This work was funded by the Bill and Melinda Gates Foundation (Grand Challenge Exploration award to SBG), the National Institutes for Health Research (NIHR), Biomedical Research Centre in Microbial Diseases, and the NIHR Comprehensive Local Research Network. We acknowledge the NWDA for infrastructural support. We thank Prof. J. N Weiser, University of Pennsylvania and Prof. P Hermans, University of Nijmegen for the pneumococcal isolates. Thanks to the EHPC Safety Committee for their support and advice, to all our volunteers for their participation and to Mathieu Bangert for taking the photos.

1. Volunteer Selection and Screening
<ol>
	<li>Healthy volunteers are recruited through poster advertisements and on the University website according to the NHS Research and Ethics Committee guidance.</li>
	<li>The inclusion criteria for participation are:
	<ol>
		<li>Adults aged 18-60 years.</li>
		<li>Speaks fluent English and is able to communicate easily by both mobile telephone and text messaging.</li>
	</ol>
	</li>
	<li>The exclusion criteria for participation are:
	<ol>
		<li>Close contact with at risk individuals (children, immunosuppressed adults, elderly, chronic ill health).</li>
		<li>Current smoker or significant smoking history (&gt;10 pack years).</li>
		<li>Asthma or other respiratory disease.</li>
		<li>Pregnancy.</li>
		<li>Allergy to penicillin or amoxicillin.</li>
		<li>Current involvement in another clinical trial unless observational or in non-interventional phase.</li>
		<li>Unable to give fully informed consent.</li>
	</ol>
	</li>
	<li>An initial screening visit, approximately one week before inoculation, includes a focused clinical history and targeted clinical examination.</li>
	<li>If a previously unrecognized abnormality is found, the volunteer will be excluded from the study and appropriate investigation will be arranged through primary care.</li>
	<li>During the initial screening visit a full blood count is obtained to ensure that the white cell count is within normal range before the inoculation visit. A nasal wash is performed to exclude natural pneumococcal carriers (see 6.1).</li>
	<li>Prior to inoculation, volunteers are educated on the risks involved with participation in the study and are provided with an emergency patient leaflet, digital thermometer, emergency telephone numbers and 3 day course of amoxicillin.</li>
</ol>
2. Preparation of Inoculation Stocks
<ol>
	<li>Preparation of the inoculation stocks must be done in a clean environment. The pipettes used should only be used for inoculum preparation. All glass and plastic ware should be sterile. We recommend that inoculation stocks be prepared in a dedicated room, using a dedicated fumehood and incubator.</li>
	<li>Pneumococcal strains are inoculated onto a blood agar plate. Plates are incubated at 37 &deg;C in 5% CO2 overnight. The following day all the bacteria is scraped from the blood agar plate and inoculated into a bead stock preservation system. These bead stocks are used for inoculation stock preparation. Test the bead stocks for contamination and colony uniformity before preparing an inoculation stock.</li>
	<li>To prepare the inoculation stock, inoculate a blood agar plate with two beads from the desired pneumococcal serotype bead stock, streaking to cover the entire plate. Incubate plates at 37 &deg;C in 5% CO2 overnight. All further incubations will use these same conditions. Also incubate the Vegitone media overnight to ensure sterility.</li>
	<li>The next morning swab half the plate, taking care not to remove any blood agar, and add to 12 ml of pre-warmed Vegitone broth. Repeat using the other half of the plate. Leave the two 12 ml vials at 37 &deg;C for 2 hr or until a change in turbidity becomes apparent, whichever comes first.</li>
	<li>Add the 12 ml to 40 ml of pre-warmed Vegitone broth and mix thoroughly. Repeat for the other 12 ml vial. This is time point = 0. Remove 100 &mu;l to read the optical density (OD) at 600 nm. Remove 20 &mu;l for quantification of bacteria by colony forming unit (CFU) count using a variation of the method by Miles and Misra (M&amp;M)7. Incubate both vials.
	<ol>
		<li>For the M&amp;M, divide a blood agar plate into six sections. Add 180 &mu;l of sterile PBS to six wells of a 96 well plate (U-bottom). Add 20 &mu;l of the broth to the top well and mix. Serially dilute the sample 1:10 six times and discard 20 &mu;l from the sixth well. Place three 10 &mu;l drops from the top well into the first section of the blood agar plate. Repeat for the five other wells. Ensure the plate is dry before it is inverted and incubated.</li>
		<li>The next day count the number of visible colonies in each dilution section and record. Using the dilution section with a count between 30 and 300, divide the number of visible colonies by three to get an average.</li>
		<li>Multiply the average number of colonies by the dilution factor and then divide by 10 (the amount of the 10 &mu;l drop). This gives CFU/&mu;l.</li>
		<li>Multiply this number by 1,000 to get CFU/ml.</li>
	</ol>
	</li>
	<li>Perform OD 600 readings and quantification of bacteria by M&amp;M method every hour until early mid-log phase is reached, an OD of 0.25.</li>
	<li>Once this OD is reached, add sterilized 10% glycerol to one vial and prepare 1 ml aliquots. Store aliquots at -80 &deg;C.</li>
	<li>For a more concentrated stock, centrifuge the other vial at 3,345 x g for 15 min and remove the supernatant. Resuspend the pellet in 22.5 ml of Vegitone medium and add 10% glycerol. Prepare 1 ml aliquots and store at -80 &deg;C.</li>
	<li>Leave stocks at -80 &deg;C for at least 48 hr before thawing and quantifying.</li>
	<li>Quantify the frozen bacterial stock by the M&amp;M method. Test three stock tubes individually to ensure reproducibility. Use the value obtained to dilute the stock to the desired inoculum concentration in saline, as described below.</li>
	<li>For an inoculation, the quantity of pneumococci in the inoculum is calculated before and after the inoculation. This quantification of bacteria by the M&amp;M method is used to determine the average number of pneumococci in the inoculum which accounts for the time it takes to complete the entire procedure.
	<ol>
		<li>Prepare an inoculum, diluting to the desired concentration, and perform quantification by the M&amp;M method for the "pre-inoculation" count.</li>
		<li>In our Clinical Research Facility it takes the team 30 min to complete the entire inoculation procedure. Pneumococcal suspensions in saline will usually show a decreased count in this interval. To account for this, we let the diluted inoculum sit at room temperature for 30 min and then perform a "post-inoculation" quantification of the bacteria by the M&amp;M method.</li>
