1. Growth of H. Capsulatum
<ol>
	<li>Prepare a biosafety cabinet for work with the fungus by cleaning the cabinet with 10% bleach followed by 20 min UV irradiation. H. capsulatum in the yeast phase requires Biosafety level (BSL) II practices, while the mould form requires BSLIII. Use yeast form in the following steps.</li>
	<li>Next prepare a 15 ml conical tube with 5 ml of PBS. Harvest a colony of H. capsulatum (strain Hc ATCC G217B) from a BHI blood agar plate [glucose 10 g/L, cysteine 0.1 g/L, penicillin-streptomycin 1% and sheep red blood cells 50 ml/L (Colorado Serum Co., Colorado, USA)] cultivated at 37&deg;C or take an aliquot from frozen yeast stock at -80&deg;C and add it to the PBS. Vortex the cells vigorously and centrifuge for 1100 x g for 10 min at room temperature. Carefully remove the supernatant without disturbing the pellet and add 10mL of fresh PBS. Repeat this procedure three times. Suspend the cells in 5mL of PBS and tranfer to a 50 mL conical tube.</li>
	<li>To disrupt aggregated cells, pass the yeast cell suspension through a 26G1/2 needle using a 10mL syringe 5 times in a biosafety cabinet (using facial protection). To isolate small, uniformly-sized yeast, centrifuge the cells at 55 x g for 1 min at room temperature and then remove the top 1 ml.</li>
	<li >Add the cell suspension to an sterile erlenmeyer flask containing 100 ml of HAM F12 medium (GIBCO) supplemented with 16g/L glucose, 1g/L glutamic acid, 8.4mg/L cystine, 6g/L glutamic acid and then grow the cells at 37&deg; C for 48 h in an incubator with 150rpm shaking.</li>
</ol>
2. Hybridoma Growth and Purification of Monoclonal Antibodies
<ol>
	<li>Transfer the selected hybridomas to DMEM medium containing 10% fetal calf serum, 10% NCTC, 1% non-essential amino acids and 1% Penicillin-Streptomycin. Grow the cells in a cell culture flask (Becton Dickenson) for 5-7 days at 37&deg;C/5%CO2.</li>
	<li>Then centrifuge the medium at 1100 x g for 10 min at room temperature and collect all the supernatant without disrupting the pellets.</li>
	<li>Next, purify the cell free supernatant using a protein A/G resin by FPLC.</li>
	<li>The mAb concentration can then be calculated by a capture ELISA (1) using an IgG isotype control as standard (Figure 1B).</li>
</ol>
3. Histoplasma Capsulatum Inoculum Preparation
<ol>
	<li>Take a 48 h Histoplasma capsulatum (strain Hc ATCC G217B) yeast phase culture grown in HAM F-12 medium.</li>
	<li>Centrifuge the cells at 1100 x g for 10 min. Discard the supernatant and add fresh PBS.</li>
	<li>Repeat the 3.2 procedure 3 times. </li>
	<li>Pass the yeast cell suspension 5 times through a 26G1/2 needle using a syringe.</li>
	<li>Centrifuge the suspension at 55 x g for 1 min to pellet residual cell clusters. Transfer the supernatant containing the single cell suspension to a new tube.</li>
	<li>Enumerate the single cell suspension using a hematocytometer.</li>
	<li>Adjust the cell concentration in order to achieve 1.25 x 107 yeast (survival) or 5.0 x 106 (CFU, cytokines and histology) in a suspention of &lt;50 &mu;L (Figure 2).</li>
</ol>
4. Intraperitoneal Administration of the MAbs 
<ol>
	<li>Pick up the mouse by its tail and the scruff of the neck (Fig 3A). Immobilize the mouse by the scruff of its neck as close to theears as possible (make sure to take up enough skin so that the mouse cannotturn its head to bite the person handling it; Figure 3B and 3C).</li>
	<li>Stabilize the tail withthe little finger gently pressed against the palm of the hand (Figure 3D).</li>
	<li>Swab the injection area with 70% ethanol before placing the needle and to aspirate to look for blood before injecting.</li>
	<li>Use the 26G1/2 to piercethe skin and abdominalmuscles to inject the mAb solution containing 500&mu;g (PBS, isotype control antibody or the mAb to be tested, diluted in 1 mL PBS) into thelower left or lower right abdominal quadrant (intraperitoneal cavity),with the animal in the head down position, taking care to avoid the diaphragm andother internal organs (Figure 3E).</li>
	<li>Wait briefly before withdrawing the needleto reduce the likelihood of leakage.</li>
	<li>Wait a minimum of two hours before proceeding to the next step.</li>
</ol>
5. Mouse Anesthesia and Intranasal Infections 
<ol>
	<li>Prepare the anesthesia using ketamine and xylazine at 100 mg/Kg and 10 mg/Kg, respectively. Perform intraperitoneal administration of PBS, isotype control antibody, or experimental antibody as described in the accompanying written protocol. Wait two hours after mAb injection before anesthetizing the mouse and proceeding with the intranasal infection. 
	 
