbers in the interval [0, 2)
separated by
steps of 0.5
)
Finds the rank of x
using gaussian elimination.
Gets the element-wise reciprocal of x
.
Equivalent to TypedArray.prototype.reduce
.
Reshapes x
Returns the value of each element of x
rounded to the nearest integer.
Adds a multiple of one row multiplied by scalar
to another inside x
.
Multiplies all elements of x
with a specified scalar
.
Accelerated with BLAS ?scal
.
Sets the element at i, j, ..., n
to value
.
Returns the sign of each element of x
, indicating
whether it is positive, negative or zero.
Returns the sine of each element of x
.
Returns the hyperbolic sine of each element of x
.
Slices x
in the corresponding dimension
Solves the equation AX = B (where A is x
and B is y
).
Accelerated with LAPACK ?gesv
.
Returns the positive square root of each element of x
.
Asserts if x
is square.
Subtracts y
from x
.
Sum of x
Swaps two rows i
and j
in x
.
Returns the tangent of each element of x
.
Returns the hyperbolic tangent of each element of x
.
Converts x
into a JavaScript array.
Converts x
into a readable formatted string.
Gets the trace of x
(the sum of all diagonal elements).
Transposes x
(mirror across the diagonal).
Returns the integer part of each element of x
,
removing any fractional digits.
Creates an array containing zeros (0
) of shape shape
Transposes current matrix (mirror across the diagonal).
Equivalent to this.get(3)
Equivalent to this.set(3, value)
Equivalent to this.get(0)
Equivalent to this.set(0, value)
Equivalent to this.get(1)
Equivalent to this.set(1, value)
Equivalent to this.get(2)
Equivalent to this.set(2, value)
Generated using TypeDoc
Returns the absolute value of each element of current array.


"x"
"1" "DAYAN,J, DEGUINGAND,S AND TRUZMAN,C, STUDY OF THE MUTAGENIC ACTIVITYOF 6 HEPATOTOXIC PHARMACEUTICAL DRUGS IN THE SALMONELLA TYPHIMURIUM MICROSOMETEST, AND THE HGPRT AND SODIUM-POTASSIUM ATPASE SYSTEM __ IN CULTURED MAMMALIANCELLS, MUTAT. RES. 157(1):1-12, 1985"


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Summer course in statistics
Last summer I taught Introduction to Psychological Measurement and Data Analysis for undergrads, which is my department’s standard intro stats course. While I had some experience teaching, it was my first experience teaching a course completely on my own. There are many things I wish I had done differently, but overall I enjoyed and learned a lot from the experience. I love trying to explain difficult concepts, so this was great practice.
One of the most useful things for me at the beginning of the course was to make a course narrative. I learned about this idea from Prof. Pachego-Vega (who you should follow on Twitter), and it helped me shape what I wanted to teach and why. When I first started I had these rather grand visions about what a statistics course should teach. I have a lot of opinions about the statistics we teach in these courses. NHST is an alright tool, but I think that teaching that perspective first hurts a student’s longterm statistical learning. This might be a blogpost in of itself… However I quickly realized that would require me to make a lot of new material for students, and would end up swamping my already high workload.
To ease my workload, I used material from other instructors, including the previous summer when another graduate student taught. Some of his material he got from the main instructor for the fall/spring courseI got a lot of content from Brent Carpenter (the previous graduate instructor) and Mark Stellmack . Creating a course from scratch takes an enormous amount of time, and while I made my own lectures it was really useful to use material others had made.
Using other material was probably my main reason for not dramatically restructuring the course. I mostly followed Statistics Unplugged in terms of content, which is a pretty decent introductory text for the standard intro stats material (it ends on ANOVA, chi-square and regression). Caldwell focuses on going through the calculations by hand, using t-tables and the like, and helping students with anxiety about The Maths. Unfortunately it focuses on a very basic functional understanding of how to use these tests without a super deep understanding of what they mean. While in itself that’s understandable (and where I attempted to fill in with my lectures), it ignores how most statistics is done via computer code these days.
source
Teaching programming was my biggest initial debate when designing the course, and I ended up not covering it. A lot of discussions I had with other grad students ended up concerned whether students would get it or not, and if it would just double the workload. I really still don’t know, especially because I think it’s so important as a skill for modern psychology work and something students can use outside of the course itself. If given the chance to teach again I likely will want to incorporate some programming at the expense of other content, possibly using material from the R for data science or Intro to Stats and Data Science via R online texts, or create my own.
One thing I did include was more time initially on graphics and communicating statistical results. I spend some time at the beginning covering Tufte and other aspects of graph making.  These assignments allowed me to teach a bit on communicating statistical results and allowed them to go from beginning to end with their newly learned skills. I really enjoyed these projects and would definitely use them again, but possibly refine. Since it was a summer course we ended up having to drop one of them due to time, so it definitely warrants playing with more.
Since my own research rarely uses hypothesis testing, I wanted to also teach some more advanced Bayesian methods, factor analysis (PCA), and regression. Focusing on the concepts, with a few examples, seemed to help them get a good sense of the scope of statistics while also seeing what is common across everything (dealing with uncertainty and patterns in data). The last day covered the replication crisis in psychology, and a bit about how meta-analyses are done, which students seemed really engaged in!
Little errors were possibly the most difficult thing for me, especially given it was a math course. Many of the students (given this is psychology) had taken very little mathematics and had to review a lot of algebra. While I have a degree in mathematics, I’m horrible at in-the-moment computations. Coming up with a new example usually resulted in me fumbling around, but students were usually satisfied by the end of it.
I would love to get the chance to teach the course again, even though it did take a large amount of effort from me. The fact that it was a summer course also meant the whole thing went incredibly quickly. Given the discussions people have been having online about how to improve psychology research, I can see both how important the instructional side is and also how much effort it will take. Given my own experience it goes beyond changing a single course, but can certainly start there.
January 2, 2018


Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbp Δ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbp Δ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.
Smallpox was one of the biggest human scourges when it was rife, with mortality rates of 30–40%. VARV is an orthopoxvirus (OPV) that causes smallpox, a disease that has killed more humans than all other infectious diseases combined [ 1– 4]. As the disease was eradicated more than 30 years ago, animal models of OPV infections have been used to establish strong causal relationships and map the requirements for protection against infection in a way that is not possible in humans. ECTV is a natural pathogen of mice that causes a generalized infection known as mousepox, an excellent small animal model to study the pathogenesis of smallpox [ 4– 7]. Both VARV and ECTV cause acute infections in their respective hosts, which either succumb to infection or clear the virus with complete recovery.
Resistance to mousepox is dependent on rapid generation of a strong polarized type 1 immune response [ 8]. ECTV-resistant C57BL/6 mice generate robust NK cell, CTL, and IFN-γ responses whereas in ECTV-susceptible BALB/c mice these responses are sub-optimal but high levels of IL-4 is produced. Loss of IFN-γ function in resistant mice [ 9, 10] or over-expression of IL-4 by recombinant ECTV encoding IL-4, which abrogates IFN-γ production [ 11, 12] results in ineffective NK cell and CTL responses and fulminant disease. The existence of virus-encoded host response modifiers that specifically target type 1 cytokines further underscores the importance of these factors in immunity to poxvirus infections [ 13– 16]. Indeed, BALB/c mice infected with a deletion mutant virus lacking vIFN-γbp (ECTV-IFN-γbp Δ) are able to contain virus replication and overcome the infection [ 17]. Absence of vIFN-γbp increases IFN-γ production and as a consequence, augments cell-mediated immunity and virus control.