		<li>Take the average of the before and after to determine the final inoculated concentration. Adjust the "sitting" time according to your clinical protocol but keep the interval as brief as possible.</li>
	</ol>
	</li>
	<li>All new stocks should be sent to a reference laboratory for confirmation of stock purity and identity.</li>
</ol>
3. Inoculum Preparation
<ol>
	<li>Preparation of the inoculum should begin 30 min before the scheduled volunteer inoculation appointment.</li>
	<li>Take one of the previously made stock tubes out of the -80 &deg;C freezer, thaw it and spin the tube in the microcentrifuge at 17,000 x g for 3 min.</li>
	<li>Warm blood agar plates in the 37 &deg;C incubator. Prepare a 96 well plate for bacterial quantification by M&amp;M method and prepare dilution tubes according to the desired dose.</li>
	<li>Remove the broth supernatant from the centrifuged tube and resuspend the pellet in 1 ml of saline. This step removes the broth from the inoculum.</li>
	<li>Repeat the centrifugation step.</li>
	<li>Remove the saline supernatant and resuspend the pellet in 1 ml of saline.</li>
	<li>Perform dilution set for desired inoculum concentration and quantify by M&amp;M method.</li>
	<li>Take the inoculum to the volunteer appointment and after inoculating the volunteers, repeat the quantification at the lab. Label the tube with the date and inoculum dose and store at -80 &deg;C.</li>
	<li>To ensure consistency from inoculation to inoculation, it is important to use the same pipettes and dedicated laboratory equipment every time.</li>
</ol>
4. Inoculation
<ol>
	<li>Volunteers should be seated comfortably in a semi-recumbent position.</li>
	<li>Using a P200 pipette and sterile filter tips draw up 100 &mu;l of the inoculum and insert the tip just inside the nasal cavity.</li>
	<li>Slowly expel the inoculum onto the nasal mucosa using a circular motion.</li>
	<li>It is crucial that the pipette tip does not come in contact with the nasal mucosa as a disruption in the integrity of the epithelium could result in the bacteria entering the bloodstream.</li>
	<li>It is also vital that the inoculum is not placed too far back or it will run down the throat; it should reside inside the nasal cavity.</li>
	<li>Repeat for the other naris.</li>
	<li>Have the volunteer remain in the semi-recumbent position for 10 min without sniffing or blowing the nose. This allows time for the bacteria to disperse across the mucosa.</li>
</ol>
5. Monitoring
<ol>
	<li>Volunteers are monitored daily following inoculation. This includes a daily text message sent by the volunteer to investigators before 2 pm.</li>
	<li>If the text is not received by 2 pm the volunteer will be contacted to ensure his/her well-being. If he/she does not respond, the allocated next of kin will be contacted to ensure the volunteer's well-being.</li>
	<li>Volunteers are asked to report on any upper respiratory tract symptoms at every appointment. Clinical staff will ask the volunteer to describe his/her symptoms and will follow up any complaints.</li>
	<li>The antibiotics given to the volunteers are only to be taken under three circumstances; in the event that they are unwell and are instructed to take them by the research team, if they are carrying pneumococcus at the end of the study, or if they are unwell and unable to contact the research team. The volunteer is encouraged to keep this emergency pack with them at all times during the study and is requested to contact the research team on a daily basis for the next 7 days.</li>
	<li>Our team is available 24/7 with nurse contact during working hours and two doctors available out of hours. All queries are dealt with by telephone call and/or immediate review. Provisions are available for immediate, direct admission to an Infectious Disease ward, if needed.</li>
</ol>
6. Nasal Wash (NW) Procedure
<ol>
	<li>Nasal washes (NW) are performed at an initial screening visit, as well as 48 hr, 7, and 14 days post- inoculation. The NW method used is taken from Naclerio et al.8 Volunteers naturally colonized by S. pneumoniae are excluded from inoculation and follow up.</li>
	<li>The volunteer is seated comfortably and the head is tilted back 30&deg; from the vertical.</li>
	<li>Ask the volunteer to take a deep breath in and hold their breath whilst pushing their tongue up and backwards against the roof of the mouth. Whilst in this position, the volunteer is asked to signal that they are ready.</li>
	<li>A syringe filled with 20 ml saline is inserted into the anterior nasal space and 5 ml of saline is expelled. The volunteer then leans forward immediately and expels the fluid by exhaling rapidly through their nose into a foil bowl.</li>
	<li>Repeat this procedure 3 more times so that each naris has been washed twice and the full 20 ml has been used.</li>
	<li>Pool all samples together in a centrifuge tube and send to the laboratory at room temperature for processing.</li>
</ol>
7. Nasal Wash Processing
<ol>
	<li>Centrifuge the samples for 10 min at 3,345 x g.</li>
	<li>Remove the supernatant and store as 1 ml aliquots in labelled Eppendorf tubes at -80 &deg;C.</li>
	<li>Add 100 &mu;l of STGG medium9 to the pellet and mix thoroughly. Ensure that the total volume in the tube at this point is determined. This dilution will be used in calculating the CFU of a carriage positive NW.</li>
	<li>Plate a 20 &mu;l drop of the STGG containing the resuspended pellet onto a blood agar plate containing gentamicin and streak the entire plate.
	<ol>
		<li>If the NW is post-inoculation, remove 10 &mu;l from the STGG containing the resuspended pellet and use for bacterial quantification by M&amp;M method.</li>
	</ol>
	</li>
	<li>Add another 800 &mu;l of STGG to the NW tube and mix thoroughly.</li>
	<li>Plate 25 &mu;l onto a blood agar plate and 25 &mu;l onto a chocolate agar plate, streaking the entire plate.</li>
	<li>Divide the remaining amount bacteria suspended in STGG medium into 2 cryovials and store at -80 &deg;C.</li>
	<li>Incubate the plates at 37 &deg;C overnight in 5% CO2.</li>
	<li>Examine the plates the next day for pneumococcus and other potential respiratory pathogens.</li>
	<li>Pneumococci are identified on plates by colony morphology. Alpha haemolytic colonies with draughtsman morphology are sub-cultured onto blood agar with an optochin disc and incubated overnight.</li>
	<li>Presumptive pneumococcal colonies that are optochin sensitive are Gram stained to confirm Gram positive diplococci and serotyped using the Statens Serum Institut Pneumotest-Latex kit.</li>
</ol>

