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## **Meet the editor**

Mag. Schmid is an Academic Research Specialist in the Department of Medicine, Division of Hematology-Oncology, at the University of California at Los Angeles and the technical director of the UCLA Flow Cytometry Shared Resource. Mag. Schmid received her degree in the Pharmaceutical Sciences from the University of Vienna, Austria. She immigrated to the United States of America in 1981 and joined the UCLA laboratory of Dr. John Fahey, a noted immunologist, in 1983. She started working in flow cytometry at that time, and in 1989, working with Dr. Janis Giorgi, a well-known HIV researcher, she established the UCLA Flow Cytometry Core Facility. Mag. Schmid published twenty-seven peer-reviewed, first author papers, reviews, and book chapters and has co-authored an additional twenty-eight papers and two chapters. Her present interests include the development of accurate methods for the flow cytometric assessment of circulating epithelial progenitor cells as biomarkers of cardiovascular disease as well as the formulation of documents that provide guidance to the flow cytometry community at large for creating Standard Operating Procedures for cell sorting that contain appropriate safeguards.

Contents

**Preface IX** 

Sara Rojas-Dotor

**T CD4+**

Chapter 1 **Effect of Monocyte Locomotion Inhibitory** 

Chapter 3 **High-Throughput Flow Cytometry for** 

**Factor (MLIF) on the Activation and Production of** 

Chapter 2 **Applications of Flow Cytometry to Clinical Microbiology 17**  Barbara Pieretti, Annamaria Masucci and Marco Moretti

> **Predicting Drug-Induced Hepatotoxicity 43**  Marion Zanese, Laura Suter, Adrian Roth, Francesca De Giorgi and François Ichas

Chapter 4 **B Cells in Health and Disease – Leveraging Flow Cytometry to Evaluate Disease Phenotype and the Impact of Treatment with Immunomodulatory Therapeutics 60**  Cherie L. Green, John Ferbas and Barbara A. Sullivan

Chapter 5 **Evaluation of the Anti-Tumoural and Immune Modulatory Activity of Natural Products by Flow Cytometry 91** 

**Cancer Stem Cells Using Flow Cytometry 107** 

**Peripheral Blood After Vaccination as a Novel Diagnostic Marker for Assessing Antibody** 

Zita Chovancova, Jiri Litzman and Jindrich Lokaj

**Responses in Patients with Hypogammaglobulinaemia 125** 

Susana Fiorentino, Claudia Urueña, Sandra Quijano, Sandra Paola Santander, John Fredy Hernandez and Claudia Cifuentes

Chapter 7 **Flow Based Enumeration of Plasmablasts in** 

Vojtech Thon, Marcela Vlkova,

Chapter 6 **Identification and Characterization of** 

Yasunari Kanda

**Intracellular Cytokine and Chemokine Receptors in Human** 

 **Lymphocytes Measured by Flow Cytometry 1** 

### Contents

#### **Preface XI**


Zita Chovancova, Jiri Litzman and Jindrich Lokaj


### Preface

Advances in patient management have often been closely linked to the development of critical quantitative analysis methods. Flow cytometry is such an important methodology. It can be applied to individual cells or organelles allowing investigators interested in obtaining information about the functional properties of cells to assess the differences among cells in a heterogeneous cell preparation or between cells from separate samples. It is characterized by the use of a select wavelength of light (or multiple ones) to interrogate cells or other particles one at a time providing statistically relevant, rapid correlated measurements of multiple parameters with excellent temporal resolution. These intrinsic attributes, as well as advances in instrumentation and fluorescent probes and reagents, have contributed to the tremendous growth of clinical applications of flow cytometry and to the world-wide expansion of laboratories which use this technology since its inception in the late 1960s.

This publication reflects these facts as indicated by the global author panel and the wide range of sample types, assays, and methodologies described. Openly accessible, the book is intended to introduce novices to this powerful technology and also provide experienced professionals with valuable insights and an opportunity to refresh or update their knowledge in various subject areas of clinical flow cytometry.

> **Ingrid Schmid**, Mag. Pharm. Department of Medicine Division of Hematology-Oncology University of California, Los Angeles USA

**1** 

Sara Rojas-Dotor

*México* 

**Effect of Monocyte Locomotion Inhibitory**

**Production of Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>**

**Lymphocytes Measured by Flow Cytometry** 

*Unidad de Investigación Médica en Inmunología, Instituto Mexicano del Seguro Social*

The supernatant of Axenically cultured Enatamoeba *histolytica* (*E. histolytica)* produces a thermostable factor that was purified and characterized by high resolution chromatography (HPLC) and mass spectrometry (MS-MS), supplemented by the methods of Edman (Edman & Begg, 1967). This revealed a pentapeptide with a molecular weight of 583 Daltons and established the aminoacid sequence (Met - Gln - Cys - Asn - Ser), which was termed Monocyte Locomotion Inhibitory Factor (MLIF). MLIF has powerful and selective antiinflammatory properties, which were established *in vitro* by Boyden chamber studies. MLIF inhibits locomotion, both random chemokinetic and chemotactic, of mononuclear phagocytes (PM) from normal human peripheral blood but not of neutrophils toward various attractants, such as C5a-Desargues lymphokine and Lymphocyte-derived chemotatic factor (LDCF) (Kretschmer et al., 1985). This factor also depresses the respiratory burst of monocytes and neutrophils activated with zymosan *in vitro*, as measured by chemiluminescence (Rico et al., 1992), and nitric oxide production in mononuclear phagocytes and human polymorphonuclear neutrophils (PMNs) (Rico et al., 2003). Such effects were not accompanied by changes in expression of CD43, a ligand critical in the initial activity of phagocytes, in the membrane of these cells, and did not affect the viability of phagocytes (Kretschmer et al., 1985). In contrast, MLIF does not affect either locomotion or the respiratory burst of zymosan-activated human PMNs (Rico et al., 1998). *In vivo*, MLIF delays the arrival of mononuclear leukocytes in Rebuck chambers applied to the skin of healthy human volunteers (Kretschmer et al., 1985), inhibits cutaneous delayed contact hypersensitivity to 1-chloro-2-4-dinitrobenzene (DNCB) in guinea pigs (Giménez-Scherer et al., 1997) and decreases expression of the adhesion molecules VLA-4 on monocytes and VCAM-1 in the vascular epithelium (Giménez-Scherer et al., 2000). MILF inhibits the expression induced in inflammatory proteins such as MIP-1α and MIP-1β in U-937 cells, which are NF-κB pathway-regulated proteins (Utrera-Barillas et al., 2003). The p65–p50 heterodimer comprises the most abundant form of NF-κB in a PMA-induced system. Temporary studies showed that MLIF induces p50 translocation, which may be explained

**1. Introduction** 

**Factor (MLIF) on the Activation and**

### **Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup> Lymphocytes Measured by Flow Cytometry**

Sara Rojas-Dotor

*Unidad de Investigación Médica en Inmunología, Instituto Mexicano del Seguro Social México* 

#### **1. Introduction**

The supernatant of Axenically cultured Enatamoeba *histolytica* (*E. histolytica)* produces a thermostable factor that was purified and characterized by high resolution chromatography (HPLC) and mass spectrometry (MS-MS), supplemented by the methods of Edman (Edman & Begg, 1967). This revealed a pentapeptide with a molecular weight of 583 Daltons and established the aminoacid sequence (Met - Gln - Cys - Asn - Ser), which was termed Monocyte Locomotion Inhibitory Factor (MLIF). MLIF has powerful and selective antiinflammatory properties, which were established *in vitro* by Boyden chamber studies. MLIF inhibits locomotion, both random chemokinetic and chemotactic, of mononuclear phagocytes (PM) from normal human peripheral blood but not of neutrophils toward various attractants, such as C5a-Desargues lymphokine and Lymphocyte-derived chemotatic factor (LDCF) (Kretschmer et al., 1985). This factor also depresses the respiratory burst of monocytes and neutrophils activated with zymosan *in vitro*, as measured by chemiluminescence (Rico et al., 1992), and nitric oxide production in mononuclear phagocytes and human polymorphonuclear neutrophils (PMNs) (Rico et al., 2003). Such effects were not accompanied by changes in expression of CD43, a ligand critical in the initial activity of phagocytes, in the membrane of these cells, and did not affect the viability of phagocytes (Kretschmer et al., 1985). In contrast, MLIF does not affect either locomotion or the respiratory burst of zymosan-activated human PMNs (Rico et al., 1998). *In vivo*, MLIF delays the arrival of mononuclear leukocytes in Rebuck chambers applied to the skin of healthy human volunteers (Kretschmer et al., 1985), inhibits cutaneous delayed contact hypersensitivity to 1-chloro-2-4-dinitrobenzene (DNCB) in guinea pigs (Giménez-Scherer et al., 1997) and decreases expression of the adhesion molecules VLA-4 on monocytes and VCAM-1 in the vascular epithelium (Giménez-Scherer et al., 2000). MILF inhibits the expression induced in inflammatory proteins such as MIP-1α and MIP-1β in U-937 cells, which are NF-κB pathway-regulated proteins (Utrera-Barillas et al., 2003). The p65–p50 heterodimer comprises the most abundant form of NF-κB in a PMA-induced system. Temporary studies showed that MLIF induces p50 translocation, which may be explained

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

Inflammation is the body's reaction against invasion by an infectious agent, an antigenic stimulus or even just physical injury. This response induces the infiltration of leukocytes and plasma molecules into regions of infection or injury. Its main effects include increased blood flow to the region, increased vascular permeability allowing the passage of large serum molecules such as immunoglobulin and leukocyte migration through the vascular endothelium toward the inflamed area. Inflammation is controlled by cytokines, factors produced by mast cells, platelets and leukocytes, chemokines and plasma enzyme systems such complement, coagulation and fibrinolysis. Cytokines stimulate the expression of adhesion molecules by endothelial cells, and these adhesion molecules bind to leukocytes and initiate their attraction to areas of infection. Microbial products, such as peptides with N-formilmetionil, chemokines, and peptides derived from complement such as C5a, and leukotrienes (B4), act on leukocytes to stimulate their migration and their microbicidal abilities. The composition of cells involved in inflammatory processes changes with time and goes from neutrophil rich to mononuclear cell rich, reflecting a change in the leukocytes attracted (Roitt, 1998; Abbas & Lichtman, 2004). Macrophages attracted to the site of infection are activated by microbial products and interferon-gamma (IFN-γ) which cause

Lymphocytes Measured … 3

Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

them to phagocytose and kill microorganisms (Figure 2).

Fig. 2. Cytokines play an important role in the development of acute or chronic

several days, it will induce chronic inflammation, recruit mast cells, eosinophils, lymphocytes and macrophages, and induce the production of antibodies and cytokines.

These cells are often found in damaged tissue. (Luscinskas & Gimbrone, 1996)

inflammatory responses. Interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α) and IL-12, in addition to cytokines and chemokines, have redundant and pleiotropic effects, which together contribute to the inflammatory response. If the antigen is eliminated, inflammatory cells become apoptotic or return to the circulation. If the antigen persists for

**2. Inflammation** 

by the ability of MLIF to induce AMPc synthesis and protein kinase A phosphorylation in NF-κB and IκB followed by NF-κB translocation (Kretschmer et al., 2004). This may also explain the atypical inflammation observed in invasive amoebiasis, in which there is decreased chemotaxis and disequilibrium in cytokine production. This is supported by *in vivo* observations that MLIF notably decreased cellular infiltration and inflammatory cytokine expression.

 The selective actions of MLIF upon a variety of cell types suggest that it disrupts an organism's pro- and anti-*inflammatory* network (Giménez-Scherer et al., 1987; Kretschmer et al., 1985, 2001; Rojas-Dotor et al., 2006). A pentapeptide with the same amino acids but in a different sequence, termed a MLIF scramble (Gln-Cys-Met-Ser-Asn), showed no antiinflammatory properties (Giménez-Scherer et al., 2004). The observed effects of MLIF could be attributed to the chemical activity of the peptide. Ongoing studies in quantum chemistry have revealed that a pharmacophore group in the MLIF sequence, Cys-Asn-Ser, could be responsible for most of the anti-inflammatory properties of the molecule (Soriano-Correa et al., 2006) Figure 1.

It is possible that MLIF is derived from a larger peptide or protein synthesized by the amoeba, which is then degraded by proteases present in the cytoplasm. The lysate of amoebae material, washed and processed according to the method of Aley (Aley et al., 1980), maintains the inhibitory activity, suggesting that the MLIF is produced by the amoeba through de novo synthesis and not due to a complex-degradation process of ingestion and regurgitation of a product present in axenic medium (Rico et al., 1997).

