PAFE in Filamentous Fungi

Pass the isolates by subculturing onto SD or potato dextrose agar for 57 d at 28 C. 2. Determine the MIC according to the NCCLS (5). 3. Prepare the stock solution of the antifungal agent at convenient starting concentration and store at -70 C until use (see Note 1). 4. Dilute the stock solution at least 50 times in RPMI-1640 plus 0.5 Tween-20 for nonwater-soluble drugs (see Note 2). 5. Prepare serial dilution from the above resulting starting drug concentration to obtain the desire...

Phagocytosis Assay General

Phagocytosis assays are performed with MQ and neutrophils after adherence to 13-mm diameter glass cover slips in 24-well tissue culture plates. DCs also may be studied after adherence to glass cover slips as described in Subheading 3.5.3. for the binding assay. This has the advantage of being able to compare the properties of MQ vs DC under identical conditions. 1. After adherence, the monolayers are washed twice with HBSA, and may be treated at this time with various reagents. 2. At the end of...

Assessment of Fungal Cell Damage

Damage of Pseudohyphae MTT Microassay MTT is a tetrazolium dye used to quantify phagocyte-mediated damage to Candida pseudohyphae. MTT is reduced to a blue-colored formazan deriva tive by the fungal dehydrogenase system via the electron-coupling agent coenzyme Q. Disruption of the dehydrogenase activity is affected by pseudohyphal damage after incubation with phagocytes. Percent hyphal damage induced is assessed colorimetrically. 1. Make a suspension of 2.5 x 104 mL blastoconidia in...

Staining and Imaging of Gels

It is common to look for proteins in the gel by staining them with dye. Many different types of staining with different characteristics and limitations in respect of sensitivity for proteins are available. All stains interact differently with different proteins thus, no stain is universal for all proteins. In our laboratory, silver nitrate staining and Coomassie blue staining are used. Silver staining is the most sensitive nonradioactive staining method. It involves a very complex, multistep...

P David Rogers

College of Pharmacy, University of Tennessee Health Sciences Center, Memphis, TN 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine is a trademark of The Humana Press Inc. All papers, comments, opinions,...

Preface To Candida

In the current medical era, fungal infections have emerged as an important clinical threat, with significant associated morbidity and mortality. Along with the emergence of fungal infections has come the development of antifungal resistance to existing antifungal agents and the development of agents directed at novel drug targets. Methods for evaluating such resistance patterns, mechanisms of resistance, and novel antifungal agents have all been successfully developed recently. This volume of...

Time Kill Curves

RPMI-MOPS medium dissolve 10.4 g of RPMI-1640 powder (with glutamine and phenol red, but without bicarbonate) in 900 mL of distilled water. Add 34.53 g of MOPS (0.165 mol L final) and adjust the pH to 7.0 with NaOH. Make up to a final volume of 1 L with distilled water. Filter sterilize and store at 4 C. 5. Sterile spreaders (Hockey sticks). 7. 25 cm2 Polystyrene tissue culture flasks. 8. Antifungal drugs an antifungal powder of known potency should be used. 3. Methods 1. Prepare concentrated...

Antifungal Susceptibility Testing

Antimicrobial susceptibility testing methods for fungi recently have been standardized by the Clinical and Laboratory Standards Institutes (CLSI) (formerly known as the National Committee for Clinical Laboratory Standards 1,2 ). The CLSI first published methods for antifungal susceptibility testing of Candida spp. and Cryptococcus neoformans in 1997, whereas the methods for filamentous fungi were approved in 2002. A macrodilution method for susceptibility testing was approved first, quickly...

Mode of Action Studies

Mode of action studies (vs mechanism of action studies in which an isolated target is assayed), can suggest the general biological process perturbed by an antifungal agent and whether that action is fungicidal or fungistatic. Basic types of mode of action whole-cell experiments include the following 1. Detection of fungicidal vs fungistatic actions. 3. Drug combination interaction assays. 3.3.1. Fungicidal vs Fungistatic Actions A fungicidal agent kills cells at concentrations similar to its...

Inoculum Preparation

Because the inoculum is a critical variable that defines the lethality and rapidity of infection in the animals, special attention should be directed towards the correct preparation of a conidial suspension. 1. A stock conidial suspension of Aspergillus should be swabbed on three to five PDA plates or 6-10 agar slants 1 wk before inoculation. 2. Plates or slants are incubated at 37 C in a humid chamber for 3-5 d or until mature growth (thick fungal lawn) is evident. 3. Inoculum preparation...

Agents TSr

David Rogers y L 121. Placenta Research Methods and Protocols Volume 1, edited by Michael J. Soares and Joan S. Hunt, 2005 120. Breast Cancer Research Protocols, edited by Susan A. Brooks and Adrian Harris, 2005 119. Human Papilloma Viruses Methods and Protocols, edited by Clare Davy and John Doorbar, 2005 118. Antifungal Agents Methods and Protocols, edited by Erika J. Ernst and P. David Rogers, 2005 117. Fibrosis Research Methods and Protocols, edited by John Varga,...

Bioautography

Bioautography is a simple method to assign activity to compounds in crude preparations often found in a natural products laboratory using a combination of bioassay and TLC (for TLC methods, see ref. 16). Two general methods are available agar diffusion (60) and direct TLC (61). 3.2.1. Agar Diffusion Bioautography The following example employs C. albicans ATCC 90028, but any other fungus may be used. 1. A suitable TLC solvent system is chosen for optimum separation of constituents in an active...