	Prepare anesthesia using ketamine and xylazine at 100 mg/Kg and 10 mg/Kg, respectively (7). 
	&nbsp;</li>
	<li>During the 2h waiting period, prepare the Histoplasma capsulum inoculum as described in the accompanying written protocol.</li>
	<li>Proceed according to the itens 5.1 to 5.5. Toe pinch the animals with a tweezer and to verify the absence of reflex and confirm that the anesthesia worked. After anaesthetization, gently suspend the mouse by its front teeth to an nylon or cotton string. Use 2 column stands to tie the strings in order to suspend the animals.</li>
	<li>Slowly administer approximately 50 &mu;l inoculum into a nare using a micropipette while closely monitoring the mouse's respiration rate. Allow the mouse to rest in this position for 2-5 min after intranasal infection to facilitate the deposition of the yeast into the animal's lungs (Figure 3F).</li>
	<li>After the mouse has been infected, maintain the mouse in a monitored, warm environment (temperature between 25 and 37&deg;C), carefully utilizing a heat lamp if necessary , until it recovers from the anesthesia. When the mouse awakes, return it to a clean cage with ad libitum access to food and water. The cages are returned to our animal facility and kept in a clean animal room (BLSI). </li>
</ol>
6. Survival Studies 
<ol>
	<li>Assess infected and mock-infected animals clinically for tachypnea, lethargy, obtundation, and weight loss. The animals should be checked twice daily by laboratory members and daily by Animal Caretakers. </li>
	<li>With murine histoplasmosis, until close to the end of life, there are typically no apparent signs of the infection other than a mild increase in respiration rate. With advanced disease, which usually happens from 10-11 days, the mice become significantly tachypneic and have diminished activity. Typically they rapidly become moribund and expire.</li>
	<li>Distressed animals should be euthanized appropriately with CO2 in a dedicated chamber, Deceased animals should be enumerated daily. </li>
</ol>
7. CFU Studies, Histology and Cytokines Studies 
<ol>
	<li>Prepare 15 ml conical tubes with 10mL PBS for each organ to be collected. </li>
	<li>Euthanize animals 7 and 14 days after infection with a sublethal inuculum (5.0 x 106) as previously shown and remove the lung, spleen and liver immediately.</li>
	<li>Remove a piece of the organ to be evaluated and perform the fixation in formalin overnight. The sections will be examined under the microscope for pathological evaluation.</li>
	<li>Prepare 50 ml conical tubes with 5mL PBS topped with 70 &mu;m cell strainers for each organ to be homogenized. Macerate each remaining portion of the organs seperately into the 50 mL tubes using sterile 5mL syringe plungers.</li>
	<li>Next, serially dilute organ homogenates (1:100, 1:1000 and 1:5000) and plate 100&mu;L of each dilution on BHI-blood agar plates (2). </li>
	<li>Add protease inhibitor tablets to the homogenates according to the manufacture's instructions (Roche, Germany).</li>
	<li>Incubate plates at 37&deg;C for up to 14 days and enumerate CFUs.</li>
	<li>Centrifuge the organ homogenates at 4000xg for 10 min and remove organ supernatants, whish will be used in used in the cytokine assays performed by standard methods according to the manufacturer. </li>
</ol>
8. Monoclonal Antibody Protection Against H. Capsulatum is Dependent on Antibody Specificity and Isotype
<ol>
	<li>MAb isotype is critical in protection against H. capsulatum, as mice treated with IgG1 or IgG2a mAbs to Hsp60 have significantly prolonged survival compared to control mice receiving an IgG2b mAb to Hsp60 or either an isotype control mAb or PBS (Figure 4).</li>
	<li>Further, in mice that received protective mAbs, there was a significant reduction in the number of pulmonary and splenic CFU at day 7 and decreases of 2 and 2.5 log units of yeast numbers in the lungs at day 14 (Figure 5). </li>
</ol>
<img src="/files/ftp_upload/2532/2532fig1.jpg" alt="Figure 1" /> Figure 1: Growth of H. capsulatum and hybridomas. (A) Liquid HAM F-12 culture preparation from a single colony obtained from H. capsulatum grown on a BHI-blood agar plates. (B) Hybridoma growth in cell culture flasks and mAb purification by protein A/G resin using affinity cromatography.
<img src="/files/ftp_upload/2532/2532fig2.jpg" alt="Figure 2" /> Figure 2: H. capsulatum inoculum preparation for intranasal infection. Cell suspension obtained after H. capsulatum agregates disruption by syringe and centrifugation is enumerated by counting in a hemocytometer. Cell concentration is adjusted to 1.25 x 107 (survival experiments) or 5 x 106 (CFUs, cytokines and histology) in &lt;50 &mu;L.
<img src="/files/ftp_upload/2532/2532fig3.jpg" alt="Figure 3" /> Figure 3: Mouse handling during mAb administration. (A) The mouse is picked up by the tail and (B and C) immobilized by holding the scruff of its neck as close to theears. (D) The tail is held between the little finger and palm. (E) The mAb solution is injected using a 26G1/2 needle. (F) The H. capsulatum inoculum is administered intranasally by gently pipetting the cell suspension into a nare.
<img src="/files/ftp_upload/2532/2532fig4.jpg" alt="Figure 4" /> Figure 4: MAbs to Hsp60 can alter the pathogenesis of histoplasmosis. i.p. injections with 500 &mu;g of IgG1 ,or IgG2a mAbs 2 h prior to infection significantly prolonged survival (p&lt;0.05, compared to controls), but an IgG2b mAb.
<img src="/files/ftp_upload/2532/2532fig5.jpg" alt="Figure 5" /> Figure 5: CFU determinations in lungs at 7 and 14 days after a sublethal intranasal challenge with 5x106 Hc yeasts of mice treated i.p. with selected mAbs to Hsp60 or irrelevant mAb showed reduction in fungal burden for IgG1 and IgG2a mAbs treated animals. Black bars represent CFUs at day 7 and white bars 14 days post-infection (*p&lt;0.001 at 7 days and **p&lt;0.001 at 14 days post infection).