The cytokine milieu and cell subset-selective transcription factors play important roles in the differentiation of uncommitted naive CD4 T cells into Th1 and Th2 subsets following antigenic stimulation [ 18, 19]. IFN-γ and IL-4 play dominant roles in the development of Th1 and Th2 subsets, respectively. IL-4 mediates its activity through the IL-4 receptor (R), which consists of a heterodimeric complex between IL-4Rα and the common γ chain [ 20, 21]. IL-4Rα also associates with the low affinity IL-13Rα1 to form a high affinity receptor for both IL-13 and IL-4. A second IL-13 receptor, IL-13Rα2, which exists in a monomeric form, can bind to IL-13 with high affinity and modulate IL-13 responses [ 22, 23]. Binding of IL-4 to IL-4R activates the transcription factor STAT-6, which in turn controls expression of GATA3, the master transcription regulator required for Th2 differentiation [ 24]. IL-13 is also capable of activating STAT-6 and shares many of the biological properties of IL-4.
A previous report pointed to a role for STAT-6 signaling and IL-4 production in contributing to susceptibility of BALB/c mice to ECTV infection [ 25]. STAT-6 deficient mice infected with ECTV-WT were better protected through generation of more effective NK cell and IFN-γ responses that allowed the otherwise susceptible animals to overcome infection. Curiously, IL-4 deficiency in BALB/c mice has no impact on disease outcome [ 8], implying that other factors like IL-13 may be sufficient to drive Th2-like responses in the absence of IL-4.
Since the generation of protective immunity and the outcome of a viral infection are influenced by both viral and host genes, we hypothesized that infection of gene knockout (GKO) BALB/c mice lacking one or more factors associated with Th2 response development with ECTV-WT or ECTV-IFN-γbp Δ would further augment the Th1 response and influence disease outcome. Contrary to our prediction, the disease was exacerbated in most groups of BALB/c mice lacking one or more components of Th2 signaling pathways compared to WT mice infected with WT virus. Conversely, the disease outcome was significantly improved in WT mice infected with the mutant virus, but absence of the IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage to the host. In fact, there were higher rates of mortality and accelerated deaths amongst mice deficient in IL-13 or STAT-6 compared with WT animals. Deficiency in IL-13 or STAT-6 dramatically reduced the antiviral CTL response. Furthermore, deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Thus, although recovery from ECTV infection is strongly influenced, in part, by a balance between the host’s ability to produce IFN-γ and the virus’ ability to dampen its effects [ 17], our data indicate that factors contributing to disease susceptibility are far more complex than a balance between Th1 and Th2 responses.
This study was performed in strict accordance with the recommendations in the Australian code of practice for the care and use of animals for scientific purposes and the Australian National Health and Medical Research Council Guidelines and Policies on Animal Ethics and approved by the Animal Ethics and Experimentation Committee of the Australian National University (Protocol numbers J.IG. 62/08, J.IG. 75.09 and A2012/041).
Gene knockout (GKO) mice lacking IL-4 [ 26], IL-13 [ 27], IL-4Rα [ 28], STAT-6 [ 29] or both IL-13 and IL-4Rα were either generated on a BALB/c background or backcrossed to the BALB/cJ strain for at least 10 generations or more. Female BALB/c WT and GKO mice designated IL-4 −/−, IL-13 −/−, STAT-6 −/−, IL-4Rα −/−, IL-13 −/−/IL-4Rα −/− and were bred under specific-pathogen-free conditions at the Australian Phenomics Facility, Australian National University and used at 6–8 weeks of age. The IL-4 −/− mice can respond to IL-13 whereas IL-13 −/− mice have intact IL-4 responses. IL-4Rα −/− mice lack responses to both IL-4 and IL-13 but the IL-13 produced in this strain can be modulated by IL-13Rα2. IL-13 −/−/IL-4Rα −/− mice have a complete absence of IL-4 and IL-13 responses. In STAT-6 −/− mice, only STAT-6-independent GATA3 expression and responses can occur.
BS-C-1, an African green monkey kidney epithelial cell line, CV-1, an African green monkey kidney fibroblast cell line, P-815 (H-2 d), a DBA/2 mouse-derived mastocytoma line and YAC-1 (H-2 a), a Moloney murine leukemia virus-induced lymphoma line from A/Sn mouse, were obtained from American Type Culture Collection (Rockville, MD). All cells were maintained in Eagle’s Minimum Essential Medium supplemented with 10% fetal calf serum, 2mM L-glutamine, 120μg/ml penicillin and 200μg/ml streptomycin and neomycin sulfate.
WT ECTV-Moscow strain [ 30], designated ECTV-WT, and a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbp Δ [ 17] were used in this study. All ECTV stocks were propagated in BS-C-1 cells and titers determined by viral plaque assay (VPA) [ 31]. In all experiments, mice were inoculated subcutaneously (s.c.) with 10 3 PFU of virus in the flank of the left hind limb (hock) under avertin anesthesia. This dose of virus was used in order to make meaningful comparisons with previous studies [ 8, 25].
ECTV-derived CD8 T cell peptide determinants [ 32] used in this study are shown in S1 Table. Peptides were synthesized at the Biomolecular Resource Facility, John Curtin School of Medical Research. They were purified via reverse-phase HPLC and the quality checked by mass spectroscopy. Peptides were stored in stock solutions at 10mM in 99.99% dimethyl sulfoxide (Sigma-Aldrich Inc. St. Louis, MO USA) and diluted in culture medium to the required concentration for use in the cellular assays.
The standard 51Chromium release assay [ 8] was used to determine ex vivo anti-ECTV CTL activity, using ECTV-infected and uninfected P815 (H-2 d) target cells. YAC-1 cells were used as targets for splenic NK cell cytotoxicity assays. The fold change in cytolytic activity of GKO splenocytes compared with WT splenocytes shown in the results is based on % specific lysis at a given effector: target ratio. As an example, in the data shown on CTL responses, the % specific lysis mediated by IL-13 −/−/IL-4Rα −/− CTL at 75:1 effector: ratio is comparable to % specific lysis mediated by WT cells at 25:1 effector: ratio. That is, at a 3-fold lower number of splenocytes, the cytolytic activity of WT cells is comparable to that of IL-13 −/−/IL-4Rα −/− cells. Similarly, the % specific lysis mediated by IL-13 −/− cells at 75:1 effector: ratio is comparable to % specific lysis mediated by WT cells at 2.8:1 effector: ratio, i.e. a 27-fold lower number of WT splenocytes were required to mediate comparable levels of cytolytic activity mediated by IL-13 −/− cells.
Total and granzyme B (GzB) expressing NK cells were quantified by flow cytometry. We used anti-CD49b-PE (clone DX5) to stain NK cells and anti-CD49b (DX-5)-PE plus anti-granzyme B-APC (clone MHGB05) to stain GzB expressing NK cells. Cells were also stained with anti-CD3-FITC (clone 17A2) to exclude CD3 + NKT cells in the analysis.