Fig. 1. Molecular Structure of Monocyte Locomotion Inhibitory Factor (Met-Gln-Cys-Asn-Ser). The pharmacophore site, Cys-Asp-Ser, is highlighted (Soriano-Correa et al., 2006)

MLIF seems to be exclusively produced by E *histolytica* and other related amebas, *E. invadens*  and *E. moshkovski*, but it is absent in *E. dispar*, as we corroborated through the gene bank in which we only found the MLIF genetic sequence in the E *histolytica*, and not in any other parasites. Infections caused by E *histolytica* induce a transitory cell-mediated immunitysuppressed state in early inflammatory stages in the amebic hepatic abscess (AHA), and a complex cytokine signaling system is activated due to invasion of the parasite (Chadee & Meerovitch, 1984).

#### **2. Inflammation**

2 Clinical Flow Cytometry – Emerging Applications

by the ability of MLIF to induce AMPc synthesis and protein kinase A phosphorylation in NF-κB and IκB followed by NF-κB translocation (Kretschmer et al., 2004). This may also explain the atypical inflammation observed in invasive amoebiasis, in which there is decreased chemotaxis and disequilibrium in cytokine production. This is supported by *in vivo* observations that MLIF notably decreased cellular infiltration and inflammatory

 The selective actions of MLIF upon a variety of cell types suggest that it disrupts an organism's pro- and anti-*inflammatory* network (Giménez-Scherer et al., 1987; Kretschmer et al., 1985, 2001; Rojas-Dotor et al., 2006). A pentapeptide with the same amino acids but in a different sequence, termed a MLIF scramble (Gln-Cys-Met-Ser-Asn), showed no antiinflammatory properties (Giménez-Scherer et al., 2004). The observed effects of MLIF could be attributed to the chemical activity of the peptide. Ongoing studies in quantum chemistry have revealed that a pharmacophore group in the MLIF sequence, Cys-Asn-Ser, could be responsible for most of the anti-inflammatory properties of the molecule (Soriano-Correa et

It is possible that MLIF is derived from a larger peptide or protein synthesized by the amoeba, which is then degraded by proteases present in the cytoplasm. The lysate of amoebae material, washed and processed according to the method of Aley (Aley et al., 1980), maintains the inhibitory activity, suggesting that the MLIF is produced by the amoeba through de novo synthesis and not due to a complex-degradation process of ingestion and

Fig. 1. Molecular Structure of Monocyte Locomotion Inhibitory Factor (Met-Gln-Cys-Asn-Ser). The pharmacophore site, Cys-Asp-Ser, is highlighted (Soriano-Correa et al., 2006)

MLIF seems to be exclusively produced by E *histolytica* and other related amebas, *E. invadens*  and *E. moshkovski*, but it is absent in *E. dispar*, as we corroborated through the gene bank in which we only found the MLIF genetic sequence in the E *histolytica*, and not in any other parasites. Infections caused by E *histolytica* induce a transitory cell-mediated immunitysuppressed state in early inflammatory stages in the amebic hepatic abscess (AHA), and a complex cytokine signaling system is activated due to invasion of the parasite (Chadee &

regurgitation of a product present in axenic medium (Rico et al., 1997).

cytokine expression.

al., 2006) Figure 1.

Meerovitch, 1984).

Inflammation is the body's reaction against invasion by an infectious agent, an antigenic stimulus or even just physical injury. This response induces the infiltration of leukocytes and plasma molecules into regions of infection or injury. Its main effects include increased blood flow to the region, increased vascular permeability allowing the passage of large serum molecules such as immunoglobulin and leukocyte migration through the vascular endothelium toward the inflamed area. Inflammation is controlled by cytokines, factors produced by mast cells, platelets and leukocytes, chemokines and plasma enzyme systems such complement, coagulation and fibrinolysis. Cytokines stimulate the expression of adhesion molecules by endothelial cells, and these adhesion molecules bind to leukocytes and initiate their attraction to areas of infection. Microbial products, such as peptides with N-formilmetionil, chemokines, and peptides derived from complement such as C5a, and leukotrienes (B4), act on leukocytes to stimulate their migration and their microbicidal abilities. The composition of cells involved in inflammatory processes changes with time and goes from neutrophil rich to mononuclear cell rich, reflecting a change in the leukocytes attracted (Roitt, 1998; Abbas & Lichtman, 2004). Macrophages attracted to the site of infection are activated by microbial products and interferon-gamma (IFN-γ) which cause them to phagocytose and kill microorganisms (Figure 2).

Fig. 2. Cytokines play an important role in the development of acute or chronic inflammatory responses. Interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α) and IL-12, in addition to cytokines and chemokines, have redundant and pleiotropic effects, which together contribute to the inflammatory response. If the antigen is eliminated, inflammatory cells become apoptotic or return to the circulation. If the antigen persists for several days, it will induce chronic inflammation, recruit mast cells, eosinophils, lymphocytes and macrophages, and induce the production of antibodies and cytokines. These cells are often found in damaged tissue. (Luscinskas & Gimbrone, 1996)

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

Fig. 3. Antigen-presenting cells (APCs) communicate with two types of helper T cells, Th1 and Th2. They first produce cytokines, such as IFN-γ, TNF-α and IL-2, which are responsible for inflammation, and Th2 cells produce cytokines involved in the production of antibodies.

proliferation. Because IL-2 is secreted by effector T cells, this provides a negative-feedback mechanism, in which inflammatory T-cell activity (e.g., by Th1 cells) is restrained by the

Lymphocyte activation, as measured early on by mitogenic assay, was used as an indicator of immune function. Mitogenic assays measure the proliferative response of isolated mononuclear cells to *in vitro* stimulation with mitogenic lectins (Phytohaemagglutinin, Concanalin A, and Pokeweed Mitogen) or certain specific antigens (Streptokinase, PPD). The proliferative index of activation is a proportion determined by the relative uptake of radiolabel nucleotides (3H-thimidine) by the mitogen-stimulated culture compared to a basal nonstimulated culture. Actively proliferating cells incorporate more radionucleotides than weakly proliferating cells. Non-proliferating cells should have little or no incorporation of radionucleotides. These assays are often 48-72 hours in length and require licensing, storage and disposal of radioactive waste. A similar flow cytometry-based assay utilizes the uptake of the non-radioactve nucleotide bromo-deoxyuridine (BrdU) and detection with a fluorescent anti-BrdU antibody. These assays are somewhat non-specific and provide little information regarding cytokine production or cell communication. These tests have recently been supplanted with flow cytometry-basad assays for measuring changes in cells surface

The balance of activation of between Th1 and Th2, maintained by IFN-γ and IL-10, determines the nature of an immune response. Th17 cells are another recently identified subset of CD4+ T helper cells. They are found at the interfaces between the external environment and the internal environment, such as the in the skin and the lining of the gastrointestinal tract. Regulatory T cells respond to the presence of IL-2 by rapid

resulting expansion of regulatory T cells (Image taken from www.imgenex.com)

Lymphocytes Measured … 5

Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

Chemokines are small polypeptides that activate and direct the migration of monocytes, neutrophils, eosinophils and activated T lymphocytes from the bloodstream to sites of infection. They also regulate pro-inflammatory signals by binding to specific receptors belonging to the superfamily of seven trans-membrane domain alpha protein-coupled G (such as trimeric guanosine triphosphate (GTP)), and these can also be used as markers to differentiate chemokines and their receptors can also be used as markers of differentiation of helper T cell populations, pro-inflammatory (Th1) or anti-inflammatory (Th2) (Mosmann & Fong, 1989). Th1 cells express on their cell surface CCR5 chemokine receptor but not CCR3, whereas Th2 cells express the chemokine receptor CCR3 but not CCR5 (Sallusto et al., 1998). It has been shown that several inflammatory chemokine receptors, such as CCR1, CCR2, CCR3, CCR5 and CXCR3, are expressed shortly after signaling through the T cell receptor (TCR) in Th1 and Th2 cells. In contrast to CCR7, CCR4 and CCR8, which are over-expressed after activation through the TCR, these changes in chemokine receptor expression can be used to modify the migratory behavior of activated Th cells, and to establish the hierarchy of action between the different chemokine receptors (Loetscher et al., 1998; Zingoni et al., 1998).

#### **2.1 Cytokines, soluble mediators**

Cytokines are small peptide proteins with hormone-like activity that play a central role in communication between cells of the immune system. They are soluble mediators and regulators of innate and specific immunity. Additionally, cytokines promote growth and differentiation of leukocytes and blood cell precursors. Cytokines are key mediators of inflammation in many diseases, such as rheumatoid arthritis, lupus erythematosus, asthma and allergies (Ruschpler & Stiehl, 2002; Ivashkiv, 2003, D'Ambrosio et al., 2002, 2003). The host defenses against infectious pathogens are highly cytokine-dependent mechanisms mediated by humoral or cellular immunity. Each mechanism preferentially acts against intra or extracellular pathogens, viruses or worms. These host defense responses are strictly regulated by cytokines secreted by T helper populations, Th1 and Th2 (Kawakami, 2002). Cytokines have autocrine activity, increasing the proliferation, differentiation and effector functions of their own cell subset, and may additionally have far ranging effects on other cell types. T helper lymphocytes, the main orchestrators of the immune response, are subdivided into T helper 1 (Th1) and T helper 2 (Th2) subsets by the range of cytokines they secrete. Th1 cells mainly secrete the cytokines that promote cellular immunity and the inflammatory process, such as Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ). (Mosmann, 1997). In contrast, Th2 cells secrete IL-4, IL-5 and IL-10, which direct the immune response toward a more humoral (antibody-mediated) response and impair differentiation toward the Th1 phenotype (Figure 3).

In the case of several infectious diseases, like-Leishmaniasis and HIV, the development of Th1-dependent immunity protects against the infectious agent. The development of Th2 dependent immunity, in contrast, was determined to protect the parasite or virus. Downregulation of the immune response is a frequent parasitic strategy. Monitoring the immune response polarization toward a Th1- or Th2-type response is important for the development of effective vaccines. Because of the interplay between cytokines and the cells that respond to them, looking at changes in levels of soluble cytokines, changes in cell surface cytokine receptor expression and expression of intracellular cytokines by individual cell subpopulations is crucial to the understanding of cytokine biology. (Clark at al., 2011; Campanelli et al., 2010).

Chemokines are small polypeptides that activate and direct the migration of monocytes, neutrophils, eosinophils and activated T lymphocytes from the bloodstream to sites of infection. They also regulate pro-inflammatory signals by binding to specific receptors belonging to the superfamily of seven trans-membrane domain alpha protein-coupled G (such as trimeric guanosine triphosphate (GTP)), and these can also be used as markers to differentiate chemokines and their receptors can also be used as markers of differentiation of helper T cell populations, pro-inflammatory (Th1) or anti-inflammatory (Th2) (Mosmann & Fong, 1989). Th1 cells express on their cell surface CCR5 chemokine receptor but not CCR3, whereas Th2 cells express the chemokine receptor CCR3 but not CCR5 (Sallusto et al., 1998). It has been shown that several inflammatory chemokine receptors, such as CCR1, CCR2, CCR3, CCR5 and CXCR3, are expressed shortly after signaling through the T cell receptor (TCR) in Th1 and Th2 cells. In contrast to CCR7, CCR4 and CCR8, which are over-expressed after activation through the TCR, these changes in chemokine receptor expression can be used to modify the migratory behavior of activated Th cells, and to establish the hierarchy of action between the different chemokine receptors (Loetscher et al., 1998; Zingoni et al., 1998).

Cytokines are small peptide proteins with hormone-like activity that play a central role in communication between cells of the immune system. They are soluble mediators and regulators of innate and specific immunity. Additionally, cytokines promote growth and differentiation of leukocytes and blood cell precursors. Cytokines are key mediators of inflammation in many diseases, such as rheumatoid arthritis, lupus erythematosus, asthma and allergies (Ruschpler & Stiehl, 2002; Ivashkiv, 2003, D'Ambrosio et al., 2002, 2003). The host defenses against infectious pathogens are highly cytokine-dependent mechanisms mediated by humoral or cellular immunity. Each mechanism preferentially acts against intra or extracellular pathogens, viruses or worms. These host defense responses are strictly regulated by cytokines secreted by T helper populations, Th1 and Th2 (Kawakami, 2002). Cytokines have autocrine activity, increasing the proliferation, differentiation and effector functions of their own cell subset, and may additionally have far ranging effects on other cell types. T helper lymphocytes, the main orchestrators of the immune response, are subdivided into T helper 1 (Th1) and T helper 2 (Th2) subsets by the range of cytokines they secrete. Th1 cells mainly secrete the cytokines that promote cellular immunity and the inflammatory process, such as Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ). (Mosmann, 1997). In contrast, Th2 cells secrete IL-4, IL-5 and IL-10, which direct the immune response toward a more humoral (antibody-mediated) response and impair differentiation

In the case of several infectious diseases, like-Leishmaniasis and HIV, the development of Th1-dependent immunity protects against the infectious agent. The development of Th2 dependent immunity, in contrast, was determined to protect the parasite or virus. Downregulation of the immune response is a frequent parasitic strategy. Monitoring the immune response polarization toward a Th1- or Th2-type response is important for the development of effective vaccines. Because of the interplay between cytokines and the cells that respond to them, looking at changes in levels of soluble cytokines, changes in cell surface cytokine receptor expression and expression of intracellular cytokines by individual cell subpopulations is crucial to the understanding of cytokine biology. (Clark at al., 2011;

**2.1 Cytokines, soluble mediators** 

toward the Th1 phenotype (Figure 3).

