FG is supported by Associazione Italiana per la Ricerca sul Cancro (IG 2016Id.18842), Cariplo Foundation (Grant 2014-0655), and Fondazione Regionale per la Ricerca Biomedica (FRRB). 
IZ is supported by NIH grant 1R01AI121066-01A1, 1R01DK115217, HDDC P30 DK034854 grant, Harvard Medical School Milton Found, CCFA Senior Research Awards (412708), the Eleanor and Miles Shore 50th Anniversary Fellowship Program, and the Cariplo Foundation (2014-0859).

All the procedures were approved under the Institutional Animal Care and Use Committee (IACUC) and operated under the supervision of the department of Animal Resources at Children&#39;s Hospital (ARCH) at Boston Children&#39;s Hospital or were approved by the Italian Ministry of Health and performed under the Institutional Animal Care Committee at the University of Milano-Bicocca.
1. C. albicans Preparation
<ol>
	<li>Culture C. albicans, strain CAF3-113, in tubes containing rich medium (yeast extract peptone dextrose (YEPD), 1% (w/v) yeast extract, 2% (w/v) of Bacto Peptone, 2% (w/v) glucose) supplemented with uridine (50 mg/L) at 25 &#176;C. Under these conditions, C. albicans grows as a yeast. 
	NOTE: Other C. albicans strains can be used, such as the clinical isolate C. albicans strain SC531413. Since we have recently demonstrated that the ulcerative process is induced by the activation of the innate immune system14, theoretically every C. albicans strain that maintains both its cell wall components and structure, as well as the ability to originate hyphal projections, could be used.</li>
	<li>Monitor the culture by counting the cellular concentration using a cell counter analyzer.</li>
	<li>Harvest the culture when it reaches a concentration of about 8 x 106 cells/mL. Alternatively, use a spectrophotometer to measure the OD600. 
	NOTE: Usually, a value of OD600 = 1 corresponds to a yeast concentration of 3 x 107 cells/mL, but titration is recommended.</li>
	<li>Resuspend the culture in YEPD-uridine medium, using HEPES (50 mM, pH 7.5) as a buffer agent and incubate the culture at 37 &#176;C to induce hyphae formation. Check hyphal formation under a microscope at 100X magnification. Hyphal formation is visible 5 h after culturing at 37 &#176;C; however, use the cells 16 h after incubation at 37 &#176;C when the hyphal percentage reaches almost 95% of the total culture.</li>
	<li>To further enrich the preparation in hyphal concentration, centrifuge aliquots of the culture at 3,300 x g for 5 min. Discard the supernatant and resuspend the pellet in sterile PBS at a concentration of 1 x 108 hyphae/mL.</li>
</ol>
2. Deep Dermal Injection
<ol>
	<li>24 h prior the infection procedure, anesthetize the mice with an intraperitoneal injection of ketamine (80 - 100 mg/kg) and xylazine (10 mg/kg), and then shave the flank of the mice using an electric clipper. 
	NOTE: After any procedure involving anesthesia, place the mice under a heating lamp until they have efficiently recovered.</li>
	<li>Rest the mice for 24 h to avoid any inflammation due to the shaving process.</li>
	<li>24 h after the shaving procedure, anesthetize the mice with an intraperitoneal injection of ketamine (80 - 100 mg/kg) and xylazine (10 mg/kg).</li>