Total ECTV-specific or ECTV determinant-specific IFN-γ producing CD8 T cells were enumerated using intracellular cytokine staining. The protocol for detection of ECTV-specific CD8 T cells has been described previously [ 17]. Briefly, 10 6 splenocytes were incubated with either 2x10 5 virus-infected P815 cells or with synthetic peptides ( S1 Table) at final concentrations of 0.1μM (K d-EVMA52
For enumeration of IFN-γ or IL-4 producing CD4 T cells, splenocytes from virus-infected mice were incubated at 37°C with media (unstimulated) or stimulated with Phorbol 12-Myristate 13-Acetate (PMA) plus Ionomycin (Calbiochem) (PMA: 0.1μg/ml; Ionomycin: 1μg/ml) for 5h as positive control. Following incubation, cells were stained with anti-CD4 (clone GK1.5), permeabilized for 20 min followed by intracellular staining with anti-IL-4 (clone 11B11) or anti-IFN-γ (clone XMG1.2) in staining buffer containing 0.1% saponin. Total events for cells were acquired using a BD LSR Fortessa cell analyzer and analyzed using FlowJo software (Tree Star Inc.). Antibodies were purchased from BD Biosciences, Biolegend and Invitrogen.
To estimate numbers of T-bet- or GATA-3-expressing CD4 T cells, 2 x10 6 spleen cells were stained without re-stimulation with anti-CD3 and anti-CD4 in a 50μl volume for 30 mins at 4°C. The cells were then washed and re-suspended in 100μl of freshly prepared fix/perm buffer (FoxP3 transcription factor staining buffer kit, e-Bioscience) and incubated for 30 mins at 4°C. The cells were then washed twice with permeablization buffer and then incubated for 40 mins at 4°C with anti-GATA3 (Clone L50–823; BD Biosciences) and anti-T-bet (Clone 4B10, Biolegend) antibodies. The cells were washed twice with permeablization buffer and resuspended in buffer just prior to analysis on BD LSR Fortessa cell analyzer.
Cell numbers in experiments were determined by multiplying the percentage of cells positive for GzmB, IFN-γ ( S1, S2, S3 Figs.), IL-4 ( S4 Fig.), T-bet or GATA-3 by the total number of spleen cells obtained from each mouse.
Organ tissue and blood were removed aseptically from infected mice and stored at −80°C until processed. Virus titers, expressed as log
Statistical analyses of experimental data, employing parametric and nonparametric tests as indicated in figure legends, were performed using GraphPad Prism (GraphPad Software, San Diego, USA). The Kaplan-Meier Log rank test was used to determine p values for survival proportions. Two-way ANOVA followed by Fisher’s Least Significant Difference (LSD) test was used to determine significance for viral load, NK, CD8 and CD4 T cell responses.
Groups of WT and GKO mice were infected with ECTV-WT or ECTV-IFN-γbp Δ and monitored for 21 days to determine whether the absence or presence of vIFN-γbp during ECTV infection in BALB/c mice lacking IL-4, IL-13, IL-4Rα, STAT-6 or both IL-13 and IL-4Rα may result in better recovery rates. We compared the outcome of infection with the 2 viruses in individual mouse strains ( Fig. 1), ECTV-WT infection in WT versus individual GKO mouse strains ( S5 Fig.) and ECTV-IFN-γbp Δ infection of WT versus individual GKO mouse strains ( S6 Fig.). The detailed statistical analysis is presented in S2 Table.
Groups of female mice were infected with ECTV-WT or ECTV-IFN-γbp Δ. Mice were monitored daily for 21 days for disease signs. Data shown are combined results obtained from two separate experiments in which 5–15 mice per strain were used ( S2 Table). P values for survival proportions were obtained by using Kaplan-Meier Log rank statistical test: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The survival rates of WT mice compared to GKO mice infected with WT or mutant virus are presented in S5 and S6 Figs., respectively and the statistical analysis presented in S2 Table.
Regardless of genotype, all groups of mice infected with ECTV-WT succumbed to mousepox with 100% mortality ( Fig. 1, S5 Fig., S2 Table). Notably, most GKO strains succumbed more rapidly than WT mice to ECTV-WT infection. Absence of one or more factors associated with Th2 response development did not provide any added advantage to hosts infected with WT virus. In contrast, all groups of mice infected with ECTV-IFN-γbp Δ had better rates of recovery compared with ECTV-WT infection ( Fig. 1, S6 Fig., S2 Table). However, even despite the improved recovery rates compared with WT virus infection, some GKO strains infected with mutant virus succumbed to mousepox significantly more rapidly than WT mice. In particular, mice deficient in IL-13 or STAT-6 had the lowest recovery rates and succumbed to disease more rapidly than WT mice infected with ECTV-IFN-γbp Δ. These experiments established that absence of vIFN-γbp significantly assisted in the recovery of WT mice from ECTV infection but did not improve recovery of the GKO strains. They also revealed that absence of IL-13, IL-4Rα, STAT-6 or both IL-13 and IL-4Rα increased susceptibility of mice to ECTV-WT infection.
Susceptibility to ECTV is associated with uncontrolled virus replication, particularly in the liver and spleen [ 5– 8]. We measured titers of ECTV-WT and ECTV-IFN-γbp Δ in livers, spleens and blood collected from animals at day 7 post-infection (p.i.) to determine whether the increased susceptibility of some strains was due to increased virus load. This time point was chosen as WT BALB/c mice infected with ECTV-WT begin to succumb to mousepox from day 7 p.i. as a result of high viral load [ 8, 17].
In the liver, ECTV-WT replicated to significantly higher levels in STAT-6 −/− mice compared to WT animals but titers were otherwise comparable across other strains ( Fig. 2, S3 Table). ECTV-IFN-γbp Δ titers on the other hand were significantly lower in IL-4 −/− and IL-13 −/−/IL-4Rα −/− mice compared to WT mice but no other differences were evident. A comparison of ECTV-WT and ECTV-IFN-γbp Δ titers indicated that the mutant virus replicated at significantly lower levels in STAT-6 −/−, IL-13 −/−, IL4-Rα −/−, and IL-13 −/−/IL-4Rα −/− mice ( S3 Table).
Groups of 5 mice were infected with either ECTV-WT or ECTV-IFN-γbp Δ and sacrificed at day 7 p.i. Viral load was determined by viral plaque assay and expressed as Log
In the spleen, the WT viral load increased significantly in STAT-6 −/−, IL-13 −/−, and IL-4Rα −/− mice compared with WT animals ( Fig. 2, S4 Table). On the other hand, ECTV-IFN-γbp Δ titers were significantly higher in IL-4 −/− and IL-4Rα −/− mice compared with WT mice. In a comparison of WT and mutant virus titers among all mouse strains, it was apparent that ECTV-IFN-γbp Δ replicated to lower levels only in the spleen of STAT-6 −/− mice ( S4 Table).
Finally, in the blood, ECTV-WT titers were increased in STAT-6 −/− mice compared to WT mice ( Fig. 2, S5 Table) but no differences in its replicative capacity were evident in the other strains. ECTV-IFN-γbp Δ replicated to higher levels in IL-13 −/− mice compared to WT animals but titers were lower than that of WT virus in STAT-6 −/− and IL-4 −/− mice.