<ol>
	<li>Ferreira, D. M., Jambo, K. C., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+human+pneumococcal+carriage+models+for+vaccine+research.">Experimental human pneumococcal carriage models for vaccine research.</a> Trends in Microbiology. 19, 464-470 (2011).</li><li>Mera, R., Miller, L. A., Fritsche, T. R., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+and+multiple+resistance+in+Streptococcus+pneumoniae+after+the+introduction+of+the+conjugate+pneumococcal+vaccine.">Serotype replacement and multiple resistance in Streptococcus pneumoniae after the introduction of the conjugate pneumococcal vaccine.</a> Microbial Drug Resistance. 14, 101-107 (2008).</li><li>Tocheva, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Declining+serotype+coverage+of+new+pneumococcal+conjugate+vaccines+relating+to+the+carriage+of+Streptococcus+pneumoniae+in+young+children.">Declining serotype coverage of new pneumococcal conjugate vaccines relating to the carriage of Streptococcus pneumoniae in young children.</a> Vaccine. 29, 4400-4404 (2011).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378 (10), 1962-1973 (2011).</li><li>Wu, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Wright, A. K. A., Ferreira, D. M., Gritzfeld, J. F., Wright, A. D., Armitage, K., Jambo, K. C., Bate, E., Batrawy, S. E. l., Collins, A., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Nasal+Challenge+with+Streptococcus+pneumoniae+is+Immunising+in+the+Absence+of+Carriage.">Human Nasal Challenge with Streptococcus pneumoniae is Immunising in the Absence of Carriage.</a> PloS Pathogens. 8 (4), e1002622 (2012).</li><li>Miles, A. A., Misra, S. S., Irwin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimation+of+the+bactericidal+power+of+the+blood.">The estimation of the bactericidal power of the blood.</a> The Journal of Hygiene. 38, 732-749 (1938).</li><li>Naclerio, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediator+release+after+nasal+airway+challenge+with+allergen.">Mediator release after nasal airway challenge with allergen.</a> Am. Rev. Respir. Dis. 128, 597-602 (1983).</li><li>O'Brien, K. L., Nohynek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+from+a+WHO+working+group:+standard+method+for+detecting+upper+respiratory+carriage+of+Streptococcus+pneumoniae.">Report from a WHO working group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae.</a> Pediatr. Infect. Dis J. 22, 133-140 (2003).</li><li>Gritzfeld, J. F., Roberts, P., Roche, L., Batrawy, S. E. l., Gordon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+nasopharyngeal+swab+and+nasal+wash,+using+culture+and+PCR,+in+the+detection+of+potential+respiratory+pathogens.">Comparison between nasopharyngeal swab and nasal wash, using culture and PCR, in the detection of potential respiratory pathogens.</a> BMC Research Notes. 4, 122 (2011).</li><li>McCool, T. L., Cate, T. R., Moy, G., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+to+pneumococcal+proteins+during+experimental+human+carriage.">The immune response to pneumococcal proteins during experimental human carriage.</a> J. Exp. Med. 195, 359-365 (2002).</li></ol>