Campanelli et al., 2010).

Fig. 3. Antigen-presenting cells (APCs) communicate with two types of helper T cells, Th1 and Th2. They first produce cytokines, such as IFN-γ, TNF-α and IL-2, which are responsible for inflammation, and Th2 cells produce cytokines involved in the production of antibodies. The balance of activation of between Th1 and Th2, maintained by IFN-γ and IL-10, determines the nature of an immune response. Th17 cells are another recently identified subset of CD4+ T helper cells. They are found at the interfaces between the external environment and the internal environment, such as the in the skin and the lining of the gastrointestinal tract. Regulatory T cells respond to the presence of IL-2 by rapid proliferation. Because IL-2 is secreted by effector T cells, this provides a negative-feedback mechanism, in which inflammatory T-cell activity (e.g., by Th1 cells) is restrained by the resulting expansion of regulatory T cells (Image taken from www.imgenex.com)

Lymphocyte activation, as measured early on by mitogenic assay, was used as an indicator of immune function. Mitogenic assays measure the proliferative response of isolated mononuclear cells to *in vitro* stimulation with mitogenic lectins (Phytohaemagglutinin, Concanalin A, and Pokeweed Mitogen) or certain specific antigens (Streptokinase, PPD). The proliferative index of activation is a proportion determined by the relative uptake of radiolabel nucleotides (3H-thimidine) by the mitogen-stimulated culture compared to a basal nonstimulated culture. Actively proliferating cells incorporate more radionucleotides than weakly proliferating cells. Non-proliferating cells should have little or no incorporation of radionucleotides. These assays are often 48-72 hours in length and require licensing, storage and disposal of radioactive waste. A similar flow cytometry-based assay utilizes the uptake of the non-radioactve nucleotide bromo-deoxyuridine (BrdU) and detection with a fluorescent anti-BrdU antibody. These assays are somewhat non-specific and provide little information regarding cytokine production or cell communication. These tests have recently been supplanted with flow cytometry-basad assays for measuring changes in cells surface

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

Louis, MO) and CD4+ T lymphocytes were obtained by negative selection technique (MACS® Reagents, Kit isolation, Human cell T CD4+). The purity of lymphocytes was analyzed by flow cytometry. The flow cytometry measures and analyzes the optical properties of individual cells pass through a laser beam. Depending on how cells interact with the laser beam, the cytometer measures five parameters for each cell: size (forward scatter, FSC), complexity (side scatter, SSC) and three fluorescence emissions (FL-1, FL-2 and FL -3). An electro-optical system converts the voltage signals, which is translated into a digital value which is stored in a computer, the data are then retrieved and analyzed with the software that combines information from different cells in statistical charts, which measure individual parameters (histograms) or two parameters at a time (dot plot, density or contour). For our study, purified lymphocytes were analyzed in a dot plot of SSC vs. FSC marking the region corresponding to lymphocytes, excluding debris and dead cells. In a dot plot is compensated for fluorescence with anti CD3-FITC and anti-CD4-PE (cluster of

Test samples of at least 10.000 events were acquired under these conditions. With this procedure, we obtained a population of CD4+ lymphocytes with 96% purity. (Figure 4)

Fig. 4. Simple analysis of CD4+ T cells obtained from healthy individuals by flow cytometry. The X-axis shows staining with fluorescein isothiocyanate (FITC), and the Y-axis shows staining for phycoerythrin (PE). a) Autofluorescence; b) Isotype control, staining with mouse

The presence or absence of chemokine receptors on cell surfaces also provides information regarding the cell's state of activation. Chemokine receptors can by analyzed by flow cytometry using fluorescently labeled anti-receptor antibodies or fluorescently-labeled chemokines. Combining these reagents with antibodies against the activation marker CD69

IgG1-FITC; c) Staining for subpopulations of CD3 coupled to FITC; d) Staining for subpopulations of CD4 coupled to PE. All stains show simple representation of the

histogram and are an example of 6 experiments ± SE

Lymphocytes Measured … 7

Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

differentiation (CD) and marker for T lymphocytes CD4+).

markers and assays for measuring the expression of intracellular cytokine. Flow cytometers are laser-based cell counters that are capable of distinguishing 3, 4, 5 or more (depending of flow cytometer), different fluorescence emissions, each associated with a particle identified by its light scatter proprieties. Fluorescence dyes with distinct fluorescence emissions are attached to monoclonal antibody that recognizes distinct cell surface antigens.

Traditionally, cytokines have been measured by radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA). Unfortunately, these techniques are limited by their detection range and an inability to simultaneously measure multiple analytes (García, 1999).

Using extremely sensitive multiparameter flow cytometers, Multiplexed Cytokine Immunoassay Kits overcome both of these limitations. Multiplexing is the simultaneous assay of many analytes in a single sample. Applications for flow cytometry are diverse, ranging from simple cell counting and viability to more complex studies of immune function, apoptosis and cancer, stem cells, separation of cells populations such as monocytes and T and B lymphocytes, measuring changes in cell surface markers, cell cycle analysis, cellular activation, and measuring the expression of intracellular cytokines (Collins et al., 1998; McHugh, 1994; Spagnoli,et al., 1993; Trask et al., 1982).

#### **3. Cell activation**

Activation of lymphocytes is a complex yet finely regulated cascade of events that results in the expression of cytokine receptors, the production and secretion of cytokines and the expression of several cell surface molecules, eventually leading to divergent immune responses. Parasite-specific immune responses are regulated by cytokines and chemokines. They modulate and direct the immune response, but may also contribute to an infection induced by the pathogenesis and parasite persistence (Talvani et al*.,* 2004). Parasitic infections frequently result in highly polarized CD4+ T cell responses, characterized by Th1 or Th2 cytokine dominated production profiles. Although it was previously thought that these infections were strictly dependent on signaling by cytokines, such as IFN-γ, IL-12 and IL-4, recent data indicate that this polarization may be primarily directed by a series of different factors intrinsic to the pathogen–antigen-presenting-cell interaction that directs T cell priming, and that all of this is influenced by the local environment (Katzman et al., 2008). The infection caused by the *E. histolytica* parasite is associated with an acute inflammatory response (Chadee & Meerovitch, 1984). However, it is not completely clear how *E. histolytica* triggers the host inflammatory response or how host-parasite interactions start, modulate, and eventually turn off the inflammatory response.

During inflammation, leukocytes are orchestrated and regulated by the mononuclear leukocyte Thl/Th2 derived cytokine network. Thus, it was interesting for us to evaluate the effects of MLIF on lymphocyte activation and Thl/Th2 cytokine production. Additionally, it has been suggested that *E. histolytica* invasion occurs within a territory where the Thl response can be inhibited, this is, in an unbalanced environment where Thl < Th2. In this experiment, we evaluated the *in vitro* effect of MLIF on the activation and production of Thl/Th2 intracellular cytokines (IL-1β, IL-2, INF-γ IL-4, and IL-10) and the relation with the chemokine receptors CCR4 and CCR5 in human CD4+ T cells. Peripheral blood samples were obtained from healthy, nonsmoking adult volunteer donors of both sexes. The peripheral blood mononuclear cells were obtained by Ficoll-Hypaque (Sigma Chemical Co.,

markers and assays for measuring the expression of intracellular cytokine. Flow cytometers are laser-based cell counters that are capable of distinguishing 3, 4, 5 or more (depending of flow cytometer), different fluorescence emissions, each associated with a particle identified by its light scatter proprieties. Fluorescence dyes with distinct fluorescence emissions are

Traditionally, cytokines have been measured by radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA). Unfortunately, these techniques are limited by their detection range and an inability to simultaneously measure multiple analytes (García, 1999). Using extremely sensitive multiparameter flow cytometers, Multiplexed Cytokine Immunoassay Kits overcome both of these limitations. Multiplexing is the simultaneous assay of many analytes in a single sample. Applications for flow cytometry are diverse, ranging from simple cell counting and viability to more complex studies of immune function, apoptosis and cancer, stem cells, separation of cells populations such as monocytes and T and B lymphocytes, measuring changes in cell surface markers, cell cycle analysis, cellular activation, and measuring the expression of intracellular cytokines (Collins et al.,

Activation of lymphocytes is a complex yet finely regulated cascade of events that results in the expression of cytokine receptors, the production and secretion of cytokines and the expression of several cell surface molecules, eventually leading to divergent immune responses. Parasite-specific immune responses are regulated by cytokines and chemokines. They modulate and direct the immune response, but may also contribute to an infection induced by the pathogenesis and parasite persistence (Talvani et al*.,* 2004). Parasitic infections frequently result in highly polarized CD4+ T cell responses, characterized by Th1 or Th2 cytokine dominated production profiles. Although it was previously thought that these infections were strictly dependent on signaling by cytokines, such as IFN-γ, IL-12 and IL-4, recent data indicate that this polarization may be primarily directed by a series of different factors intrinsic to the pathogen–antigen-presenting-cell interaction that directs T cell priming, and that all of this is influenced by the local environment (Katzman et al., 2008). The infection caused by the *E. histolytica* parasite is associated with an acute inflammatory response (Chadee & Meerovitch, 1984). However, it is not completely clear how *E. histolytica* triggers the host inflammatory response or how host-parasite interactions

During inflammation, leukocytes are orchestrated and regulated by the mononuclear leukocyte Thl/Th2 derived cytokine network. Thus, it was interesting for us to evaluate the effects of MLIF on lymphocyte activation and Thl/Th2 cytokine production. Additionally, it has been suggested that *E. histolytica* invasion occurs within a territory where the Thl response can be inhibited, this is, in an unbalanced environment where Thl < Th2. In this experiment, we evaluated the *in vitro* effect of MLIF on the activation and production of Thl/Th2 intracellular cytokines (IL-1β, IL-2, INF-γ IL-4, and IL-10) and the relation with the chemokine receptors CCR4 and CCR5 in human CD4+ T cells. Peripheral blood samples were obtained from healthy, nonsmoking adult volunteer donors of both sexes. The peripheral blood mononuclear cells were obtained by Ficoll-Hypaque (Sigma Chemical Co.,

attached to monoclonal antibody that recognizes distinct cell surface antigens.

1998; McHugh, 1994; Spagnoli,et al., 1993; Trask et al., 1982).

start, modulate, and eventually turn off the inflammatory response.

**3. Cell activation** 

Louis, MO) and CD4+ T lymphocytes were obtained by negative selection technique (MACS® Reagents, Kit isolation, Human cell T CD4+). The purity of lymphocytes was analyzed by flow cytometry. The flow cytometry measures and analyzes the optical properties of individual cells pass through a laser beam. Depending on how cells interact with the laser beam, the cytometer measures five parameters for each cell: size (forward scatter, FSC), complexity (side scatter, SSC) and three fluorescence emissions (FL-1, FL-2 and FL -3). An electro-optical system converts the voltage signals, which is translated into a digital value which is stored in a computer, the data are then retrieved and analyzed with the software that combines information from different cells in statistical charts, which measure individual parameters (histograms) or two parameters at a time (dot plot, density or contour). For our study, purified lymphocytes were analyzed in a dot plot of SSC vs. FSC marking the region corresponding to lymphocytes, excluding debris and dead cells. In a dot plot is compensated for fluorescence with anti CD3-FITC and anti-CD4-PE (cluster of differentiation (CD) and marker for T lymphocytes CD4+).