Taken together, with the exception of STAT-6 −/− mice in which ECTV-WT titers were significantly increased in all organs compared to WT mice, there was no consistent correlation between increased susceptibility of individual mouse strains and viral load in visceral organs or blood.
NK cells play an important role in virus control during the acute phase of ECTV infection and recovery of mice [ 5, 17, 33, 34]. We measured the splenic NK cell activity at the peak of the response at day 5 p.i. in order to determine whether the absence of vIFN-γbp and/or factors associated with Th2 responses augmented these responses following infection.
ECTV-WT induced weak splenic NK cell activity in WT, IL-13 −/−, IL-4Rα −/− and IL-13 −/−/IL-4Rα −/− mice ( Fig. 3A). The only exceptions were the IL-4 −/− and STAT-6 −/− strains in which the responses were about 3-fold higher than in WT animals. ECTV-IFN-γbp Δ induced a 3-fold higher NK cell cytolytic activity compared with ECTV-WT in WT and IL-4Rα −/− mice but did not affect responses in the other strains ( Fig. 3B). The magnitude of NK cell responses to both mutant and WT viruses were similar in IL-4 −/−, IL-13 −/−, STAT-6 −/− and IL-13 −/−/IL-4Rα −/− mice. For the purpose of comparison, the NK cell responses to both WT and mutant mouse strains at 100: 1 effector to target cell ratio is shown in Fig. 3C.
Groups of 5 mice were infected with ECTV-WT or ECTV-IFN-γbp Δ, sacrificed 5 days later and the cytolytic activities of splenic NK cells measured. Data shown are % specific lysis of 51Cr-labelled YAC-1 target cells by splenic NK cells from (A) ECTV-WT-infected or (B) ECTV-IFN-γbp Δ-infected WT and GKO mouse strains. (C) Percent specific lysis of targets by splenocytes from the various strains infected with ECTV-WT or ECTV-IFN-γbp Δ at 100:1 effector-to-target ratio. (D) Numbers of GzB + NK cells. Data shown are means SEM of results obtained from one of 3 separate experiments. Statistical significance was determined by 2-way ANOVA followed by Fisher’s LSD test for significance between groups. For panel C, (i) NK cell responses to ECTV-WT vs. ECTV-IFN-γbp Δ in WT mice ( p = 0.0024) and IL-4Rα −/− mice ( p < 0.0023); (ii) NK cell responses to ECTV-WT in WT mice vs IL-4 −/− ( p = 0.009), WT mice vs STAT-6 −/− mice (** p = 0.0476); (iii) NK cell response to ECTV-IFN-γbp Δ in WT vs IL-13 −/− ( p = 0.0024), WT vs IL-13 −/−/IL-4Rα −/− mice (* p = 0.02). For panel D, (i) GzB + NK cell numbers in ECTV-WT vs. ECTV-IFN-γbp Δ infected strains: WT mice ( p = 0.02), IL-4 −/− mice ( p = 0.02), STAT-6 −/−( p = 0.0002), IL-13 −/− (p = 0.0068) and IL-13 −/−/IL-4Rα −/− mice ( p = 0.008); (ii) GzB + NK cell numbers in ECTV-WT infected WT mice vs IL-4 −/− mice (p = 0.0006) and WT mice vs STAT-6 −/− mice (p<0.0001); (iii) GzB + NK cell numbers in ECTV-IFN-γbp Δ infected WT mice vs IL-4 −/− mice (p = 0.0007) and WT mice vs STAT-6 −/− mice (p<0.0001). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
The total numbers of DX5 + NK cells were not altered significantly in GKO strains compared with WT mice following infection with ECTV-WT, with the exception of the IL-13 −/−/IL-4Rα −/− mice in which the numbers were about 5-fold lower ( Fig. 3D). Numbers of NK cells increased significantly in most GKO strains following infection with the mutant virus except for WT and IL-13 −/−/IL-4Rα −/− mice. We also assessed the expression of granzyme B (GzB), a serine protease found in granules of cytotoxic lymphocytes, to determine whether there was any association between the cytolytic activity of NK cells and GzB expression. Flow cytometric analysis revealed that WT virus infection increased GzB + NK cell (CD3-DX5 +) numbers in IL-4 −/− and STAT-6 −/− mice compared to WT mice ( Fig. 3E). In contrast, mutant virus infection increased numbers of GzB + cells in all strains compared to WT virus. Nonetheless, increases in GzB + cell numbers did not always result in a corresponding increase in cytolytic activity ( Fig. 3A-3C). As an example, infection of STAT-6 −/− mice with ECTV-WT resulted in increased GzB + cell numbers over and above those in WT mice and infection with the mutant virus further increased numbers. However, regardless of the type of virus used, the cytolytic activities of NK cells in WT and STAT-6 −/− mice were comparable ( Fig. 3A-3C).
CD8 T cells are an important source of IFN-γ production in mousepox beginning at day 6 p.i. The levels of IFN-γ produced by these cells closely parallel the magnitude of their cytolytic activity [ 8] and both responses peak between days 7–8 p.i. We assessed whether deletion of vIFN-γbp augmented IFN-γ production and CTL activity in CD8 T cells in WT and GKO mice at day 7 p.i.
Absence of IL-4Rα signaling did not affect CTL responses since WT and IL-4Rα −/− mice infected with ECTV-WT generated similar levels of killing ( Fig. 4A). The responses in IL-4 −/− and IL-13 −/−/IL-4Rα −/− mice were about 3-fold lower than WT mice. However, the most striking differences were evident in STAT-6 −/− and IL-13 −/− mice in which the responses were about 9-fold and 27-fold lower, respectively, compared to responses in WT mice. The significant reduction in CTL response in IL-13 −/− mice was partially reversed when IL-4Rα was also absent. The data indicated that IL-4, STAT-6 and IL-13, which are normally associated with development of Th2 responses, play important and non-redundant roles in the generation of optimal antiviral CTL responses.
Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp and sacrificed 7 days later to measure splenic CTL activity. Shown is % specific lysis of 51Cr-labelled, ECTV-infected P815 target cells by splenocytes from (A) ECTV-WT- or (B) ECTV-IFN-γbp Δ-infected WT and GKO mice. (C) Percent specific lysis of targets by splenocytes at 75:1 effector-to-target ratio. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis: (i) CTL response to ECTV-WT vs. ECTV-IFN-γbp Δ in IL-13 −/− (p < 0.0001) and STAT-6 −/− (p < 0.0001) (ii) CTL response to ECTV-WT in WT vs IL-4 −/− (p = 0.0083), vs STAT-6 −/− (p = 0.0003), vs IL-13 −/− (p = 0.0001), vs IL-13 −/−/IL-4Rα −/− (p = 0.0037) mice; (iii) CTL response to ECTV-IFN-γbp in WT vs STAT-6 −/− (p = 0.0002); vs IL-13 −/− (p = 0.0478); vs IL-4Rα −/− (p = 0.0174) and IL-13 −/−/IL-4Rα −/− (p = 0.000046) mice. ****, p<0.0001. (D—F) Spleen cells from mice infected mice were re-stimulated for 5 h with ECTV-infected P815 cells or H-2 d 8 T cell peptide determinants prior to intracellular IFN-γ staining followed by flow cytometry. Data shown are means of absolute numbers of ECTV-specific IFN-γ + 8 T cells in spleens of ECTV-WT- vs. ECTV-IFN-γbp Δ -infected mice (D) and determinant-specific IFN-γ + 8 T cells following infection with ECTV-WT (E) or ECTV-IFN-γbp (F) viruses. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis. For panels D, E and F, IFN-γ + 8 T cell numbers in WT mice were significantly higher (p<0.0001) than numbers in all GKO strains. Data shown for D-F are representative of one of three independent experiments with similar results. *, p< 0.05; **, p < 0.01; ***, p < 0.0001.