No conflicts of interest declared.

The EHPC platform has a number of potential uses including use as a mucosal vaccine model and as a surrogate of protection for testing novel protein vaccines. The method is dependent upon a consistent inoculation technique and a high quality NW.
Reproducibility can be an issue with the inoculum. Owing to the variable nature of the frozen bacterial aliquots, it can be difficult to replicate the desired dose. A halving or doubling of the desired dose is considered within range. It is crucial that the bacterial quantification be done immediately prior to- and following inoculation, in order to accurately determine quantification of bacteria by CFU count. For this reason it may be helpful if volunteer appointments occur simultaneously so that only one inoculum need be prepared and the quantification plating done prior to and after inoculation happens no more than 30 min apart.
The NW method requires the cooperation of the volunteer and would not be an ideal method in children. If the yield is less than 5 ml, the procedure can be repeated using up to an extra 20 ml. Wearing dentures may reduce the NW yield. If the volunteer has a blocked/congested nose and the saline is running out anteriorly after insertion, blow the nose, insert the syringe a little further back, and tilt the head back more if needed.
The NW is a technique that gets better with practice. Some volume will usually be lost so the aim is for a return of at least 10 ml. If the volunteer can taste saline during the NW then the connection between the posterior nasopharynx and oropharynx has not been adequately closed by the volunteer's tongue. If the volunteer swallows most of the saline, explain the procedure again, emphasizing the pushing of the tongue against the roof of the mouth. Then repeat the procedure to increase the volume returned.
If the NW contains a substantial amount of mucus, it is best to remove it before centrifugation. Vortex the sample so as to loosen anything that might be bound to the mucus and then remove as much of the large pieces as possible while removing as little saline as possible.
A previous EHPC study in the U.S.A. was completed safely and had no adverse events11. To ensure the continued safety of the EHPC platform, we have set up an emergency protocol which is based around 24 hr access to the research team. Volunteers are given contact details, allowing them access to the research team 24 hr a day. Any medical care needed is provided in the Royal Liverpool University Hospital. As a further measure of protection, a safety committee consisting of clinicians and scientists from outside the research group receives a weekly safety report. The report contains the bacterial dose each volunteer received and whether any symptoms or illness were reported. This report allows the safety of the study to be externally critiqued without bias. The safety committee are also available 24 hr a day in the unlikely event of an emergency.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Cat#</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Fisher</td>
 <td>G/0650/08 </td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride</td>
 <td>B. Braun</td>
 <td>3627667</td>
 </tr>
 <tr>
 <td>Skim milk powder</td>
 <td>Fluka</td>
 <td>70166</td>
 </tr>
 <tr>
 <td>Tryptone soya broth</td>
 <td>Becton Dickinson</td>
 <td>211768</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>BDH</td>
 <td>284504S</td>
 </tr>
 <tr>
 <td>Columbia Agar with chocolated horse blood</td>
 <td>Oxoid</td>
 <td>PB0124</td>
 </tr>
 <tr>
 <td> Pneumotest-latex kit</td>
 <td>Statens Serum Institut</td>
 <td>51823</td>
 </tr>
 </tbody>
</table>