Test samples of at least 10.000 events were acquired under these conditions. With this procedure, we obtained a population of CD4+ lymphocytes with 96% purity. (Figure 4)

Fig. 4. Simple analysis of CD4+ T cells obtained from healthy individuals by flow cytometry. The X-axis shows staining with fluorescein isothiocyanate (FITC), and the Y-axis shows staining for phycoerythrin (PE). a) Autofluorescence; b) Isotype control, staining with mouse IgG1-FITC; c) Staining for subpopulations of CD3 coupled to FITC; d) Staining for subpopulations of CD4 coupled to PE. All stains show simple representation of the histogram and are an example of 6 experiments ± SE

The presence or absence of chemokine receptors on cell surfaces also provides information regarding the cell's state of activation. Chemokine receptors can by analyzed by flow cytometry using fluorescently labeled anti-receptor antibodies or fluorescently-labeled chemokines. Combining these reagents with antibodies against the activation marker CD69

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

levels in response to MLIF and, when co-expressed, the increase was even greater, demonstrating that MLIF possessed an additive effect on these markers (Figure 6) (Rojas-

Fig. 6. Expression profiles of CCR4, CCR5, and CCR4/CCR5 on isolated CD4+ T cells. 5 × 105 CD4+ T lymphocytes were cultured for 24 h with RPMI or MLIF (50 μg/mL). Cells were stained with PE or FITC anti- human CCR4, anti-human CCR5, or anti-human CCR4/CCR5 mAbs. Box plots represent range, 25th and 75th percentiles, and vertical lines represent the 10th and 90th percentiles of data. Horizontal bars show significant statistical differences among the different groups. NS = no significant difference. Values (p) were calculated using a Mann-Whitney Test. Dot plots show the co-expression of CCR4/CCR5, and bold numbers

The effect of MLIF upon the production of intracellular cytokines was evaluated using a quantitative method of flow cytometry. This was used to assess the production of IL-lβ, IL-2, IFN-γ, IL-4, and IL-10. CD4+ T cells were cultured in 24-well plates in RPMI-1640 medium (supplemented with fetal calf serum (FCS), L-glutamine, streptomycin, gentamicin, and sodium pyruvate) with PMA alone or in conjunction with MLIF for 24 h at 37 ºC with 5% CO2. Cell viability was ≥ 90% determined by trypan blue dye (Sigma) exclusion. Once CD4+ T lymphocytes were activated, we determined if the effect of MLIF on cytokine production was related to a Th1 or Th2 cytokine pattern. To stain for intracellular cytokine expression, lymphocytes are labeled with anti-CD antibodies to identify cells by their subset, such as helper lymphocytes, B lymphocytes and cytotoxic lymphocytes. The cells are then stabilized by fixation with formaldehyde. Holes are punched in the cell membrane by detergent to enable the passage of anti-cytokine antibodies to the interior of the cells. By three-color flow cytometry analysis, activated T- lymphocytes can be subdivided into several different populations according to their staining characteristics. CD4 and CD3 positive and negative cells populations are identified using a FITC or PE-labeled anti-CD4 or CD3 antibody, which labels the cell surface. Following the permeabilization step, intracellular cytokines are stained with anti-human mAbs directed against IL-1β, IL-2, IFN-γ, IL-4, and IL-10, and Th1 and Th2-associated cytokine-producing lymphocytes can be counted on a flow cytometer. This procedure helps to differentiate between Th1 (IFN-γ producing) and Th2 (IL-4 producing)

Lymphocytes Measured … 9

Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

are the mean of three independent experiments

**5. Intracellular cytokines** 

Dotor et al., 2009).

enables analysis of cell activation within specific cell population. Figure 5 shows that the best activation was obtained with 50 ng of phorbol 12-myristate 13-acetate (PMA) and 50 μg of MLIF.

CD69 is a cell surface activation marker expressed on T cells, B cells, and activated NK cells. MLIF is able to induce expression of this marker, suggesting that it activates CD4+ T lymphocytes. T-lymphocyte activation is also associated with an up-regulation of cell surface chemokine receptors. (Figure 5)

Fig. 5. Expression of the chemokine receptor CXCR3 and the activation marker CD69

Cell surface expression of the chemokine receptor CXCR3 and the activation marker CD69 on CD4+ T cells after 24 hours of treatment with RPMI medium alone or activation with PMA and MLIF at different concentrations. The cell population positive for both CXCR3 and CD69 were identified using a FITC-labeled anti-CD69.

#### **4. Cell surface molecules**

Cellular activation may modify the expression of chemokines and chemokine receptors, which are essential for leukocyte recruitment during inflammation. Once activated, T lymphocytes acquire different migratory capacities and are necessary for efficient immune response regulation (Mackay, 1993; Katakai et al., 2002). CCR5 is a receptor that regulates normal activation, and it was expressed along with the tested Th1 cytokines. However, MLIF exposure inhibited these cells and induced significant decreases in production of IFNγ and IL-1β. IFN-γ exerted a strong influence on Th1/Th2 polarization, and also affected chemokine receptor expression. MLIF induced an increase in CCR5 and CCR4 expression; however, this increase was only significant for the first. The observed CCR5 increase was greater in CCR4+ cells than in CCR4- cells (31% vs. 7%). The increases in CCR5 expression cannot be considered as a pro-Th1 response. The chemokine receptors, which are key factors in immune regulation, are influenced by MLIF. Th2 cells exhibited high CCR4 expression levels in response to MLIF and, when co-expressed, the increase was even greater, demonstrating that MLIF possessed an additive effect on these markers (Figure 6) (Rojas-Dotor et al., 2009).

Fig. 6. Expression profiles of CCR4, CCR5, and CCR4/CCR5 on isolated CD4+ T cells. 5 × 105 CD4+ T lymphocytes were cultured for 24 h with RPMI or MLIF (50 μg/mL). Cells were stained with PE or FITC anti- human CCR4, anti-human CCR5, or anti-human CCR4/CCR5 mAbs. Box plots represent range, 25th and 75th percentiles, and vertical lines represent the 10th and 90th percentiles of data. Horizontal bars show significant statistical differences among the different groups. NS = no significant difference. Values (p) were calculated using a Mann-Whitney Test. Dot plots show the co-expression of CCR4/CCR5, and bold numbers are the mean of three independent experiments

#### **5. Intracellular cytokines**

8 Clinical Flow Cytometry – Emerging Applications

enables analysis of cell activation within specific cell population. Figure 5 shows that the best activation was obtained with 50 ng of phorbol 12-myristate 13-acetate (PMA) and 50 μg

CD69 is a cell surface activation marker expressed on T cells, B cells, and activated NK cells. MLIF is able to induce expression of this marker, suggesting that it activates CD4+ T lymphocytes. T-lymphocyte activation is also associated with an up-regulation of cell

Fig. 5. Expression of the chemokine receptor CXCR3 and the activation marker CD69

CD69 were identified using a FITC-labeled anti-CD69.

**4. Cell surface molecules** 

Cell surface expression of the chemokine receptor CXCR3 and the activation marker CD69 on CD4+ T cells after 24 hours of treatment with RPMI medium alone or activation with PMA and MLIF at different concentrations. The cell population positive for both CXCR3 and

Cellular activation may modify the expression of chemokines and chemokine receptors, which are essential for leukocyte recruitment during inflammation. Once activated, T lymphocytes acquire different migratory capacities and are necessary for efficient immune response regulation (Mackay, 1993; Katakai et al., 2002). CCR5 is a receptor that regulates normal activation, and it was expressed along with the tested Th1 cytokines. However, MLIF exposure inhibited these cells and induced significant decreases in production of IFNγ and IL-1β. IFN-γ exerted a strong influence on Th1/Th2 polarization, and also affected chemokine receptor expression. MLIF induced an increase in CCR5 and CCR4 expression; however, this increase was only significant for the first. The observed CCR5 increase was greater in CCR4+ cells than in CCR4- cells (31% vs. 7%). The increases in CCR5 expression cannot be considered as a pro-Th1 response. The chemokine receptors, which are key factors in immune regulation, are influenced by MLIF. Th2 cells exhibited high CCR4 expression

of MLIF.

surface chemokine receptors. (Figure 5)

The effect of MLIF upon the production of intracellular cytokines was evaluated using a quantitative method of flow cytometry. This was used to assess the production of IL-lβ, IL-2, IFN-γ, IL-4, and IL-10. CD4+ T cells were cultured in 24-well plates in RPMI-1640 medium (supplemented with fetal calf serum (FCS), L-glutamine, streptomycin, gentamicin, and sodium pyruvate) with PMA alone or in conjunction with MLIF for 24 h at 37 ºC with 5% CO2. Cell viability was ≥ 90% determined by trypan blue dye (Sigma) exclusion. Once CD4+ T lymphocytes were activated, we determined if the effect of MLIF on cytokine production was related to a Th1 or Th2 cytokine pattern. To stain for intracellular cytokine expression, lymphocytes are labeled with anti-CD antibodies to identify cells by their subset, such as helper lymphocytes, B lymphocytes and cytotoxic lymphocytes. The cells are then stabilized by fixation with formaldehyde. Holes are punched in the cell membrane by detergent to enable the passage of anti-cytokine antibodies to the interior of the cells. By three-color flow cytometry analysis, activated T- lymphocytes can be subdivided into several different populations according to their staining characteristics. CD4 and CD3 positive and negative cells populations are identified using a FITC or PE-labeled anti-CD4 or CD3 antibody, which labels the cell surface. Following the permeabilization step, intracellular cytokines are stained with anti-human mAbs directed against IL-1β, IL-2, IFN-γ, IL-4, and IL-10, and Th1 and Th2-associated cytokine-producing lymphocytes can be counted on a flow cytometer. This procedure helps to differentiate between Th1 (IFN-γ producing) and Th2 (IL-4 producing)

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

\**p* <0.05 (Mann-Whitney Test). Bold numbers (dot plots) represent the mean.

(white), MLIF (diagonals), PMA (dotted), and PMAM+ MLIF (black) treated cells and untreated cells represent mean values ± SEM. Asterisk shows comparison among groups,

The presence and regulation of cytokines and chemokines receptors were studied with MLIF. The cells were also stained to detect chemokine receptors and cytokine with the following combinations of mAb: anti-IL-1βPE/anti-CCR5FITC, anti-IL2FITC/anti-CCR5PE, anti-IFNγ PE/anti-CCR5FITC, anti-IL-4PE/anti-CCR4FITC and anti-IL-10FITC/anti-CCR4PE (PharMingen). 5 X105 CD4+ T cells from each group were incubated in 24-well plates for 24 h; 10 μg/ml brefeldin A were added and incubated for the last 6 hours. After incubation, cells were centrifuged for 5 min at 400g and supernatants were aspirated without disturbing pellets. Cells were washed with PBS/0.5% albumin/2mM EDTA then they were marked with mAb, and incubated for 20 min at 4°C in the dark, and fixed with 1% p-formaldehyde according to the manufacturer's instructions (PharMingen). Acquisition of 10,000 events was conducted in flow cytometry FACScan (BD Biosciences, Sa Jose, USA). For analysis, Facs Diva and Win MDI 2.8 software were used. The results showed that CD4+ T cells control 2% co-expressed IL-lβ/CCR5, IL-2/CCR5, and IFN-γ/CCR5, while 3% co-expressed IL-4/CCR4, and 1% co-expressed IL-10/CCR4. After stimulating CD4+ T cells with MLIF, 15% cells co-expressed IL-1β/CCR5, 21% IL-2/CCR5, and 16% IFN-γ/CCR5, while 18% co-expressed IL-4/CCR4 and 16% IL-10/CCR4. PMA increased the expression of all of them (24%, 28%, 23%, 32%, and 31% respectively) and the combination PMA+MLIF showed that MLIF inhibited significant IL-1β/CCR5 (p<0.05) and IFN-γ/CCR5 (p< 0.002) induced by

Lymphocytes Measured … 11

Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

**6. Cytokines and chemokine receptors** 

Fig. 8. Cytokine and chemokine receptor co-expression

PMA (figure 8).

cells in specific cell populations. MLIF increased the expression of IL-lβ, IL-2, IFN-γ, IL-4, and IL-10. Following PMA+MLIF treatment, the production of IFN-γ and IL-1β was inhibited compared to treatment with PMA alone. MLIF possessed the ability to nonspecifically activate CD4+ T cells, and it induced an increase in pro- and antiinflammatory cytokine production (IL-1β, IL-2, IFN-γ, IL-4, and IL-10) (Rojas-Dotor et al., 2006). In contrast, in PMA +MLIF-incubated cells, we found that IFN-γ and IL-1β production was inhibited and production of IL-10, the prototypical anti-inflammatory cytokine, was increased (Figure 7) (Rojas-Dotor et al., 2009). It is probable that MILF induces a signaling cascade, which results in the activation of transcription factors, such as nuclear factor kB (NF-kB) (Kretschmer et al., 2004). After its translocation into the nucleus, NF-kB binds to genomic sites that regulate a large number of genes implicated in cytokine production. In this way, *E. histolytica* could potentially first establish an acute transitory reaction involving pro-inflammatory cytokines, followed by an increase and dominant pattern of antiinflammatory signals mainly through increased IL-10. IL-10 could cause the decreased inflammatory reaction observed in the advanced states of invasive amoebiasis (Kretschmer et al., 1985).

Fig. 7. Intracellular cytokine production

5 × 105 CD4+ T lymphocytes were cultured for 24 h in the presence of RPMI, MLIF, PMA, or PMA+MLIF. Brefeldin A, a cellular transport inhibitor was added during the last 6 h of culture . Cells were permeabilized and stained with anti-human cytokine mAbs (IL-1β, IL-2, IFNγ, IL-4, and IL-10) or mouse anti-IgG as an isotype control. FACScan dot plots are representative of control and treated cells. The numbers in each quadrant indicate the mean of the 6 independent experiments. In A, B, C, D, and E, the histograms represent control (white), MLIF (diagonals), PMA (dotted), and PMAM+ MLIF (black) treated cells and untreated cells represent mean values ± SEM. Asterisk shows comparison among groups, \**p* <0.05 (Mann-Whitney Test). Bold numbers (dot plots) represent the mean.