The vIFN-γbp had minimal impact on CTL activity in WT, IL-4 −/− and IL-4Rα −/− mice as these strains generated comparable responses to WT or mutant virus infection ( Fig. 4A-4C). The most dramatic effects of infection with ECTV-IFN-γbp Δ in the GKO mouse strains were evident in STAT-6 −/− and IL-13 −/− mice. These groups had significantly diminished CTL responses to WT virus infection ( Fig. 4A and 4C). However, mutant virus infection in both mouse strains induced robust CTL activities, which were almost as high as the magnitude in WT mice ( Fig. 4B and 4C). Thus, vIFN-γbp had a greater deleterious effect on CD8 T responses in STAT-6 −/− and IL-13 −/− mice compared with the other GKO strains.
ECTV-WT induced significantly higher (p<0.0001) numbers of virus-specific IFN-γ-producing CD8 T cells in WT mice compared to all GKO strains, in which numbers were reduced by 20–40-fold ( Fig. 4D). While ECTV-IFN-γbp Δ induced comparable numbers of IFN-γ-producing CD8 T cells as WT virus in WT mice, absence of vIFN-γbp only marginally increased numbers of IFN-γ + CD8 cells in each GKO strain ( Fig. 4D).
The MHC class I L d-restricted response generally dominates at day 7 p.i. in BALB/c mice. Numbers of L d-, K d- and D d-restricted determinant-specific IFN-γ + cells were significantly higher (p<0.0001) in WT mice infected with ECTV-WT compared to all GKO strains ( Fig. 4E). The reduction in virus-specific IFN-γ-producing CD8 T cell numbers seen in GKO strains ( Fig. 4D) was clearly reflected in the determinant-specific responses, which were nonetheless varied ( Fig. 4E). Curiously, there was a complete lack of reactivity to the three H-2d-restricted viral determinants in the STAT-6 −/− strain. Although we do not have an explanation for this finding, it nonetheless suggested the possibility that the response might have been directed against other H-2d determinants.
In WT mice, ECTV-IFN-γbp Δ infection generated determinant-specific IFN-γ + CD8 T cells ( Fig. 4F) that were comparable in numbers to ECTV-WT infection. Mutant virus infection increased numbers of determinant-specific IFN-γ + CD8 T cells in all GKO strains ( Fig. 4F), over and above those induced by ECTV-WT ( Fig. 4E). In particular, the biggest increases were seen in STAT-6 −/− mice and IL-13 −/− mice ( Fig. 4F). The mechanism(s) though which absence of vIFN-γbp at least partially reconstituted the determinant-specific IFN-γ + CD8 T cell responses in STAT-6 −/− mice or increased both the total ( Fig. 4D) and determinant-specific responses ( Fig. 4F) in the other GKO strains is currently unknown but merits investigation.
The preceding data established that deficiencies in specific Th2 cytokine signaling significantly reduced the numbers of IFN-γ + CD8 T cells and that vIFN-γbp partially modulated the response. It was therefore of interest to determine whether the combined deficiencies in Th2 cytokines and vIFN-γbp promoted Th1 responses in CD4 T cells.
Intriguingly, WT mice infected with ECTV-WT or ECTV-IFN-γbp Δ generated the highest numbers of IFN-γ + CD4 T cells whereas the numbers in all GKO strains were significantly (p<0.0001) reduced ( Fig. 5A). Of note, STAT-6 −/− mice infected with WT virus had the lowest number of IFN-γ producing CD4 T cells but it was the only strain in which mutant virus infection increased numbers significantly by nearly 90-fold. Similarly, in the case of IL-4 production, WT mice infected with WT or mutant virus generated the largest number of IL-4 + CD4 T cells but numbers were significantly lower (p<0.01) in all GKO strains of mice ( Fig. 5B). Data shown in Fig. 5A and 5B are using non-stimulated cells. CD4 T cells from IL-4 −/− mice did not produce IL-4 as expected but IL-4 + CD4 T cell numbers were also minimal in STAT-6 −/− and IL-13 −/− mice. In vitro stimulation of splenocytes with PMA + ionomycin increased the numbers of IFN-γ ( Fig. 5C) and IL-4 ( Fig. 5D) producing CD4 T cells in all groups but the response in WT cells was still much higher than in GKO cells.
Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp Δ, sacrificed on day 7 p.i. and splenocytes used for intracellular staining for IFN-γ, IL-4, T-bet or GATA-3 without stimulation (A and B) or with PMA + ionomycin stimulation (C and D). Shown are means of absolute numbers of IFN-γ +CD4 T cells with no stimulation (A) or PMA + ionomycin stimulation (C), IL-4 +CD4 T cells with no stimulation (B) or PMA + ionomycin stimulation (D). Also shown are means of absolute numbers of T-bet + and GATA-3 + CD4 T cells following ECTV-WT (E) or ECTV-IFN-γbp Δ (F) infection. Two-way ANOVA followed by Fisher’s LSD test for significance was used. For panel A, numbers of IFN-γ +CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001) compared to numbers in all GKO strains. In IL-4 −/− and STAT-6 −/− mice, IFN-γ +CD4 T cell numbers generated by ECTV-IFN-γbp Δ infection were significantly higher ( p = 0.0005 and p = 0.0013, respectively) compared to WT virus infection. In IL-13 −/− mice, IFN-γ +CD4 T cell numbers generated by WT virus infection were significantly higher ( p = 0.0034) compared to ECTV-IFN-γbp Δ infection. For panel B, numbers of IL-4 +CD4 T cells in WT mice for both viruses were significantly higher (p<0.01) compared to numbers in all GKO strains. No other significant differences were found. For panel C, numbers of IFN-γ +CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001 in ECTV-WT and p < 0.001 in ECTV-IFN-γbp Δ) compared to numbers in all GKO strains. In IL-4 −/−, STAT-6 −/− IL-4Rα −/− and IL-13 −/− / IL-4Rα −/− mice, IFN-γ +CD4 T cell numbers generated by ECTV-IFN-γbp Δ infection were significantly higher compared to WT virus infection. For panel D, numbers of IL-4 +CD4 T cells in WT mice for ECTV-WT virus was significantly higher (p<0.05) compared to numbers in all GKO strains. In WT mice, IL-4 +CD4 T cell numbers generated by WT virus infection were significantly higher ( p<0.05) compared to ECTV-IFN-γbp Δ infection. For panel E, T-bet + CD4 T cell numbers were significantly increased (p<0.01) in STAT-6 −/− and IL-13 −/− mice compared to WT animals. For panel F, no significant differences were found between strains. Data shown for panels A and B are from one of two independent experiments with similar results. Data shown for panels C-F are from one experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.0001.