experimental human pneumococcal carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

We have experience inoculating 159 people, of which 35 have carried pneumococcus. The lowest dose we achieved carriage with using serotype 6B was 11,100 CFU/100 &mu;l per naris, the highest dose was 313,000 CFU/100 &mu;l per naris. The lowest dose we achieved carriage with using serotype 23F was 9,000 CFU/100 &mu;l per naris, the highest dose was 84,500 CFU/100 &mu;l per naris.
Our method of carriage assessment is a nasal wash, chosen because a recent study10 showed that 91% of volunteers found nasal wash to be more comfortable than nasopharyngeal swab; the nasal wash was also more likely to detect pathogens using microbiological culture. Microbial flora recovered on blood agar plates inoculated with NW can be variable and difficult to read. Gentamicin is used on the first blood agar plate to select for pneumococcus. The chocolate agar plate and the second blood agar plate are used to detect other potential respiratory pathogens which may help or hinder pneumococcal colonization. An example of a blood agar plate inoculated with NW that is easy to read is shown in Figure 1a; a difficult plate containing numerous kinds of flora is shown in Figure 1b.
At every appointment volunteers were asked to describe any upper respiratory tract symptoms, if present, and comparisons between carriers and non-carriers were highlighted. Of the 145 people recently inoculated with pneumococcus, 28 complained of symptoms (Table 1). Eight of these were carriers, twenty were not. The most common symptoms were non-specific nasal symptoms (Table 1). Of the cases in which there were reported systemic symptoms, including fever and/or malaise, none of the investigations undertaken showed evidence of pneumococcal disease and symptoms were consistent with concurrent viral infection except in one case of tonsillitis. Two pneumococcal positive volunteers were treated with antibiotics. One complained of sore throat and ear ache and, after meeting with the study doctor, was advised to take antibiotics as a precaution. The other volunteer was diagnosed clinically with tonsillitis. All volunteers had rapid resolution of symptoms.
<img alt="Figure 1" src="/files/ftp_upload/50115/50115fig1.jpg" /> 
 Figure 1. Blood agar plate inoculated with NW. Blood agar plates inoculated with NW can be difficult to read. 1a is an example of a plate that is easy to read with pneumococci clearly visible. 1b is a plate with lots of co-colonizing flora making it more difficult to detect if pneumococci is present.
<table border="1">
	<tbody>
		<tr>
			<td>Symptoms</td>
			<td>&nbsp;</td>
			<td>Carriage (n=32)</td>
			<td>No Carriage (n=112)</td>
		</tr>
		<tr>
			<td>All symptoms</td>
			<td>&nbsp;</td>
			<td>24%</td>
			<td>18%</td>
		</tr>
		<tr>
			<td>Systemic</td>
			<td>Fever</td>
			<td>3%</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Malaise*</td>
			<td>9%</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>Local</td>
			<td>Ears</td>
			<td>9%</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Nose</td>
			<td>0</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Throat</td>
			<td>12%</td>
			<td>3%</td>
		</tr>
	</tbody>
</table>
Table 1. Characterization of volunteer reported symptoms following S. pneumoniae 6B inoculation. All complaints were reviewed by the external safety committee and, following full investigations, no symptoms were attributed to pneumococcal disease. The most common symptoms were sore throat, stuffed nose and flu-like illness.

Watch this Scientific Journal Video about Experimental Human Pneumococcal Carriage at JoVE.com

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

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15.3K Views.  Liverpool School of Tropical Medicine. Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Video: Experimental Human Pneumococcal Carriage

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.