Methods

The bulk of the literature regarding natural product screening in the late 20th century primarily describes agar well and diffusion methods. These techniques do detect active extracts, but in a medium- to high-throughput antifun-gal screening laboratory these methods are relatively cumbersome. Along with broth macrodilution techniques, agar methods require relatively large amounts of sample, which may limit the use of natural products (17,18). These methods are therefore not described here but...

Assessment of Cytokine and Chemokine Production by MNCs Using RTPCR

Phagocytosis Notes Flow Chart

Immunomodulators expressed in phagocytes in response to Candida infection can be detected and quantified either at the ribonucleic acid (RNA) level by RT-PCR or at the protein level by the quantitative sandwich enzyme immunosorbent assay (Fig. 2). The method of RT-PCR described in Subheading 3.4.2. combines both complimentary deoxyribonucleic acid (cDNA) synthesis and PCR in a single tube using gene-specific primers and the polymerase mixture Superscript II HRT and Platinum Taq polymerase. PCR...

Spot Identification Through Custom Database Searching

Proteins can be identified by searching in appropriate databases with pep-tide mass fragmentation data. Mass fingerprints for unknown proteins are generated using MALDI-TOF MS. PROWL software (formerly Proteometrics, Inc.) is used to search protein databases with mass spectrometry data. A custom database for PROWL has been constructed in our laboratory from a database of Candida albicans open reading frames (ORFs) DNA sequence (http genolist.pasteur.fr CandidaDB ), and is used to match unknown...

Antifungal Activity and Cytokine Expression Assays Maria Simitsopoulou and Emmanuel Roilides

Candida albicans and non-albicans Candida spp. can cause serious infections in hospitalized and immunocompromised patients. A large number of antifungal agents and immunomodulators have been developed and may interact with both polymorphonuclear and mononuclear phagocytes against blastoconidia and pseudohyphae of Candida spp. Blastoconidia are predominantly destroyed by phagocytosis, whereas pseudohyphal killing is initiated after the attachment of phagocytes to the cell wall of the organism...

Invasive Candida Infections

Invasive candidiasis is one of the leading causes of morbidity and mortality in hospitalized and immunocompromised patients nowadays (1). Although Candida albicans has been historically the most frequent cause of invasive candidiasis, non-albicans Candida spp. are becoming increasingly important being more resistant to mainstream antifungal agents than C. albicans (2). Hence, the intrinsic host response against Candida spp. and the effects of the immunomodulatory cytokine network on it need to...

Antifungal Susceptibility Testing Methods

Broth dilution methods for antifungals can be performed by macrodilution or micordilution methods (2). The macrodilution method is prepared in test tubes in 1-mL volumes. This method has been established as the basis for comparing all other methods of susceptibility testing for yeast. The macrodilution method has largely been replaced by the microdilution method that is performed in a 96-well microdilution plate in volumes of 200 L per well. Both methods use a starting inoculum on 0.5-2.5 x 103...

Construction of the Disruption Cassette

The MPAr flipper cassette in plasmid pSFIl was constructed in the vector pBluescript II KS and was designed to contain several unique restriction sites on the left (ApaI, Xhol) and right (NotI, Sacll, SacI) borders. These sites can be used for cloning flanking sequences of the target gene that serve for specific genomic integration by homologous recombination. For example, an upstream fragment is amplified by polymerase chain reaction (PCR) with primers introducing a distal ApaI site and a...

Katherine S Barker and P David Rogers Summary

The near completion of sequencing the Candida albicans genome has made it possible to employ genomic technologies, such as microarray analysis, to aid in identifying key genes involved in such clinical problems as the acquisition of high-level resistance to azole antifun-gal agents. Here, we outline in detail the methodologies utilized in our laboratory to culture clinical isolates of C. albicans, isolate ribonucleic acid from such cultures, synthesize labeled complimentary deoxyribonucleic...

Gordon Ramage and Jos Luis Lopez Ribot Summary

Candida albicans is capable of forming biofilms on a variety of inert and biological surfaces. Cells in biofilms display phenotypic properties that are radically different from their free-floating planktonic counterparts, including their recalcitrance to antimicrobial agents. Consequently, Candida biofilm-associated infections are difficult to treat and to fully eradicate with standard antifungal therapy. Here, we describe a simple, fast, inexpensive and highly reproducible microtiter...

Animal Infection Model

Six-week-old, specific pathogen-free female Swiss ICR (CD1) mice can be purchased from Harlan Sprague-Dawley (Indianapolis, IN see Note 3 3-6,812,27,28,37-40 ). 2. Before the study, the animals should be housed in groups of five per cage and allowed to acclimate 7 d before the initiation of experiments. 3. Mice should be allowed free access to food and water. 4. At the time of study, the animals should weigh 23-27 g. 5. All of our animal studies were approved by our local animal research...

General Microplate Based Whole Cell Screening

Sample Preparation and Sample Dilution Table 2 illustrates the general methods recommended by the NCCLS for antifungal susceptibility testing (35,36). Each step may be adapted to encompass a natural product screening laboratory's desired outcome. Unfortunately, when dealing with crude extracts and column fractions of natural products, purity and potency of the sample are not applicable. However, careful attention to consistency of sample preparation (collection, weighing, extraction,...

Massoumeh Z Hooshdaran George M Hilliard and P David Rogers

The sequencing of the Candida albicans genome and recent refinements in protein resolution and identification techniques have greatly enhanced the application of proteomics for the study of this fungal pathogen. Proteome analysis includes the separation and isolation of proteins by two-dimensional polyacrylamide gel electrophoresis and subsequent protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This...