We interrogated CD4 T cells by flow cytometry to detect expression of intracellular T-bet and GATA-3, the signature Th1 and Th2 transcription factors, respectively. In WT mice infected with ECTV-WT, CD4 T cells were predominantly GATA-3 + ( Fig. 5E). IL-4 deficiency further increased GATA-3 + CD4 T cell numbers whereas STAT-6 or IL-13 deficiency increased T-bet + cell numbers over and above those in WT mice. On the other hand, ECTV-IFN-γbp Δ infection did not significantly change T-bet + or GATA-3 + cell numbers in GKO strains compared to those in WT mice. However, in comparing numbers induced by ECTV-WT infection ( Fig. 5C), both T-bet + and GATA-3 + cell numbers were increased in all strains following infection with ECTV-IFN-γbp Δ ( Fig. 5D). Increases in T-bet + cell numbers did not necessarily translate to increased numbers of IFN-γ + CD4 T cells ( Fig. 5A and 5C) and likewise, increases in GATA-3 + cells did not result in increases in IL-4 + CD4 T cells ( Fig. 5, panels B and D). On the contrary, deficiency in specific Th2 cytokines, transcription factor or cytokine receptor resulted in reduced numbers of IFN-γ + and IL-4 + CD4 T cells. vIFN-γbp modulated the IFN-γ response only in STAT-6 −/− mice but that did not assist in the recovery of this strain from mutant virus infection. Deficiency in Th2 cytokine signaling did not bias CD4 T cells towards a Th1 response.
A type 1/Th1-like cytokine response coupled with robust cell-mediated immunity is critical for recovery from mousepox whereas a type 2/Th2-like response associated with an absent or weak IFN-γ response and cell-mediated immunity is associated with susceptibility [ 8]. Deficiency in IFN-γ [ 9, 10] or its suppression through over-expression of IL-4 by recombinant ECTV in resistant mice results in generation of poor cell-mediated immunity, overcomes resistance to mousepox and results in fulminant disease [ 11, 12]. While it may be speculated that type 1-like responses were important for recovery of humans from smallpox, there is circumstantial evidence that Th2-biased responses can significantly exacerbate the disease and result in increased mortality.
In non-vaccinated humans, about 90% of cases were of the ordinary-type smallpox with a case fatality rate of about 30–40% [ 35]. A more severe form of the disease, hemorrhagic-type smallpox, characterized by mucosal and skin hemorrhage, was mainly seen in pregnant women. Early hemorrhagic-type smallpox was associated with almost 100% mortality regardless of vaccination status and pregnant women with smallpox had 3–4 times higher case fatality rates than non-pregnant women and men of the same age groups [ 35, 36]. Although the reasons for higher case fatality rates were not known at the time when smallpox was endemic, it is now recognized that an immune bias towards a Th2-like response associated with pregnancy [ 37– 39] may have been responsible. That Th2-biased response can exacerbate smallpox even in vaccinated individuals is significant. In this regard, the demonstration that infection of previously vaccinated mice with recombinant ECTV encoding IL-4 can completely overcome protective immunity and genetic resistance [ 11, 12] helped establish that over exuberant Th2-biased responses increase susceptibility to OPV infections. This raises the question of whether defective or sub-optimal Th2 responses allow for the generation of more effective antiviral immunity. Our results indicate they do not.
Deficiencies in IL-4, STAT-6, IL-13 and/or IL-4Rα signaling on immune response generation were varied and further modulated by vIFN-γbp but 3 broad categories of outcomes were evident. In the first, the IL-4 −/− mice had comparable recovery rates to WT mice regardless of whether they were infected with WT or mutant virus. In the second, the IL-4Rα −/− and IL-13 −/−/IL-4Rα −/− mice exhibited increased susceptibility to WT virus infection but the absence of vIFN-γbp during infection mitigated the increased susceptibility. Finally, mice deficient in IL-13 or STAT-6 were significantly more susceptible than WT animals in response to WT or mutant virus infection. Susceptibility to ECTV is attributed to uncontrolled generalized virus replication in visceral organs, including in the liver [ 6, 7], spleen and blood [ 40]. However, in the current study, viral load in organs and blood 7 days after infection did not consistently correlate with increased susceptibility, with the exception of STAT-6 −/− mice. This outcome is surprising and might have been a consequence of the extent to which deficiencies in IL-4, IL-13 and/or IL-4Rα, combined with immunomodulation by vIFN-γbp impacted on inflammation, the immune response and virus control. Viral load in the liver and spleen generally increase exponentially over time if not effectively controlled by the immune response. However, the blood viral load is usually only increased following primary, secondary and subsequent occurrence of viremia following release of virions from the visceral organs. It is possible that kinetics of virus replication in the different GKO mouse strains was different and measurement of viral load at a single time-point might not have revealed the extent to which the viruses replicated and spread. However, the fact that most GKO mice succumbed to mousepox at or after 6 days post-infection suggests that measurement of viral titers at day 7 should have indicated if uncontrolled virus replication was the cause of death. Another finding of note is that deficiency in IL-13 or IL-4Rα resulted in comparable viral titers in liver, spleen and blood but titers were lower in the liver and blood in IL-13 −/−/IL-4Rα −/− mice. While the reason for this is unclear, it might be explained, at least in part by the CTL responses generated in the absence of IL-13 or both IL-13 and IL-4Rα (see below).
Absence of IL-4, STAT-6, IL-13 or IL-4Rα signaling clearly had a significant impact on both CD4 and CD8 T cell responses, which were modulated to some extent by vIFN-γbp. Contrary to our prediction, the numbers of IFN-γ + CD4 and CD8 T cells as well as IL-4 + CD4 T cells were decreased in all GKO strains compared to WT mice. Absence of vIFN-γbp during infection had minimal effects on CD4 T cell responses with the exception of STAT-6 −/− mice in which mutant virus infection significantly increased IFN-γ + CD4 T cell numbers. The most striking reductions in the anti-ECTV CTL responses were seen with WT virus infected STAT-6 −/− and IL-13 −/− mice, which were almost completely reversed when these strains were infected with ECTV-IFN-γbp Δ. Deficiency in the IL-4Rα alone did not have any impact on the CTL response but in the IL-13 −/− mice, the significant reduction in CTL activity was partially reversed when IL-4Rα was also absent. This is an interesting finding and although the mechanisms involved are not known, it suggests that in the absence of IL-13, IL-4 signaling through the IL-Rα was far more detrimental to induction of antiviral CTL responses than in the complete absence of IL-4 and IL-13 responses in IL-13 −/−/IL-4Rα −/− mice. We speculate that IL-13 might modulate the IL-4 response in induction of ECTV-specific CTL responses. Another important finding in the STAT-6 −/− mice is the complete lack of reactivity of CD8 T cells to any of the H-2d determinants that were tested. One explanation for the complete lack of reactivity to the three H-2 d-restricted viral determinants in the STAT-6 −/− strain is that the response might have been directed against other determinants. It is also possible that results could be influenced by subtle changes in reactivity of cells maturing in a STAT-6-deficient environment. Such changes in immune responses may be due to altered peptide expression that might be negligible or difficult to identify or quantify, but should nonetheless be considered for investigations in our future studies. It is curious why Th2-response linked genes influence the reactivity of CD8 T responses but may be related, at least in part, to potential defects in antigen presentation by dendritic cells as discussed below.