#### **6. Cytokines and chemokine receptors**

10 Clinical Flow Cytometry – Emerging Applications

cells in specific cell populations. MLIF increased the expression of IL-lβ, IL-2, IFN-γ, IL-4, and IL-10. Following PMA+MLIF treatment, the production of IFN-γ and IL-1β was inhibited compared to treatment with PMA alone. MLIF possessed the ability to nonspecifically activate CD4+ T cells, and it induced an increase in pro- and antiinflammatory cytokine production (IL-1β, IL-2, IFN-γ, IL-4, and IL-10) (Rojas-Dotor et al., 2006). In contrast, in PMA +MLIF-incubated cells, we found that IFN-γ and IL-1β production was inhibited and production of IL-10, the prototypical anti-inflammatory cytokine, was increased (Figure 7) (Rojas-Dotor et al., 2009). It is probable that MILF induces a signaling cascade, which results in the activation of transcription factors, such as nuclear factor kB (NF-kB) (Kretschmer et al., 2004). After its translocation into the nucleus, NF-kB binds to genomic sites that regulate a large number of genes implicated in cytokine production. In this way, *E. histolytica* could potentially first establish an acute transitory reaction involving pro-inflammatory cytokines, followed by an increase and dominant pattern of antiinflammatory signals mainly through increased IL-10. IL-10 could cause the decreased inflammatory reaction observed in the advanced states of invasive amoebiasis (Kretschmer

5 × 105 CD4+ T lymphocytes were cultured for 24 h in the presence of RPMI, MLIF, PMA, or PMA+MLIF. Brefeldin A, a cellular transport inhibitor was added during the last 6 h of culture . Cells were permeabilized and stained with anti-human cytokine mAbs (IL-1β, IL-2, IFNγ, IL-4, and IL-10) or mouse anti-IgG as an isotype control. FACScan dot plots are representative of control and treated cells. The numbers in each quadrant indicate the mean of the 6 independent experiments. In A, B, C, D, and E, the histograms represent control

et al., 1985).

Fig. 7. Intracellular cytokine production

The presence and regulation of cytokines and chemokines receptors were studied with MLIF. The cells were also stained to detect chemokine receptors and cytokine with the following combinations of mAb: anti-IL-1βPE/anti-CCR5FITC, anti-IL2FITC/anti-CCR5PE, anti-IFNγ PE/anti-CCR5FITC, anti-IL-4PE/anti-CCR4FITC and anti-IL-10FITC/anti-CCR4PE (PharMingen). 5 X105 CD4+ T cells from each group were incubated in 24-well plates for 24 h; 10 μg/ml brefeldin A were added and incubated for the last 6 hours. After incubation, cells were centrifuged for 5 min at 400g and supernatants were aspirated without disturbing pellets. Cells were washed with PBS/0.5% albumin/2mM EDTA then they were marked with mAb, and incubated for 20 min at 4°C in the dark, and fixed with 1% p-formaldehyde according to the manufacturer's instructions (PharMingen). Acquisition of 10,000 events was conducted in flow cytometry FACScan (BD Biosciences, Sa Jose, USA). For analysis, Facs Diva and Win MDI 2.8 software were used. The results showed that CD4+ T cells control 2% co-expressed IL-lβ/CCR5, IL-2/CCR5, and IFN-γ/CCR5, while 3% co-expressed IL-4/CCR4, and 1% co-expressed IL-10/CCR4. After stimulating CD4+ T cells with MLIF, 15% cells co-expressed IL-1β/CCR5, 21% IL-2/CCR5, and 16% IFN-γ/CCR5, while 18% co-expressed IL-4/CCR4 and 16% IL-10/CCR4. PMA increased the expression of all of them (24%, 28%, 23%, 32%, and 31% respectively) and the combination PMA+MLIF showed that MLIF inhibited significant IL-1β/CCR5 (p<0.05) and IFN-γ/CCR5 (p< 0.002) induced by PMA (figure 8).

Fig. 8. Cytokine and chemokine receptor co-expression

Effect of Monocyte Locomotion Inhibitory Factor (MLIF) on the Activation and Production of

MLIF acts at the beginning of the inflammatory process as a nonspecific activator, inducing the production of both pro and anti-inflammatory cytokines. As inflammation progresses, Th2 cytokine production prevails, which may inhibit Th1 cytokines. The observed effect of MLIF in this study could be explained by Th1 inhibition, as decreases in IFN-γ, IL-1β, cytokine and IL-1β/CCR5, IFN-γ/CCR5 cytokine and chemokine receptor co-expression were observed along with increases in the Th2 factors IL-4/CCR4 and IL-10/CCR4,

The effects of MLIF on the expression of cell surface molecules and intracellular cytokine expression was made possible by the availability of a range of monoclonal anti- antibodies

The research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), México (No. 38104-M). We also wish to acknowledge American Journal Experts (AJE) for the

Abbas, A. & Lichtman, A. Ed. Saunders. (2004). *Cellular and molecular immunology*. ISBN

Aley, SB.; Scott, WA. & Cohn, ZA. (1980). Plasma membrane of Entamoeba histolytica. *J Exp* 

Campanelli, AP., Brodskyn, CI., Boaventura, V., Silva, C., Roselino, AM., Costa, J., Saldanha,

Chadee, K. & Meerovitch, E. (1984). The pathogenesis of experimentally induced amebic

Clark, S., Page, E., Ford, T., Metcalf, R., Pozniak, A., Nelson, M., Henderson, DC., Asboe, D.,

Collins, DP., Luebering, BJ. & Shaut, DM. (1998). T-lymphocyte functionality assessed by

D´Ambrosio, D. (2002). Role of chemokine receptors in allergic inflammation and new

D'Ambrosio, D., Panina-Bordignon, P. & Sinigaglia, F. (2003). Chemokine receptors in

AC., de Freitas, LA., De Oliveira, CI., Barral-Netto, M., Silva, JS. & Barral, A. (2010). Chemokines and chemokine receptors coordinate the inflammatory immune response in human cutaneous leishmaniasis. *Hum Immunol,* Vol. 71, No. 12, (Dec),

liver abscess in the gerbil (Meriones unguiculatus). *Am J Pathol*, Vol. 117, No. 1,

Gotch, F., Gazzard, BG. & Kelleher, P. (2011). Reduced T(H)1/T(H)17 CD4 T-cell numbers are associated with impaired purified protein derivative-specific cytokine responses in patients with HIV-1 infection. *Allergy Clin Immunol*. Vol. 128, No. 4,

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Intracellular Cytokine and Chemokine Receptors in Human T CD4<sup>+</sup>

resulting in a predominantly anti-inflammatory Th1<Th2 pattern.

critical review of the manuscript in English (EE.UU).

978848174710-2, Elsevier, EEUU.

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pp. 346-350

ISSN:0022-1759

*Cytometry*, Vol. 33, No. 2, (Oct), pp.249-55.

**8. Acknowledgements** 

**9. References** 

Cells were cultured with RPMI, MLIF, PMA, or PMA+MLIF for 24 h at the previously mentioned concentrations. Brefeldin A was added during the last 6 h of culture. The cells were first stained to detect the surface cell molecules with anti- human CCR5 or CCR4 mAbs. They were then permeabilized and stained with mAbs directed against IL-1β and CCR5, IL-2 and CCR5, IFNγ and CCR5, IL-4 and CCR4, or IL-10 and CCR4 and were analyzed on a flow cytometer. A, B, C, D, and E. FACScan dot plots are representative staining of the control and the treated cells, bold numbers represent the mean of the 6 additional experiments. The histograms represent control (white), MLIF (diagonals), PMA (dotted), and PMAM+ MLIF (black) treated cells and untreated cells represent mean values ± SEM. Asterisks indicate significant differences between the groups, \*p <0.05, \*\*p<0.002 (Mann-Whitney Test).

The precise mechanisms through which MLIF causes these biological effects are unknown, but it is known that MLIF interacts with human leukocytes by means of a mannosecontaining receptor (Kretschmer et al., 1991), and that it causes an increase in the number of pericentriolar microtubules and cytoplasmic AMPc concentration without concomitant GMPc diminution (Rico et al., 1995). Recent studies show that the MLIF does not interfere with programmed cell death or necrosis (Rojas-Dotor et al., 2011).

 Given the level of activity of the studied cytokines, we observed that MLIF acted to promote cell populations that express IL-2/IL-10 or IFN-γ/IL-10 and CCR4/CCR5 chemokine receptor. These effects have been previously reported and are associated with pro- and anti-inflammatory functions (Katsikis et al., 1995). In previous work, MLIF was found to inhibit the induction of CC, MIP-1α, MIP-1β, and I-309 chemokines, the CCR1 receptor (Utrera-Barrillas et al., 2003), and the IL-1β, IL-5, and IL-6 cytokines (Rojas-Dotor et al., 2006). This behavior may be associated with the atypical inflammation observed in invasive amoebiasis in which there is a decrease in chemotaxis and disequilibrium in cytokine production. This conclusion is supported by observations *in vivo* in which MLIF notably decreased cellular infiltration and inflammatory cytokine expression.

#### **7. Conclusion**

Entamoeba *histolytica* produces Monocyte Locomotion Inhibitory Factor (MLIF), a pentapeptide with proven anti-inflammatory properties both *in vitro* and *in vivo*. MLIF may contribute to the exiguous inflammation observed in late amebic liver abscess, through effects exerted directly on monocytes, such as decreased locomotion and respiratory burst, or indirectly by modulating the production and expression of cytokines involved in mononuclear cell recruitment to the inflammatory focus. We evaluated the effect of MLIF on the expression of pro and anti-inflammatory CD4+ T cells after 24 h of incubation with RPMI, MLIF, PMA or PMA + MLIF. MLIF treatment increased expression of CD69 by these cells, from which we can infer that MLIF acts as an inducer or activator of CD4+ cells under these experimental conditions. The expression of the cytokines IL-1β, IFN-γ, IL-2, IL-4 and IL-10 and co-localization with the chemokine receptors IL-1 β/CCR5, IFN-γ/CCR5, IL-2/CCR5, IL-4/CCR4 and IL-10/CCR4 are induced by MLIF. While PMA-induced production of IL-1 β and IFN- γ was inhibited by MLIF, IL-2 production was not affected, in contrast to the expression of IL-10, which was increased by MLIF. The inhibitory effect of MLIF could be explained by two different and independent mechanisms: inhibition of proinflammatory cytokines such as IL-1 β and IFN-γ or increased expression of IL-10, with the concomitant increase in the suppressive effects attributed to IL-10.

MLIF acts at the beginning of the inflammatory process as a nonspecific activator, inducing the production of both pro and anti-inflammatory cytokines. As inflammation progresses, Th2 cytokine production prevails, which may inhibit Th1 cytokines. The observed effect of MLIF in this study could be explained by Th1 inhibition, as decreases in IFN-γ, IL-1β, cytokine and IL-1β/CCR5, IFN-γ/CCR5 cytokine and chemokine receptor co-expression were observed along with increases in the Th2 factors IL-4/CCR4 and IL-10/CCR4, resulting in a predominantly anti-inflammatory Th1<Th2 pattern.

The effects of MLIF on the expression of cell surface molecules and intracellular cytokine expression was made possible by the availability of a range of monoclonal anti- antibodies coupled to fluorochromes, such as FITC or PE, and analysis by flow cytometry.

#### **8. Acknowledgements**

The research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), México (No. 38104-M). We also wish to acknowledge American Journal Experts (AJE) for the critical review of the manuscript in English (EE.UU).