	<li>Check the paw reflex to ensure that the mice are deeply anesthetized. Assess the paw reflex by firmly pinching the paw. If the anesthesia is achieved, the animal does not feel pain and does not move.</li>
	<li>Inject C. albicans hyphae in the deep derma using a 0.3 mL insulin syringe with a 30 G x 8 mm needle in a final volume of 50 &#181;L/injection, corresponding to 5 x 106 hyphae/injection.
	<ol>
		<li>Pull the skin of the shaved flank taut using two fingers in order to inject the volume directly in the deep derma. Insert the needle with an angle of 10 - 15&#176; with the bevel of the needle facing up.</li>
		<li>With this angle, the pathogen will be injected with a depth of approximately 300 - 500 &#181;m. To ensure the injection is well performed, check that the injected volume forms a bump that is absorbed a few min after the injection. 
		NOTE: The bump formed after the injection will be of approximately 1 cm2. This is the area that will be removed at the designated time point for either molecular or histological analysis.</li>
		<li>For time points of less than 24 h (recommended) mark the area of infection with a marker pen, since the bump formed with the injection persists for a few minutes, after which it is absorbed. For time points of 24 h or more, this step is not required, because either an ulcer or a cyst will be clearly visible.</li>
	</ol>
	</li>
</ol>
3. Sample Collection and Preparation for Histological Staining
<ol>
	<li>Euthanize the mice according to institutional procedures.</li>
	<li>Using surgical forceps, pull the skin at the border of the injected site, previously marked with the marker pen. Using surgical scissors, excise the infected site by cutting the skin following the marked line. 
	NOTE: Be careful to separate the skin from the peritoneum using round tip scissors.</li>
	<li>Immerse the skin in a disposable base mold filled with optimal cutting temperature (OCT) compound, with the internal side of the skin facing up. Freeze the sample in liquid nitrogen until the compound becomes white. Do not let the liquid nitrogen cover the sample before it is completely frozen as this would damage the sample. 
	NOTE: Caution! During step 3.3, wear a face shield for protection from liquid nitrogen.</li>
	<li>If the samples are not used immediately for slide preparation, rapidly store them at -80 &#176;C. 
	NOTE: Samples can be stored at -80 &#176;C indefinitely.</li>
</ol>
4. Preparation of Slides
<ol>
	<li>Cut the sample in half to prepare the slices for histology starting from the center of the sample.</li>
	<li>Cut 5-&#181;m thick slices with a cryostat. 
	NOTE: For skin samples, the temperature of the cryostat should not be higher than -25 &#176;C. A higher temperature would cause a partial melting of the H-OCT and therefore the samples would not be maintained in the right position during the cutting procedure.</li>
	<li>Use positively charged microscope slides to collect the slices.</li>
	<li>If the collected slices are not used soon after the preparation, store them at -20 &#176;C. At this point, slices can be stored at -20 &#176;C indefinitely.</li>