Although IFN-γ impacts on the quality and magnitude of the anti-ECTV CTL response [ 8], it was evident that the augmented CTL activity in IL-13 −/− and STAT-6 −/− mice was not related to the marginally increased numbers of virus-specific IFN-γ + CD8 cells, which occurred in all GKO strains. It is also possible that factors other than CD8 T cell-derived IFN-γ influenced the cytolytic activity of CTL in IL-13 −/− and STAT-6 −/− mice. As CD4 T cells are important for generation of optimal CTL responses to ECTV infection [ 41], we considered the possibility that IFN-γ-producing CD4 T cells may have impacted on the magnitude of CTL responses. While this might have been the case in STAT-6 −/− animals ( Fig. 5A) in which numbers of IFN-γ + CD4 cells substantially increased following infection with the mutant virus, it did not hold true for the IL-13 −/− mice. There is therefore little or no evidence for any consistent correlation between the numbers of IFN-γ + CD4 T cells and the magnitude of the CD8 T cell-mediated CTL responses in individual mouse strains.
STAT-6-dependent IL-4 production has been purported to be associated with susceptibility of BALB/c mice to ECTV infection [ 25] but IL-4 deficiency in BALB/c mice has no impact on disease outcome [ 8]. Since IL-4 −/− mice can respond to IL-13, we hypothesized that the cytokine might compensate for absence of IL-4 to drive Th2-like responses. Our data established that this was not the case and that the responses to, and outcome of infection with, ECTV in IL-4 −/−, IL-13 −/− and STAT-6 −/−mice are very different. First, IL-13 −/− and STAT-6 −/− mice were far more susceptible to WT or mutant virus infection than IL-4 −/− mice. Second, the CTL response to WT virus was marginally reduced in IL-4 −/− mice compared to WT animals, but the effect was far more significant in IL-13 −/− and STAT-6 −/− mice. This is an important and novel finding in that factors normally associated with Th2 responses play critical and non-redundant roles in the generation of optimal antiviral CD8 CTL responses. The mechanism(s) through which IL-13 or STAT-6 impact on CTL responses generation during ECTV infection is currently unknown but merit a detailed investigation.
Naïve CD8 + T cells in IL-4 −/−, IL-13 −/− and STAT6 −/− mice express lower levels of IL-4Rα making them less responsive to IL-4 and IL-13 during T cell priming [ 42]. It is therefore possible that in IL-13 −/− mice, IL-4 signaling alone through activation of STAT-6 may promote sufficient down-regulation of CD8 co-receptor densities on naïve CD8 + T cells following cognate antigen encounter during ECTV infection, and lessen the anti-viral capacity of effector CD8 + T cells by reducing avidity and polyfunctionality [ 43– 45]. In relation to the role of STAT-6 in CTL responses, it has been reported that the activation and function of CTL may be impaired in STAT-6 −/− mice during infection with the intracellular parasite Toxoplasma gondii, likely as a consequence of reduced levels of CD86 expression on DC [ 46]. The demonstration that activation of CD8 T cells in STAT-6 −/− mice was restored after transfer of splenic adherent cells or bone-marrow derived DC from WT mice whereas transfer of naïve WT CD8 T cells into STAT-6 −/− mice were not activated suggested a defect in antigen presentation [ 46]. It is known that in vitro, IL-13, like IL-4, in combination with GM-CSF can induce generation of functional DC [ 47], suggesting that IL-13 may be important in differentiation of DC via STAT-6 signaling. We speculate that IL-4R/IL-13R-associated STAT-6 signaling might be important in CD8 T cell activation and function during ECTV infection, possibly through maturation of antigen presenting cells, such as dendritic cells (DC). Apart from promoting the expression of several cell surface molecules responsible for antigen presentation; including MHC II, CD80, and CD86, IL-4/STAT-6 signaling regulates T-cell proliferation by decreasing the expression of p27 Kip1, a known cyclin-dependent kinase inhibitor. In CD8 cells, STAT6 is required for Tc2 differentiation as the production of IL-4 and IL-5 is completely lost with STAT6 deficiency (reviewed in [ 48]). Taken together, STAT6 is required for IL-4-stimulated T-cell functions.
That STAT-6 deficiency further exacerbates susceptibility of BALB/c mice to ECTV infection is in stark contrast to a previous report in which STAT-6 −/− mice were found to be more resistant than WT BALB/c mice due to augmented NK cell and IFN-γ responses [ 25]. In the current study, apart from a 2–3-fold increase in the cytolytic activity of NK cells in STAT-6 −/− mice, all other responses were either far worse or no different to WT mice. We believe that there may be a simple explanation for this discrepancy. In the present study we used STAT-6 −/− mice that were backcrossed to the BALB/c background for at least 10 generations [ 29]. In the previous study, STAT-6 −/− mice generated on a 129 background [ 49] and backcrossed to the BALB/c background for only 6 generations were used. Given that several of the 129 sub-strains are resistant to mousepox [ 10], we believe that the 129/Sv background genes may have influenced the outcome of infection in that study. It is likely that STAT-6 deficiency in ECTV-resistant C57BL/6 or 129 mouse strains might further augment Th1 responses but this is not the case in the ECTV-susceptible BALB/c mice. Consistent with this proposition, we have found that C57BL/6 mice deficient in IL-4 generate more robust CTL and IFN-γ responses which correlated with more efficient virus control compared to WT littermate controls (data not shown). Furthermore, when the STAT-6 −/− mice used in the previous study [ 25, 49] were backcrossed to the BALB/c background 10–12 times, they were found to be more susceptible to ECTV-WT infection than WT littermates (data not shown). Clearly, STAT-6 deficiency does not increase the resistance of BALB/c mice to ECTV infection. On the contrary, STAT-6 deficiency further increased susceptibility of the BALB/c strain to ECTV.
Our finding that deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells indicates an absence of a switch to a Th1-like response and might be unique to this viral model. For example, Th1/Th2 cytokine production by IL-13 −/− cells is varied depending on in vitro or in vivo stimulation conditions and the type of cells involved [ 27]. IL-13 deficient splenocytes or CD4 T cells stimulated with mitogens produce reduced levels of IL-4 but similar levels of IFN-γ compared to WT cells. Under Th1/Th2 culture conditions, IL-13 −/− CD4 T cells produce dramatically reduced levels of IL-4 but only a 2-fold higher amount of IFN-γ. In contrast, mast cells from IL-13 −/− and WT mice stimulated with PMA and ionomycin produce comparable levels of IL-4. In vivo, challenge of IL-13 −/− mice with schistosome eggs reduced IL-4 levels only by about 3-fold whereas challenge with the parasite N. brasiliensis had no impact on Th2 response generation or IL-4 production by lymph node cells or purified CD4 T cells.