#### **9. References**

12 Clinical Flow Cytometry – Emerging Applications

Cells were cultured with RPMI, MLIF, PMA, or PMA+MLIF for 24 h at the previously mentioned concentrations. Brefeldin A was added during the last 6 h of culture. The cells were first stained to detect the surface cell molecules with anti- human CCR5 or CCR4 mAbs. They were then permeabilized and stained with mAbs directed against IL-1β and CCR5, IL-2 and CCR5, IFNγ and CCR5, IL-4 and CCR4, or IL-10 and CCR4 and were analyzed on a flow cytometer. A, B, C, D, and E. FACScan dot plots are representative staining of the control and the treated cells, bold numbers represent the mean of the 6 additional experiments. The histograms represent control (white), MLIF (diagonals), PMA (dotted), and PMAM+ MLIF (black) treated cells and untreated cells represent mean values ± SEM. Asterisks indicate significant differences between the groups, \*p <0.05, \*\*p<0.002

The precise mechanisms through which MLIF causes these biological effects are unknown, but it is known that MLIF interacts with human leukocytes by means of a mannosecontaining receptor (Kretschmer et al., 1991), and that it causes an increase in the number of pericentriolar microtubules and cytoplasmic AMPc concentration without concomitant GMPc diminution (Rico et al., 1995). Recent studies show that the MLIF does not interfere

 Given the level of activity of the studied cytokines, we observed that MLIF acted to promote cell populations that express IL-2/IL-10 or IFN-γ/IL-10 and CCR4/CCR5 chemokine receptor. These effects have been previously reported and are associated with pro- and anti-inflammatory functions (Katsikis et al., 1995). In previous work, MLIF was found to inhibit the induction of CC, MIP-1α, MIP-1β, and I-309 chemokines, the CCR1 receptor (Utrera-Barrillas et al., 2003), and the IL-1β, IL-5, and IL-6 cytokines (Rojas-Dotor et al., 2006). This behavior may be associated with the atypical inflammation observed in invasive amoebiasis in which there is a decrease in chemotaxis and disequilibrium in cytokine production. This conclusion is supported by observations *in vivo* in which MLIF

Entamoeba *histolytica* produces Monocyte Locomotion Inhibitory Factor (MLIF), a pentapeptide with proven anti-inflammatory properties both *in vitro* and *in vivo*. MLIF may contribute to the exiguous inflammation observed in late amebic liver abscess, through effects exerted directly on monocytes, such as decreased locomotion and respiratory burst, or indirectly by modulating the production and expression of cytokines involved in mononuclear cell recruitment to the inflammatory focus. We evaluated the effect of MLIF on the expression of pro and anti-inflammatory CD4+ T cells after 24 h of incubation with RPMI, MLIF, PMA or PMA + MLIF. MLIF treatment increased expression of CD69 by these cells, from which we can infer that MLIF acts as an inducer or activator of CD4+ cells under these experimental conditions. The expression of the cytokines IL-1β, IFN-γ, IL-2, IL-4 and IL-10 and co-localization with the chemokine receptors IL-1 β/CCR5, IFN-γ/CCR5, IL-2/CCR5, IL-4/CCR4 and IL-10/CCR4 are induced by MLIF. While PMA-induced production of IL-1 β and IFN- γ was inhibited by MLIF, IL-2 production was not affected, in contrast to the expression of IL-10, which was increased by MLIF. The inhibitory effect of MLIF could be explained by two different and independent mechanisms: inhibition of proinflammatory cytokines such as IL-1 β and IFN-γ or increased expression of IL-10, with the

with programmed cell death or necrosis (Rojas-Dotor et al., 2011).

notably decreased cellular infiltration and inflammatory cytokine expression.

concomitant increase in the suppressive effects attributed to IL-10.

(Mann-Whitney Test).

**7. Conclusion** 


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**2** 

*Fano (PU), Italy* 

**Applications of Flow** 

**Cytometry to Clinical Microbiology** 

*Laboratorio di Patologia Clinica, Ospedale S. Croce Fano A.O.R.M.N. Azienda Ospedali Riuniti Marche Nord* 

Barbara Pieretti, Annamaria Masucci and Marco Moretti

Microbiology in general and clinical microbiology in particular have witnessed important changes during the last few years. Traditional methods of bacteriology and mycology require the isolation of the organism prior to identification and other possible testing. In most cases, culture results are available in 48 to 72 h. Virus isolation in cell cultures and detection of specific antibodies have been widely used for the diagnosis of viral infections (*Weinstein, 2007*). These methods are sensitive and specific, but, the time required for virus isolation is quite long and is governed by viral replication times. Additionally, serological assays on serum from infected patients have often most limits in specificity and sensitivity. Life-threatening infections require prompt antimicrobial therapy and therefore need rapid and accurate diagnostic tests. Procedures which do not require culture and which detect the presence of antigens or the host's specific immune response have shortened the diagnostic time. More recently, the emergence of molecular biology techniques, particularly those based on nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. These techniques have led to a revolutionary change in many of the traditional routine tests used in clinical microbiology

The current organization of clinical microbiology laboratories is now subject to increased use of automation exemplified by systems used for detecting bacteremia, screening of urinary tract infections, antimicrobial susceptibility testing and antibody detection. To obtain better sensitivity and speed, manufacturers continuously modify all these systems. Nevertheless, the equipment needed for all these approaches is different, and therefore the initial costs,

Indeed, in recent years microbiological techniques have been increasingly complemented by

We have gotten used to consider the flow cytometry applicable only in the field of hematology, then associate it with clinical microbiology makes it even more mysterious.

Over the past forty years we have witnessed several attempts of application of the flow

cytometry to microbiology, with good results but also with many difficulties.

**1. Introduction** 

laboratories.

both in equipment and materials, are high.

technologies such as those provided by flow cytometry.


### **Applications of Flow Cytometry to Clinical Microbiology**

Barbara Pieretti, Annamaria Masucci and Marco Moretti *Laboratorio di Patologia Clinica, Ospedale S. Croce Fano A.O.R.M.N. Azienda Ospedali Riuniti Marche Nord Fano (PU), Italy* 

**1. Introduction** 

16 Clinical Flow Cytometry – Emerging Applications

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Microbiology in general and clinical microbiology in particular have witnessed important changes during the last few years. Traditional methods of bacteriology and mycology require the isolation of the organism prior to identification and other possible testing. In most cases, culture results are available in 48 to 72 h. Virus isolation in cell cultures and detection of specific antibodies have been widely used for the diagnosis of viral infections (*Weinstein, 2007*). These methods are sensitive and specific, but, the time required for virus isolation is quite long and is governed by viral replication times. Additionally, serological assays on serum from infected patients have often most limits in specificity and sensitivity. Life-threatening infections require prompt antimicrobial therapy and therefore need rapid and accurate diagnostic tests. Procedures which do not require culture and which detect the presence of antigens or the host's specific immune response have shortened the diagnostic time. More recently, the emergence of molecular biology techniques, particularly those based on nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. These techniques have led to a revolutionary change in many of the traditional routine tests used in clinical microbiology laboratories.

The current organization of clinical microbiology laboratories is now subject to increased use of automation exemplified by systems used for detecting bacteremia, screening of urinary tract infections, antimicrobial susceptibility testing and antibody detection. To obtain better sensitivity and speed, manufacturers continuously modify all these systems. Nevertheless, the equipment needed for all these approaches is different, and therefore the initial costs, both in equipment and materials, are high.

Indeed, in recent years microbiological techniques have been increasingly complemented by technologies such as those provided by flow cytometry.

We have gotten used to consider the flow cytometry applicable only in the field of hematology, then associate it with clinical microbiology makes it even more mysterious.

Over the past forty years we have witnessed several attempts of application of the flow cytometry to microbiology, with good results but also with many difficulties.

Applications of Flow Cytometry to Clinical Microbiology 19

cytometers). Some flow cytometers are able to physically separate cell subsets (sorting)

Fluorochromes can be classified according to their mechanism of action: those whose fluorescence increases with binding to specific cell compounds such as proteins (fluorescein isothiocyanate [FITC]), nucleic acids (propidium iodide [PI]), and lipids (Nile Red); those whose fluorescence depends on cellular physiological parameters (pH, membrane potential, etc.); and those whose fluorescence depends on enzymatic activity (fluorogenic substrates) such as esterases, peroxidases, and peptidases. Fluorochromes can also be conjugated to antibodies or nucleotide probes to directly detect microbial antigens or DNA and RNA

Several articles of literature propose flow cytometry as rapid diagnostic tool in the fight against infection (*Alvarez-Barrientos, 2000*). In fact this methodology can be used in the isolation of microbes and their identification, in the determination of antibodies to a particular pathogen in different stages of the disease and in direct detection of essential microbial components such as nucleic acids and proteins directly in clinical specimens (tissues, body fluids, etc.) and for evaluation of effectiveness of antimicrobial therapy in

Recently, the Sysmex UF-100 flow cytometer has been developed to automate urinalysis. *Penders et al*. have valuated this instrument to explore the possibilities of flow cytometry in the analysis of peritoneal dialysis fluid and have compared the obtained data with those of counting chamber techniques, biochemical analysis and bacterial culture (*Penders, 2004*); while *Pieretti et al.* have applied this technology at diagnosis of bacteriuria, for example

Several studies are reported in the literature concerning the use of flow cytometry to determine the presence of bacteria, viruses, parasites, etc, in a biological sample. In this

Microorganisms are small and they are very different in structure and function, and both

Conventionally, microorganisms are studied at the population scale because cultures of microbes are considered to be uniform populations which can be adequately described by average values. However, the availability of tools such as flow cytometry and image analysis which allow measurements to be made on individual cells has changed our perception of microbes within both the laboratory and the natural environment. Only a small proportion of the diversity of microorganisms has been identified and a smaller

Microbes cannot be investigated without technological assistance, meaning that methods such as microscopy and flow cytometry with appropriate fluorochomes are essential for the acquisition of both qualitative and quantitative information. Although these methods have become conventional tools in microbial cell biology and in the analysis of environmental samples, their use in investigations of bacteria is limited by the physical constraint of optical resolution. Application of cell markers is also a challenge, simply because the cells have only

these factors lead to technological and methodological problems in studying them.

based on their cytometric characteristics (cell sorters).

**3.1 Direct detection of bacteria, fungi, parasites, viruses** 

section we describe the techniques used for this purpose.

proportion still has been characterized through laboratory studies.

sequences (*see Table 1*).

general.

(*Pieretti, 2010*).

In particular, the problems encountered relate the difficulty of measuring microbes by flow due to their small size and point towards the development of instrumentation that has managed to overcome this limitation of standard instrumentation used for routine flow cytometry in different fields from microbiology.

The aim of this chapter is to provide a complete overview of the applications of flow cytometry in microbiology, referring mainly to what is published in the literature. Will be presented innovative methods and practical examples of applications of flow cytometry in different areas of microbiology following the scheme outlined in paragraphs listed below.

The authors report in paragraph "References" articles that offer important points of discussion to make useful chapter to the various professionals in the targeted book.

### **2. Flow cytometry and microbiology**

Flow cytometry is a powerful fluorescence based diagnostic tool that enables the rapid analysis of entire cell populations on the basis of single-cell characteristics (*Brehm-Stecher, 2004*). Flow cytometry (FCM) could be successfully applied in bacteremia and bacteriuria, for rapidly microorganism's detection on the basis of its cytometric characteristics. Although FCM offers a broad range of potential applications for susceptibility testing, a major contribution would be in testing for slow-growing microorganisms, such as mycobacteria and fungi.

This technique could also be applied to study the immune response in patients, in detection of specific antibodies and monitor clinical status after antimicrobial treatments.

In the last years of the 1990s, the applications of FCM in microbiology have significantly increased (*Fouchet, 1993*).

Earlier works had demonstrated the applicability of dual-parameter analysis (light scattered vs fluorescence coupled to cellular components as protein and DNA or auto-fluorescence) to discriminate among different bacteria in the same sample.

FCM has also been used in metabolic studies of microorganisms (es. autofluorescence due to NADPH and flavins as metabolic status markers), in DNA's analysis, protein, peroxide production, and intracellular pH, for count of live and dead bacteria and/or yeasts, and for the discrimination of gram-positive from gram-negative bacteria on the basis of the fluorescence emitted when the organisms are stained with two fluorochromes.

Also it offers the possibility to investigate in yeasts and bacteria the respective gene expression (*Alvarez-Barrientos, 2000*).

#### **3. Applications of flow cytometry to clinical microbiology**

FCM is an analytical method that allows the rapid measurement of light scattered (intrinsic parameters: cell size and complexity) and fluorescence emission produced by suitably illuminated cells (fluorochromes). The cells, or particles, are suspended in liquid and produce signals when they flow individually through a beam of light, and the results represent cumulative individual cytometric characteristics. An important analytical feature of flow cytometers is their ability to measure multiple cellular parameters (analytical flow

In particular, the problems encountered relate the difficulty of measuring microbes by flow due to their small size and point towards the development of instrumentation that has managed to overcome this limitation of standard instrumentation used for routine flow

The aim of this chapter is to provide a complete overview of the applications of flow cytometry in microbiology, referring mainly to what is published in the literature. Will be presented innovative methods and practical examples of applications of flow cytometry in different areas of microbiology following the scheme outlined in paragraphs listed below. The authors report in paragraph "References" articles that offer important points of

Flow cytometry is a powerful fluorescence based diagnostic tool that enables the rapid analysis of entire cell populations on the basis of single-cell characteristics (*Brehm-Stecher, 2004*). Flow cytometry (FCM) could be successfully applied in bacteremia and bacteriuria, for rapidly microorganism's detection on the basis of its cytometric characteristics. Although FCM offers a broad range of potential applications for susceptibility testing, a major contribution would be in testing for slow-growing microorganisms, such as mycobacteria

This technique could also be applied to study the immune response in patients, in detection

In the last years of the 1990s, the applications of FCM in microbiology have significantly

Earlier works had demonstrated the applicability of dual-parameter analysis (light scattered vs fluorescence coupled to cellular components as protein and DNA or auto-fluorescence) to

FCM has also been used in metabolic studies of microorganisms (es. autofluorescence due to NADPH and flavins as metabolic status markers), in DNA's analysis, protein, peroxide production, and intracellular pH, for count of live and dead bacteria and/or yeasts, and for the discrimination of gram-positive from gram-negative bacteria on the basis of the

Also it offers the possibility to investigate in yeasts and bacteria the respective gene

FCM is an analytical method that allows the rapid measurement of light scattered (intrinsic parameters: cell size and complexity) and fluorescence emission produced by suitably illuminated cells (fluorochromes). The cells, or particles, are suspended in liquid and produce signals when they flow individually through a beam of light, and the results represent cumulative individual cytometric characteristics. An important analytical feature of flow cytometers is their ability to measure multiple cellular parameters (analytical flow

of specific antibodies and monitor clinical status after antimicrobial treatments.

fluorescence emitted when the organisms are stained with two fluorochromes.