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

No conflicts of interest declared.

The protocol presented here demonstrates that mAb's to H. capsulatum can modify the course of experimental murine histoplasmosis. MAbs to cell surface antigens of pathogens can modify the complex dynamics that occur during the interplay between a host and a pathogen. This study establishes that mAbs mediate protection in a murine model of lethal histoplasmosis when injected intraperitoneally, and it suggests candidate proteins for vaccine development, such as H2B, M antigen (surface catalase)(3) and Hsp60. In vi...

<table border="1">
<tbody>
 <tr>
 <th>Name of Material</th>
 <th>Company</th>
 </tr>
 <tr>
 <td>Biological safety cabinet (BSL2)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 mL conical tubes </td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>HAM F-12 medium</td>
 <td>Gibco</td>
 </tr>
 <tr>
 <td>37&deg;C shaker</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL conical tubes</td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>26G1/2 needle </td>
 <td>BD</td>
 </tr>
 <tr>
 <td>10 mL syringe</td>
 <td>BD</td>
 </tr>
 <tr>
 <td>250 mL flasks</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FPLC (Fast protein liquid chromatography) system</td>
 <td>GE Helthcare</td>
 </tr>
 <tr>
 <td>ELISA reader </td>
 <td>BioTek</td>
 </tr>
 <tr>
 <td>Anesthesia (ketamine and Xylazine)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Column stands</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon string</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heat lamp</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>70&mu;m cell strainers</td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>BHI agar plates </td>
 <td>Gibco</td>
 </tr>
 </tbody>
</table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

passive-administration-monoclonal-antibodies-against-h-capsulatum

Research

JoVE Journal

Immunology and Infection

11.9K vistas.  Albert Einstein College of Medicine. Ratones C57BL / 6 se han utilizado para estudiar la patogénesis Hc y el mejor modelo. Estamos explorando los beneficios potenciales de la inmunidad humoral contra este hongo y generó varias Acm [de la histona H2B y 60kDa proteína de choque térmico] que la prueba de su eficacia protectora tras la administración intraperitoneal.

Video: Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...