</ol>
5. Hematoxylin &#38; Eosin Staining
<ol>
	<li>Stain the slides by immersion in Gill&#39;s hematoxylin for 4 min. Wash the slides in running tap water for 5 min.</li>
	<li>Stain the slides by immersion in Eosin Y solution for 1 min. Wash the slides in running tap water for 5 min. Rinse the slides using distilled water before continuing the protocol.</li>
	<li>Dehydrate by immersion in serially increasing concentrations of ethanol solutions (50%, 70%, 80%, 95%, 100%). Immerse the slides for 15 s in each ethanol solution.</li>
	<li>Clear the slides for at least 2 min by immersion in a histological clearing agent.</li>
	<li>Mount the slides using mounting medium and cover slips.</li>
	<li>Acquire images of the slides with a microscope slide scanner at a 40X magnification.</li>
</ol>
6. Periodic Acid-Schiff (PAS) Staining
<ol>
	<li>Fix the slides with &#8805;99.5% acetone at room temperature for 1 min. Wash the slides in running tap water for 1 min.</li>
	<li>Stain the slides by immersion in periodic acid solution (1 g/dL) for 5 min. Wash the slides in 3 changes of distilled water.</li>
	<li>Stain the slides by immersion in Schiff&#39;s reagent for 15 min. Wash the slides in running tap water for 5 min.</li>
	<li>Stain the slides by immersion in Gill&#39;s hematoxylin for 5 min. Wash the slides in running tap water for 30 s.</li>
	<li>Dehydrate the slides by immersion in 3 changes of 100% ethanol. Immerse the slides for 15 s each time.</li>
	<li>Clear the slides for at least 2 min by immersion in a histological clearing agent.</li>
	<li>Mount the slides using mounting medium and cover slips.</li>
	<li>Acquire images of the slides with a microscope slide scanner.</li>
</ol>
7. Sample Collection and Preparation for RNA Extraction
<ol>
	<li>Euthanize the mice according to institutional procedures. Remove the skin samples as in step 3.2.</li>
	<li>Immerse the samples in a 2 mL snap-cap tube with 1 mL of reagent for acid guanidinium thiocyanate-phenol-chloroform extraction. 
	NOTE: The protocol can be paused here and the samples can be stored at -80 &#176;C.</li>
	<li>Cut the sample into pieces of approximately 0.2 cm2 using surgical forceps. 
	NOTE: Caution! Most of the reagents for RNA isolation are toxic and mutagenic. Steps 7.3 and 7.4 must be done under a fume hood.</li>
	<li>Smash the sample with a bead mill at a speed of 20 oscillations/s for 20 min. Alternatively, a mechanical potter can be used.</li>
	<li>Check the effective homogenization of the samples by looking for the presence of intact pieces of skin. Repeat this step if the samples are not completely homogenized.</li>
	<li>Centrifuge the samples at 16,000 x g for 1 min. Keep the supernatants for RNA extraction and discard the pellet. This step is performed to eliminate hair and debris that could block the extraction columns.</li>
	<li>Proceed with RNA extraction using RNA purification columns14.</li>
	<li>Use a spectrophotometer to assess RNA purification and concentration. If RNA extracts are not used immediately for cDNA preparation, store the samples at -80 &#176;C.</li>
</ol>

<ol>
	<li>Belkaid, Y., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialogue+between+skin+microbiota+and+immunity.">Dialogue between skin microbiota and immunity.</a> Science. 346 (6212), 954-959 (2014).</li><li>Kashem, S. W., Kaplan, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+Immunity+to+Candida+albicans.">Skin Immunity to Candida albicans.</a> Trends Immunol. 37 (7), 440-450 (2016).</li><li>Havlickova, B., Czaika, V. A., Friedrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+trends+in+skin+mycoses+worldwide.">Epidemiological trends in skin mycoses worldwide.</a> Mycoses. 51, 2-15 (2008).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+Bloodstream+Infections+in+US+Hospitals:+Analysis+of+24,179+Cases+from+a+Prospective+Nationwide+Surveillance+Study.">Nosocomial Bloodstream Infections in US Hospitals: Analysis of 24,179 Cases from a Prospective Nationwide Surveillance Study.</a> Clin Infect Dis. 39 (3), 309-317 (2004).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">Immunity to fungal infections.</a> Nat Rev Immunol. 4 (1), 11-24 (2004).</li><li>Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Candida+infections.">Pathogenesis of Candida infections.</a> J Am Acad Dermatol. 31 (3), S2-S5 (1994).</li><li>MacCallum, D. M., Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+events+in+the+intravenous+challenge+model+for+experimental+Candida+albicans+infections+in+female+mice.">Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice.</a> Mycoses. 48 (3), 151-161 (2005).</li><li>Dai, T., Kharkwal, G. B., Tanaka, M., Huang, Y. -. Y., Bil de Arce, V. J., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+external+traumatic+wound+infections.">Animal models of external traumatic wound infections.</a> Virulence. 2 (4), 296-315 (2018).</li><li>Gaspari, A. A., Burns, R., Nasir, A., Ramirez, D., Barth, R. K., Haidaris, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD86+(B7-2),+but+not+CD80+(B7-1),+expression+in+the+epidermis+of+transgenic+mice+enhances+the+immunogenicity+of+primary+cutaneous+Candida+albicans+infections.">CD86 (B7-2), but not CD80 (B7-1), expression in the epidermis of transgenic mice enhances the immunogenicity of primary cutaneous Candida albicans infections.</a> Infect Immun. 66 (9), 4440-4449 (1998).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+Candida+gastrointestinal+colonization+and+dissemination.">Murine models of Candida gastrointestinal colonization and dissemination.</a> Eukaryot Cell. 12 (11), 1416-1422 (2013).</li><li>Mear, J. B., 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=Candida+albicans+Airway+Exposure+Primes+the+Lung+Innate+Immune+Response+against+Pseudomonas+aeruginosa+Infection+through+Innate+Lymphoid+Cell+Recruitment+and+Interleukin-22-Associated+Mucosal+Response.">Candida albicans Airway Exposure Primes the Lung Innate Immune Response against Pseudomonas aeruginosa Infection through Innate Lymphoid Cell Recruitment and Interleukin-22-Associated Mucosal Response.</a> Infect Immun. 82 (1), 306-315 (2014).</li><li>Leoni, G., Neumann, P. -. A., Sumagin, R., Denning, T. L., Nusrat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair:+role+of+immune-epithelial+interactions.">Wound repair: role of immune-epithelial interactions.</a> Mucosal Immunol. 8 (5), 959-968 (2015).</li><li>Fonzi, W. A., Irwin, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isogenic+strain+construction+and+gene+mapping+in+Candida+albicans.">Isogenic strain construction and gene mapping in Candida albicans.</a> Genetics. 134 (3), 717-728 (1993).</li><li>Santus, W., 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=Skin+infections+are+eliminated+by+cooperation+of+the+fibrinolytic+and+innate+immune+systems.">Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems.</a> Sci Immunol. 2 (15), (2017).</li><li>Florescu, D. F., Brostrom, S. E., Dumitru, I., Kalil, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+Skin+Abscess+in+a+Heart+Transplant+Recipient.">Candida albicans Skin Abscess in a Heart Transplant Recipient.</a> Infect Dis Clin Pract. 18 (4), 243-246 (2010).</li><li>Pradeu, T., Cooper, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+danger+theory:+20+years+later.">The danger theory: 20 years later.</a> Front Immunol. 3, 287 (2012).</li><li>Cheng, A. G., DeDent, A. C., Schneewind, O., Missiakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+play+in+four+acts:+Staphylococcus+aureus+abscess+formation.">A play in four acts: Staphylococcus aureus abscess formation.</a> Trends Microbiol. 19 (5), 225-232 (2011).</li></ol>

The authors have nothing to disclose.

Here we described a method of C. albicans infection to study the inflammatory process that is initiated upon fungal entrance in the deep derma.
Even though skin abscess formation is a relatively rare event upon C. albicans infection15, injection of the fungus directly in the deep derma allows not only the study of fungal-driven abscess formation, but also the analysis of specific immune cells that participate to contain fungal spread. With the method d...

The human body is covered with an extremely high number of microorganisms. The skin surface is the habitat of almost one million bacteria per square centimeter1. This number, however, does not reflect the full variety of microorganisms that colonizes the skin. In addition to bacteria, the human body is colonized by many fungal species, including C. albicans, which is able to survive both at the mucosal and the skin level2.
In recent years, the percentage of people diagnosed with fungal infections has enormously increased. This is mainly due to the higher number of immunocompromised persons, i.e., HIV-positive patients and patients that have gone through either chemotherapy or immunosuppressive drugs after transplantation3. In a surveillance study performed in the United States, Wisplinghoff et al. showed that 9.5% of the nosocomial bloodstream infections were caused by Candida species4. Due to the increased occurrence of fungal infections, and especially because of the elevated percentage of Candida species found during bloodstream infections, understanding how this pathogen escapes the control of the immune system is extremely important.
C. albicans is a dimorphic fungus that grows in different morphological forms such as yeast, blastospores, pseudohyphae, and hyphae depending on the environmental conditions5. In its hyphal form, C. albicans shows its highest invasiveness capacity and has the ability to penetrate the epithelium6.
C. albicans infections have been studied using several experimental approaches. The most common model of infection is the intravenous injection of C. albicans yeast7. However, this model does not take into consideration all the processes that happen before the fungus manages to spread into the bloodstream. Another model takes advantage of the ability of C. albicans to invade the epithelium. This method, also known as the sand paper model8, was developed by Gaspari et al. in 19989, and consists of using sand paper to abrade the skin, thus eliminating the stratum corneum before applying C. albicans. This procedure allows the fungus to penetrate the epithelium, thus enabling the analysis of the invasive abilities of this pathogen. Finally, other models of infections for the gastrointestinal10 and respiratory tracts11 have been used in different studies.
The formation of a wound (as in the sand paper model) causes the activation of several pathways, including immune cell recruitment and activation, in order to promote the healing process12. This can either alter or mask the immune response specifically elicited against the pathogen, thus leading to confounding results.
Here we describe a method of skin infection that avoids initial wound formation and the induction of a basal inflammatory environment. To maintain the intact epithelial structure, we directly inject C. albicans in its hyphal form in the deep derma. Even though a single injection can elicit mild inflammation, the amount of inflammation is limited and restricted compared to the formation of an open wound as in the sand paper model. The approach that we describe here allows the study of the immune response to fungal infection and spread while avoiding the excessive and pre-existing inflammatory environment caused by mechanical damage.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Euroclone</td>
			<td>ECB4053L</td>
			<td>warm in 37 &#176;C bath before use</td>
		</tr>
		<tr>
			<td>H-OCT compound</td>
			<td>histo-line laboratories</td>
			<td>R0030</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill&#39;s Hematoxilyn</td>
			<td>histo-line laboratories</td>
			<td>09-178-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution, alcoholic</td>
			<td>histo-line laboratories</td>
			<td>09-209-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>scharlau</td>
			<td>ET00232500</td>
			<td></td>
		</tr>
		<tr>
			<td>Citro-HISTOCLEAR</td>
			<td>histo-line laboratories</td>
			<td>R0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt, mounting medium</td>
			<td>bio-optica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>sigma-aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>PAS staining system</td>
			<td>sigma-aldrich</td>
			<td>395B-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous for molecular biology</td>
			<td>Applichem PanReac</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Merck Millipore</td>
			<td>8451</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Applichem PanReac</td>
			<td>A1070,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock tubes 2 mL</td>
			<td>eppendorf</td>
			<td>30121597</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life Technologies</td>
			<td>15596018</td>
			<td>Toxic, corrosive and mutagen. Use all precaution needed</td>
		</tr>
		<tr>
			<td>Rneasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>Wear eye protection</td>
		</tr>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter Counter-Particle Count</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415 R</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MC 3000 Microtome Cryostat</td>
			<td>histo-line laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLiser</td>
			<td>QIAGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.3 ml Insulin Syringe with a 30G x 8mm needle&#160;</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base mould disposable</td>
			<td>histo-line laboratories</td>
			<td>2781</td>
			<td></td>
		</tr>
		<tr>
			<td>Positively charged bio microscope slides</td>
			<td>bio-optica</td>
			<td>09-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips 24 x 50 mm</td>
			<td>thermo scientific</td>
			<td>11911998</td>
			<td></td>
		</tr>
	</tbody>
</table>

deep dermal injection as a model of candida albicans skin infection for histological analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Through the injection of the pathogen directly in the deep derma, the structural morphology of the tissue remains intact (Figure 1A).
The maintenance of skin structural integrity allows the detection of immune cells and their localization at the site of infection. The higher magnification shown in Figure 1B reveals that the abscess is mainly composed of polymorphonuclea...

Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

deep-dermal-injection-as-model-candida-albicans-skin-infection-for

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JoVE Journal

Immunology and Infection

10.6K Views.  University of Milano-Bicocca. Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Video: Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...