**3. Applications of flow cytometry to clinical microbiology** 

discriminate among different bacteria in the same sample.

discussion to make useful chapter to the various professionals in the targeted book.

cytometry in different fields from microbiology.

**2. Flow cytometry and microbiology** 

and fungi.

increased (*Fouchet, 1993*).

expression (*Alvarez-Barrientos, 2000*).

cytometers). Some flow cytometers are able to physically separate cell subsets (sorting) based on their cytometric characteristics (cell sorters).

Fluorochromes can be classified according to their mechanism of action: those whose fluorescence increases with binding to specific cell compounds such as proteins (fluorescein isothiocyanate [FITC]), nucleic acids (propidium iodide [PI]), and lipids (Nile Red); those whose fluorescence depends on cellular physiological parameters (pH, membrane potential, etc.); and those whose fluorescence depends on enzymatic activity (fluorogenic substrates) such as esterases, peroxidases, and peptidases. Fluorochromes can also be conjugated to antibodies or nucleotide probes to directly detect microbial antigens or DNA and RNA sequences (*see Table 1*).

Several articles of literature propose flow cytometry as rapid diagnostic tool in the fight against infection (*Alvarez-Barrientos, 2000*). In fact this methodology can be used in the isolation of microbes and their identification, in the determination of antibodies to a particular pathogen in different stages of the disease and in direct detection of essential microbial components such as nucleic acids and proteins directly in clinical specimens (tissues, body fluids, etc.) and for evaluation of effectiveness of antimicrobial therapy in general.

Recently, the Sysmex UF-100 flow cytometer has been developed to automate urinalysis. *Penders et al*. have valuated this instrument to explore the possibilities of flow cytometry in the analysis of peritoneal dialysis fluid and have compared the obtained data with those of counting chamber techniques, biochemical analysis and bacterial culture (*Penders, 2004*); while *Pieretti et al.* have applied this technology at diagnosis of bacteriuria, for example (*Pieretti, 2010*).

#### **3.1 Direct detection of bacteria, fungi, parasites, viruses**

Several studies are reported in the literature concerning the use of flow cytometry to determine the presence of bacteria, viruses, parasites, etc, in a biological sample. In this section we describe the techniques used for this purpose.

Microorganisms are small and they are very different in structure and function, and both these factors lead to technological and methodological problems in studying them.

Conventionally, microorganisms are studied at the population scale because cultures of microbes are considered to be uniform populations which can be adequately described by average values. However, the availability of tools such as flow cytometry and image analysis which allow measurements to be made on individual cells has changed our perception of microbes within both the laboratory and the natural environment. Only a small proportion of the diversity of microorganisms has been identified and a smaller proportion still has been characterized through laboratory studies.

Microbes cannot be investigated without technological assistance, meaning that methods such as microscopy and flow cytometry with appropriate fluorochomes are essential for the acquisition of both qualitative and quantitative information. Although these methods have become conventional tools in microbial cell biology and in the analysis of environmental samples, their use in investigations of bacteria is limited by the physical constraint of optical resolution. Application of cell markers is also a challenge, simply because the cells have only


Table 1. Features of same fluorescent molecules used in flow cytometry (*modified from Alvarez-Barrientos 2000*)

Applications of Flow Cytometry to Clinical Microbiology 21

a thousandth of the volume of a normal blood cell and correspondingly small amounts of cellular constituents. This is the reason why multicolor approaches in bacteria with small cell volumes will not work, as the close spatial interaction of the dyes prevents quantitative

*Mueller and Davey* (*2009*) have proposed a bibliometric analysis of flow cytometric studies in last forty-years in which appear that the role of flow cytometry in microbiology is steadily

A survey was made of the Web of Science database of the Institute for Scientific Information counting all papers whose topic database field contained the words flow and cytometr\* (es. citometry, citometric, etc) plus one or more of the following words: bacteri\*, microorganism, procaryot\* or yeast. The percentage of flow cytometry papers in general shows a steady growth after the 1990s, and in particular 8% of flow cytometry articles includes studies of

Earlier works had demonstrated the applicability of dual-parameter analysis to discriminate among different bacteria in the same sample. One parameter was light scattered (size), and the other was either fluorescence emission from fluorochromes coupled to cellular components (protein and DNA) or autofluorescence, or light scattered acquired from another angle. For example dual-parameter analysis of forward light scatter and red fluorescence signals (FSC-H vs FL3-H) allowed the discrimination between two species of *Candida,* as *Candida lusitaniae* and *Candida maltosa*, based on different fluorochrome staining backgrounds. These yeast species are indistinguishable by monoparametric analysis of

In addition it is possible the quantification of different protein amounts (measured as FITC fluorescence) to distinguish different microorganisms (bacteria and/or yeasts) present in mixed cultures by histogram representation (FITC fluorescence vs number of events); or use dual-fluorescence to discrimination of specific fungal spores. For example, *Alvarez-Barrientos*  et al. (2000) have proposed Calcofluor fluorescence vs PI fuorescence for detection of *Aspergillus*, *Mucor*, *Cladosporium*, and *Fusarium*. In particular, Calcofluor binds chitin in the spore wall, while PI stains nucleic acids. However, the use of several fluorochromes for direct staining or through antibody or oligonucleotide conjugates plus size detection is the

The simple and rapid assessment of the viability of a microorganism is another important aspect of FCM. The effect of environmental stress or starvation on the membrane potential of bacteria has been studied by several groups using fluorochromes that distinguish among

FCM has also been used in metabolic studies of microorganisms using autofluorescence due to NADPH and flavins as metabolic status markers. Other authors studied DNA, proteins, peroxide production, and intracellular pH, detection of live and dead bacteria and fungi, detection of gram-positive and gram-negative on the basis of the fluorescence emitted when

FCM has been extensively used for studying virus-cell interactions for cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, human immunodeficiency virus (HIV),

analysis (*Muller, 2009*).

forward light scatter or red autofluorescence.

nonviable, viable, and dormant cells.

and hepatitis B virus (HBV).

simplest way to visualize or identify microorganisms by FCM.

the organisms are stained with two fluorochromes, and gene expression.

increasing.

microbes.

**Applications Substrate Dye Excitation/ Emission Wavelength (λmax)nm** *SYTOX Green a,b*

*Propidium Iodide (PI)*

**Viability** *a* **DNA quantification**

*Alvarez-Barrientos 2000*)

 *b*

**RNA quantification** *c*

**Cell cycle studies** *d*

*Ethidium bromide b,d* 510-595 DNA-RNA *SYTO 13 a,b,d* 488-509 DNA (GC pairs) *Hoechst 33258/33342*

 *d*

340-450 DNA *Mithramycin d* 425-550

RNA *Pyronine Y c* 497-563 *Fluorescein isothiocyanate (FITC)* 495-525 *Texas Red* 580-620 Proteins

*Oregon Green Isothiocyanate* 496-526 Antigens *Antibodies labeled with flurochromes* 

Nucleotide sequences *Fluorescently labeled oligonucleotides Depends on fluorochrome conjugated Indo-1* 340-(398–485) *Fura-2* 340-549

*Fluor-3* 469-545 *BCECF* (460–510)–(520–610)

**Metabolic variations** pH *SNARF-1* 510-(587–635) *DIOC6(3)* 484-501 *Oxonol [DiBAC4(3)]* 488-525 **Antibiotic susceptibility Metabolic variations**

Membrane potential *Rhodamine 123* 507-529

**Cell wall composition Microbe detection**

Membrane oligosaccharides *Lectins*

Enzyme activities *Substrates linked to fluorochromes* Lipids *Nile Red e* (490–550)-(540–630) Vacuolar enzyme activity *Fun-1 e* 508-(525–590)

Chitin and other carbohydrate polymers *Calcofluor white f* 347-436

*Depends on fluorochrome conjugated* 

**Metabolic activity**

**Yeast metabolic state** *e*

**Fungal detection** *f*

**Microbe detection**

Table 1. Features of same fluorescent molecules used in flow cytometry (*modified from* 

**Ca2+ mobilization**

Ca2+

 *a,b,d*

504-525

536-625

a thousandth of the volume of a normal blood cell and correspondingly small amounts of cellular constituents. This is the reason why multicolor approaches in bacteria with small cell volumes will not work, as the close spatial interaction of the dyes prevents quantitative analysis (*Muller, 2009*).

*Mueller and Davey* (*2009*) have proposed a bibliometric analysis of flow cytometric studies in last forty-years in which appear that the role of flow cytometry in microbiology is steadily increasing.

A survey was made of the Web of Science database of the Institute for Scientific Information counting all papers whose topic database field contained the words flow and cytometr\* (es. citometry, citometric, etc) plus one or more of the following words: bacteri\*, microorganism, procaryot\* or yeast. The percentage of flow cytometry papers in general shows a steady growth after the 1990s, and in particular 8% of flow cytometry articles includes studies of microbes.

Earlier works had demonstrated the applicability of dual-parameter analysis to discriminate among different bacteria in the same sample. One parameter was light scattered (size), and the other was either fluorescence emission from fluorochromes coupled to cellular components (protein and DNA) or autofluorescence, or light scattered acquired from another angle. For example dual-parameter analysis of forward light scatter and red fluorescence signals (FSC-H vs FL3-H) allowed the discrimination between two species of *Candida,* as *Candida lusitaniae* and *Candida maltosa*, based on different fluorochrome staining backgrounds. These yeast species are indistinguishable by monoparametric analysis of forward light scatter or red autofluorescence.

In addition it is possible the quantification of different protein amounts (measured as FITC fluorescence) to distinguish different microorganisms (bacteria and/or yeasts) present in mixed cultures by histogram representation (FITC fluorescence vs number of events); or use dual-fluorescence to discrimination of specific fungal spores. For example, *Alvarez-Barrientos*  et al. (2000) have proposed Calcofluor fluorescence vs PI fuorescence for detection of *Aspergillus*, *Mucor*, *Cladosporium*, and *Fusarium*. In particular, Calcofluor binds chitin in the spore wall, while PI stains nucleic acids. However, the use of several fluorochromes for direct staining or through antibody or oligonucleotide conjugates plus size detection is the simplest way to visualize or identify microorganisms by FCM.

The simple and rapid assessment of the viability of a microorganism is another important aspect of FCM. The effect of environmental stress or starvation on the membrane potential of bacteria has been studied by several groups using fluorochromes that distinguish among nonviable, viable, and dormant cells.

FCM has also been used in metabolic studies of microorganisms using autofluorescence due to NADPH and flavins as metabolic status markers. Other authors studied DNA, proteins, peroxide production, and intracellular pH, detection of live and dead bacteria and fungi, detection of gram-positive and gram-negative on the basis of the fluorescence emitted when the organisms are stained with two fluorochromes, and gene expression.

FCM has been extensively used for studying virus-cell interactions for cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, human immunodeficiency virus (HIV), and hepatitis B virus (HBV).

Applications of Flow Cytometry to Clinical Microbiology 23

light scattering discrimination they detect the temporal aspects of sporulation, accurately quantify the proportion of the population participating in sporulation, and sort cultures into enriched populations for subsequent analysis. By coupling with nucleic acid staining (SYTO-9 plus PI), they effectively discriminated between different sporulation-associated phenotypes,

Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria (reproductively viable, metabolically active, intact, permeabilized) and can be readily applied to the enumeration of viable bacteria in a biological sample (*Khan, 2010*). Accurate determination of live, dead, and total bacteria is important in many microbiology

Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid

FCM makes specificity of different fluorochrome-labeled antibodies to binding at specific antigens present in the surface of microorganisms for their identification in short period of

The first fluorochome used to detect bacteria was ethidium bromide in association with

Live cells have intact membranes and are impermeable to dyes such as PI which only leaks into cells with compromised membranes, while thiazole orange (TO) is a permeant dye and enters all cells, live and dead, to varying degrees. With gram-negative organisms, depletion of the lipopolysaccharide layer with EDTA greatly facilitates TO uptake. Thus a combination of these two dyes provides a rapid and reliable method for discriminating live and dead bacteria. An intermediate or "injured" population can often be observed between

It is possible to create a gating strategy for bacterial populations (es. *Escherichia coli*) staining the sample with thiazole orange (TO) and propidium iodide (PI), and analyze FSC vs SSC dot plot. You can set liberally a region (R1) around the target population and another (R2) around the beads. Then you can analyze FL2 vs SSC dot plot setting another region (R3) around the stained bacteria. At this point you can observed FL1 vs FL3 dot plot gated on (R1 or R2) and R3, with regions set around the live, "injured" and dead bacterial populations. Very interesting is the work that *Khan et al.* have proposed in 2010 on enumeration of viable but non-culturable and viable-culturable Gram-Negative Bacteria using flow cytometry.