It is conceivable that regulation of Th2 response generation in vivo during infection with a virus is far more complex than how it is understood to occur in in vitro culture conditions. Various stimuli, cell types and signalling pathways are involved in orchestrating Th2 responses of which there are also variants [ 19, 50– 52]. Indeed, Th2 differentiation has been shown to occur in mice that are deficient in IL-4, IL-13, IL-4Rα or STAT-6 and cytokines like thymic stromal lymphopoietin, IL-25 and IL-33 can induce Th2 responses [ 19, 27, 51, 52]. There is consensus, however, that GATA3 is required for this process, even if expressed at low levels [ 53– 55]. In the current study, we found that numbers of GATA3 + CD4 T cells were highest in both WT and IL-4 −/− mice infected with WT virus but numbers were reduced in the GKO strains. These strains also had the highest number of T-bet + CD4 T cells. T-bet can negatively regulate GATA3 [ 56] and in STAT-6 −/− mice, T-bet + CD4 T cell numbers were increased with a corresponding decrease in GATA-3 + CD4 T cells. These changes, however, did not translate to an increase in IFN-γ + CD4 T cells. Overall, there was little correlation between GATA-3 and IL-4 expression or T-bet and IFN-γ expression in CD4 T cells regardless of the type of infecting virus. We believe that the use of transgenic CD4 and CD8 T cells, specific for a model antigen, and deficient in IL-4, IL-13, IL-4Rα or STAT-6 might help better understand the molecular mechanisms involved in driving Th2 responses in BALB/c mice infected with ECTV. The adoptive transfer of such transgenic cells into recipient mice combined with the use of recombinant ECTV encoding the model antigen for infection is an approach that we propose to undertake.
We have established that deficiency in specific factors associated with Th2 response development and/or the absence of vIFN-γ-bp during infection of BALB/c mice with ECTV does not bias development of a Th1 response. IL-4, STAT-6 and IL-13, which are normally associated with development of Th2 responses, play important and non-redundant roles in the generation of optimal antiviral CTL responses. Our data also established that the absence of IL-13 and STAT-6 further increased the susceptibility of BALB/c mice to ECTV infection. We conclude that host resistance or susceptibility to mousepox is far more complex and goes beyond the presence or absence of select genetic factors that control Th differentiation.
Data are from one of the three separate experiments showing intracellular IFN-γ expression (Y-axis) by splenic 8 T cells (X-axis) from WT and GKO mice. The numbers in the upper right quadrants in individual panels indicate percentages of IFN-γ-producing 8 T cells after stimulation with (from left) the irrelevant control (HSV-1 gB) peptide (first column), whole virus (ECTV) (second column), L d-EVM026 (third column), K d-EVMA52 (forth column) or D d-EVM043 peptides (fifth column). Absolute numbers of IFN-γ + 8 T were obtained by multiplying the percentage of cells with the total number of splenocytes from each mouse for each strain.
(TIF)
Click here for additional data file.
Data are from one of the three separate experiments showing intracellular IFN-γ expression (Y-axis) by splenic 8 T cells (X-axis) from WT and GKO mice. The numbers in the upper right quadrants in individual panels indicate percentages of IFN-γ-producing 8 T cells after stimulation with (from left) the irrelevant control (HSV-1 gB) peptide (first column), whole virus (ECTV) (second column), L d-EVM026 (third column), K d-EVMA52 (fourth column) or D d-EVM043 peptides (fifth column). Absolute numbers of IFN-γ + 8 T were obtained by multiplying the percentage of cells with the total number of splenocytes from each mouse for each strain.
(TIF)
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Data are from one of three separate experiments showing intracellular IFN-γ expression (Y-axis) in unstimulated splenic CD4 T cells (X-axis) from naïve (first column), ECTV-WT- (second column) or ECTV-IFN-γbp Δ- (third column) infected WT and GKO mice. The numbers in the upper right quadrants in individual panels indicate percentages of IFN-γ-producing CD4 T cells.
(TIF)
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Data are from one of three separate experiments showing intracellular IL-4 expression (Y-axis) in unstimulated splenic CD4 T cells (X-axis) from naïve (first column), ECTV-WT- (second column) or ECTV-IFN-γbp Δ- (right column) infected WT and GKO mice. The numbers in the upper right quadrants in individual panels indicate percentages of IL-4-producing CD4 T cells.
(TIF)
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Data in this figure is the same as in Fig. 1 but presented to compare survival curves of each GKO strain with wild type BALB/c mice. P values were obtained by using Kaplan-Meier Log rank statistical test: *, p < 0.05.
(TIF)
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Data in this figure is the same as in Fig. 1 but presented to compare survival curves of each GKO strain with wild type BALB/c mice. P values were obtained by using Kaplan-Meier Log rank statistical test: *, p < 0.05.
(TIF)
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a EVM represents nomenclature for ECTV-specific 8 T cell determinants.
(DOCX)
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a ECTV-WT vs. ECTV-IFN-γbp Δ P value using Logrank (Mantel-Cox) test. For extremely significant (****) P < 0.0001; extremely significant (***) 0.0001< P <0.001; very significant (**) 0.001< P <0.01; significant (*) 0.01< P <0.05; not significant (ns) P ≥ 0.05. b Number in brackets is the median survival time in days. c Number of animals in group. d BALB/c.WT vs. GKO strain P value using Logrank (Mantel-Cox) test. e undefined
(DOCX)
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a To evaluate significant differences between groups, viral titers were log transformed and 2-way ANOVA performed followed by Fisher’s LSD test. For extremely significant (****) P < 0.0001; extremely significant (***) 0.0001< P <0.001; very significant (**) 0.001< P <0.01; significant (*) 0.01< P <0.05; not significant (ns) P ≥ 0.05. b ECTV-WT vs. ECTV-IFN-γbp Δ. c BALB/c.WT vs. GKO strain.
(DOCX)
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a To evaluate significant differences between groups, viral titers were log transformed and 2-way ANOVA performed followed by Fisher’s LSD test. For extremely significant (****) P < 0.0001; extremely significant (***) 0.0001< P <0.001; very significant (**) 0.001< P <0.01; significant (*) 0.01< P <0.05; not significant (ns) P ≥ 0.05. b ECTV-WT vs. ECTV-IFN-γbp Δ. c BALB/c.WT vs. GKO.
(DOCX)
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a To evaluate significant differences between groups, viral titres were log transformed and 2-way ANOVA performed followed by Fisher’s LSD test. For extremely significant (****) P < 0.0001; extremely significant (***) 0.0001< P <0.001; very significant (**) 0.001< P <0.01; significant (*) 0.01< P <0.05; not significant (ns) P ≥ 0.05. b ECTV-WT vs. ECTV-IFN-γbp Δ. c BALB/c.WT vs. GKO strain.
(DOCX)
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We thank the Australian National University Animal Breeding and Research Facility and the John Curtin School of Medical Research Microscopy and Flow Cytometry Research Facility.


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The 2nd International Conference on Health Sciences
Eastparc Hotel, Yogyakarta. 11–12 July 2017The 2nd International Conference on South East Asia Studies
Eastparc Hotel, Yogyakarta. 27–28 September 2017The 2nd International Conference on Tropical Agriculture
Eastparc Hotel, Yogyakarta. 26–27 October 2017