The traditional culture methods for detecting indicator and pathogenic bacteria in food and water may underestimate numbers due to sub-lethal environmental injury, inability of target bacteria to take up nutrient components in the medium, and other physiological factors which reduce culturability; however, these methods are also time-consuming and cannot detect non-culturable (VBNC) cells. An issue of critical about microbiology is the ability to detect viable but non-culturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods)

and by using FACS they were able to enrich for the various sporulation phenotypes.

**3.1.1.1 Bacterial detection and live/dead discrimination by flow cytometry** 

time (less than 2 h), but with the extent of availability of specific antibodies.

growth medium and to proliferate in liquid nutrient broths.

light-scatter signal, and the second was propidium iodide (PI).

applications.

the live and dead populations.

#### **3.1.1 Bacteria**

*Pianetti et al.* (*2005*) compared traditional methods (spectrophotometric and plate count) used in bacteria counting cells with FCM for the determination of the viability of *Aeromonas hydrophila* in different types of water. They studied the presence of a strain of *Aeromonas hydrophila* in river water, spring water, brackish water and mineral water.

Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The plate count method is a widely used technique for determining the bacterial charge, but it supplies information related only to viability and growth capacity; while the absorbance method have a sensitivity who appears to be correlated with microbiological culture density.

The flow cytometric nucleic acid double-staining protocol is based on simultaneous use of permeable fluorescent probes (SYBR Green dyes) and an impermeable fluorescent probe (PI) and can distinguish viable, membrane-damaged, and membrane-compromised cells.

The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to their results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

Similar study was proposed to *McHugh et al.* (*2007*) who investigated FCM for the detection of bacteria in cell culture production medium, using a nucleic acid stain, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. They analyzed different bacteria: Gram positive (*Microbacterium species*) and Gram negative (*Acinetobacter species, Burkholderia cepacia, Enterobacter cloacae, Stenotrophomonas maltophilia*) vegetative bacteria, and Gram positive spore former (*Bacillus cereus*).

*Loehfelm T.W.* (*2008*) proposed a new application of FCM: identification and characterization of protein associated to biofilm in *Acinetobacter baumannii*, an opportunistic pathogen that is particularly successful at colonizing and persisting in the hospital environment, able to resist desiccation and survive on inanimate surfaces for months (*Kramer, 2006*). The authors have identified a new *A. baumannii* protein, Bap, expressed on the surface of these bacteria that is involved in biofilm formation in static culture, and that is detectable with FCM applied the following settings: forward scatter voltage, E02 (log); side scatter voltage, 582 (log); FL1 voltage, 665 (log); event threshold, forward scatter 434 and side scatter 380.

*Weiss Nielsen and collaborators* (*2011*) proposed an interesting video-protocol for detection of *Pseudomonas aeruginosa* and *Saccharomyces cerevisiae* present in biofilm by flow cell system.

*Tracy et al.* (*2008)* described the development and application of flow-cytometric and fluorescence assisted cell-sorting (FACS) techniques for study endospore-forming bacteria. In particular, they showed that by combining flow-cytometry light scattering with nucleic acid staining it's possible discriminate, quantify, and enrich all sporulation associated morphologies exhibited by the endospore-forming anaerobe *Clostridium acetobutylicum*. By

*Pianetti et al.* (*2005*) compared traditional methods (spectrophotometric and plate count) used in bacteria counting cells with FCM for the determination of the viability of *Aeromonas hydrophila* in different types of water. They studied the presence of a strain of *Aeromonas* 

Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The plate count method is a widely used technique for determining the bacterial charge, but it supplies information related only to viability and growth capacity; while the absorbance method have a sensitivity who appears to be

The flow cytometric nucleic acid double-staining protocol is based on simultaneous use of permeable fluorescent probes (SYBR Green dyes) and an impermeable fluorescent probe (PI)

The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to their results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a

Similar study was proposed to *McHugh et al.* (*2007*) who investigated FCM for the detection of bacteria in cell culture production medium, using a nucleic acid stain, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. They analyzed different bacteria: Gram positive (*Microbacterium species*) and Gram negative (*Acinetobacter species, Burkholderia cepacia, Enterobacter cloacae, Stenotrophomonas maltophilia*) vegetative bacteria, and

*Loehfelm T.W.* (*2008*) proposed a new application of FCM: identification and characterization of protein associated to biofilm in *Acinetobacter baumannii*, an opportunistic pathogen that is particularly successful at colonizing and persisting in the hospital environment, able to resist desiccation and survive on inanimate surfaces for months (*Kramer, 2006*). The authors have identified a new *A. baumannii* protein, Bap, expressed on the surface of these bacteria that is involved in biofilm formation in static culture, and that is detectable with FCM applied the following settings: forward scatter voltage, E02 (log); side scatter voltage, 582 (log); FL1 voltage, 665 (log); event threshold, forward scatter 434 and side scatter 380.

*Weiss Nielsen and collaborators* (*2011*) proposed an interesting video-protocol for detection of *Pseudomonas aeruginosa* and *Saccharomyces cerevisiae* present in biofilm by flow cell system. *Tracy et al.* (*2008)* described the development and application of flow-cytometric and fluorescence assisted cell-sorting (FACS) techniques for study endospore-forming bacteria. In particular, they showed that by combining flow-cytometry light scattering with nucleic acid staining it's possible discriminate, quantify, and enrich all sporulation associated morphologies exhibited by the endospore-forming anaerobe *Clostridium acetobutylicum*. By

and can distinguish viable, membrane-damaged, and membrane-compromised cells.

viable but nonculturable state, which are not detectable by conventional methods.

*hydrophila* in river water, spring water, brackish water and mineral water.

correlated with microbiological culture density.

Gram positive spore former (*Bacillus cereus*).

**3.1.1 Bacteria** 

light scattering discrimination they detect the temporal aspects of sporulation, accurately quantify the proportion of the population participating in sporulation, and sort cultures into enriched populations for subsequent analysis. By coupling with nucleic acid staining (SYTO-9 plus PI), they effectively discriminated between different sporulation-associated phenotypes, and by using FACS they were able to enrich for the various sporulation phenotypes.

#### **3.1.1.1 Bacterial detection and live/dead discrimination by flow cytometry**

Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria (reproductively viable, metabolically active, intact, permeabilized) and can be readily applied to the enumeration of viable bacteria in a biological sample (*Khan, 2010*).

Accurate determination of live, dead, and total bacteria is important in many microbiology applications.

Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth medium and to proliferate in liquid nutrient broths.

FCM makes specificity of different fluorochrome-labeled antibodies to binding at specific antigens present in the surface of microorganisms for their identification in short period of time (less than 2 h), but with the extent of availability of specific antibodies.

The first fluorochome used to detect bacteria was ethidium bromide in association with light-scatter signal, and the second was propidium iodide (PI).

Live cells have intact membranes and are impermeable to dyes such as PI which only leaks into cells with compromised membranes, while thiazole orange (TO) is a permeant dye and enters all cells, live and dead, to varying degrees. With gram-negative organisms, depletion of the lipopolysaccharide layer with EDTA greatly facilitates TO uptake. Thus a combination of these two dyes provides a rapid and reliable method for discriminating live and dead bacteria. An intermediate or "injured" population can often be observed between the live and dead populations.

It is possible to create a gating strategy for bacterial populations (es. *Escherichia coli*) staining the sample with thiazole orange (TO) and propidium iodide (PI), and analyze FSC vs SSC dot plot. You can set liberally a region (R1) around the target population and another (R2) around the beads. Then you can analyze FL2 vs SSC dot plot setting another region (R3) around the stained bacteria. At this point you can observed FL1 vs FL3 dot plot gated on (R1 or R2) and R3, with regions set around the live, "injured" and dead bacterial populations.

Very interesting is the work that *Khan et al.* have proposed in 2010 on enumeration of viable but non-culturable and viable-culturable Gram-Negative Bacteria using flow cytometry.

The traditional culture methods for detecting indicator and pathogenic bacteria in food and water may underestimate numbers due to sub-lethal environmental injury, inability of target bacteria to take up nutrient components in the medium, and other physiological factors which reduce culturability; however, these methods are also time-consuming and cannot detect non-culturable (VBNC) cells. An issue of critical about microbiology is the ability to detect viable but non-culturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods)

Applications of Flow Cytometry to Clinical Microbiology 25

results were compared with established standard methods such as cell enumeration with fluorescence microscopy and colony-forming units on selective agar plates. Furthermore, the whole method was tested with spiked tap water, and the detection limit was determined.

Use of fluorescent stains or fluorogenic substrates in combination with FCM allows the detection and discrimination of viable culturable, viable nonculturable, and nonviable organisms, can be used to microbial analysis of milk. *Gunasekera et al.* (*2000*) have demonstrated the potential application of flow cytometers in milk analyses developing a rapid method (less than 60 minutes) for detecting of total bacteria (*Gunasekera, 2000* ). The authors have considered as potential contaminants of milk for represent gram-negative rods

Pure populations of *E. coli* and *S. aureus* were easily detected by FCM when they were suspended in phosphate-buffered saline (PBS), but when they were inoculated into ultraheat-treated (UHT) milk, no distinct separation appeared. This is due to the presence of proteins and lipid globules that can bind nonspecifically to fluorescent stains and interfere with staining and detection of bacteria. Treatment of milk by centrifugation to remove lipids without also treating samples with proteases was insufficient to allow definition of bacteria. For these reason the authors have applied enzymatic treatment with protease K or savinase to remove or modify proteins and thereby enable distinction of bacteria by flow cytometry. The FCM procedure described estimates numbers of total bacteria in the processed sample,

This study demonstrates the ability of FCM to determine total bacterial numbers after clearing of milk and staining of bacteria with a reaily available fluorescent stain (SSC vs green fluorescence). The sensitivity of the FCM procedure was ≤104 total bacteria ml of milk-1.

*Pianetti et al.* (*2005*) proposed a protocol for the determination of the viability of *Aeromonas hydrophila* in different types of water by flow cytometry and compared this results with classical methods as spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria, using simultaneous permeable (SYBR Green dyes) and impermeable fluorescent probe (PI). The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. Furthermore, it permits fast detection of bacteria that are in a viable but

FCM can be used to demonstrate multiplexed detection of bacteria and toxins using

Antibodies specific for selected bacteria and toxins were conjugated to the coded microspheres to achieve sensitive and selective binding and detection. The respective limits of detection for bacteria and toxin are different (*Kim, 2009*). The microflow cytometer can detect for *Escherichia coli*, *Listeria*, and *Salmonella* 103, 105, and 104 cfu/mL, respectively, while the limits of detection for the toxins as cholera toxin, staphylococcal enterotoxin B, and ricin

*Escherichia coli* and for gram-positive cocci *Staphylococcus aureus*.

since SYTO BC binds to live culturable, live non-culturable, and dead cells.

nonculturable state, which are not detectable by conventional methods.

were 1.6, 0.064, and 1.6 ng/mL respectively (*Kim, 2009*).

fluorescent coded microspheres.

are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed (*Davey, 1996*).

Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min (*Khan 2010*), using SYTO dyes with different fluorescent probes (SYTO 9, SYTO 13, SYTO 17, SYTO 40) for detection of total cells and PI for detection of dead cells.

*Khan et al*. (*2010*) reported a study using FCM methods to detect cells with intact and damaged membranes. They assumed that cells having intact membranes are live (VC) and those with damaged membranes are dead or theoretically dead (VBNC).

The main objective of this study was to establish the quickest, most accurate, and easiest ways to estimate the proportions of VBNC and VC states and dead cells, as indicated by membrane integrity of these four Gram-negative bacteria: *Escherichia coli* O157:H7, *Pseudomonas aeruginosa*, *Pseudomonas syringae*, and *Salmonella enterica* serovar *Typhimurium* (*Khan, 2010*).

The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

Similar analysis was proposed by McHugh (2007) for detection of Gram positive and Gram negative vegetative bacteria (*Acinetobacter species, Burkholderia cepacia, Enterobacter cloacae, Stenotrophomonas maltophilia, Mycobacterium species, and Bacillus cereus*).

Another way in which FCM can achieve direct diagnosis is by use of different-sized fluorescent microspheres coated with antibodies against microbes. In this case is possible determine the absolute count of bacteria per unit of volume present in the sample analyses using following equation:
