DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

IgG-aggregates rapidly upregulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in the generation of reactive oxygen species.

Autoimmune complexes are an important feature of several autoimmune diseases such as lupus, as they contribute to tissue damage through the activation of immune cells. Neutrophils, key players in lupus, interact with immune complexes through Fc gamma receptors (FcgR). Incubation of neutrophils with aggregated-IgGs caused degranulation and increased the surface expression of FcgRI within minutes in a concentration-dependent fashion. After 30 minutes, IgG aggregates (1 mg/mL) upregulated FcgRI by 4.95 ± 0.45-fold. FcgRI-positive neutrophils reached 67.24% ± 6.88% on HA-IgGs stimulated neutrophils, from 3.12% ± 1.62% in non-stimulated cells, ranking IgG-aggregates among the most potent known agonists. FcgRIIa, and possibly FcgRIIIa, appeared to mediate this upregulation. Also, FcgRI-dependent signaling proved necessary for reactive oxygen species (ROS) production in response to IgG-aggregates. Finally, combinations of bacterial materials with aggregates dramatically boosted ROS production. This work suggests FcgRI as an essential component in the response of human neutrophils to immune complexes leading to the production of ROS, which may help explain how neutrophils contribute to tissue damage associated with immune complex-associated diseases, such as lupus.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation.

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

TNF-α expression in neutrophils and its regulation by glycogen synthase kinase-3: a potentiating role for lithium.

Glycogen synthase kinase 3 (GSK-3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK-3 inhibitor. GSK-3α is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK-3 inhibition on the production of TNF-α by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF-α release. In lipopolysaccharide-stimulated human neutrophils, GSK-3 inhibitors mimicked the effect of LiCl, each potentiating TNF-α release after 4 h, in a concentration-dependent fashion, by up to a 3-fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK-3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK-3 inhibition markedly increases (by more than 3-fold) de novo TNF-α protein synthesis. Our findings shed light on a novel mechanism of control of TNF-α expression in neutrophils with GSK-3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases.

Characterization of the Na/HCO3(-) cotransport in human neutrophils.

BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

A tool to assist rheumatologists to engage their lupus patients: the Purple Butterfly.

Objective: Translating the highly technical medical jargon of SLE into understandable concepts for patients, their families and individuals without expertise in SLE is a serious challenge. To facilitate communication and enable self-management in SLE, we aimed to create an innovative visual tool, the Purple Butterfly. Methods: We selected clinically representative criteria for SLE and transposed them as graphical features in an attractive and meaningful visual. We developed a script in R programming language that automatically transposes clinical data into this visualization. We asked SLE patients from a local cohort about the relevance, usefulness and acceptability of this visual tool in an online pilot survey. Results: The innovative Purple Butterfly features 11 key clinical criteria: age; sex; organ damage; disease activity; comorbidities; use of antimalarials, prednisone, immunosuppressants and biologics; and patient-reported physical and mental health-related quality of life. Each Purple Butterfly provides the health portrait of one SLE patient at one medical visit, and the automatic compilation of the butterflies can illustrate a patient's clinical journey over time. All survey participants agreed that they would like to use the Purple Butterfly to visualize the course of their SLE over time, and 9 of 10 agreed it should be used during their medical consultations. Conclusion: The Purple Butterfly nurtures effective doctor-patient communication by providing concise visual summaries of lupus patients' health conditions. We believe the Purple Butterfly has the potential to empower patients to take charge of their condition, enhance healthcare coordination and raise awareness about SLE.

Pharmacological evidence that the inhibitory effects of prostaglandin E2 are mediated by the EP2 and EP4 receptors in human neutrophils.

Prostaglandin E2 (PGE2) is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis, and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of ROS production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Oncostatin M decreases interleukin-1 ß secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo.

Oncostatin M (OSM) is a pleiotropic cytokine of the IL-6 family and displays both pro-inflammatory and anti-inflammatory activities. We studied the impact of OSM on the gene activation profile of human synovial cells, which play a central role in the progression of inflammatory responses in joints. In synovial cells stimulated with lipopolysaccharide and recombinant human granulocyte-macrophage colony-stimulating factor, recombinant human OSM and native OSM secreted by human granulocytes both reduced the gene expression and secretion of IL-1ß and CXCL8, but increased that of IL-6 and CCL2. This impact on synovial cell activation was not obtained using IL-6 or leukaemia inhibitory factor. Signal transducer and activator of transcription-1 appeared to mediate the effects of OSM on stimulated human synovial fibroblasts. In the murine dorsal air pouch model of inflammation, OSM reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α in lining tissues, and their presence in the cavity. These results as a whole suggest an anti-inflammatory role for OSM, guiding inflammatory processes towards resolution.

Tibouchina granulosa Leaves Present Anti-Inflammatory Effect.

The ethanol extract (EE) prepared from the leaves of Tibouchina granulosa, and its fraction in ethyl acetate (fEA) were evaluated concerning their capacity to reduce inflammation in different experimental models. fEA was also studied concerning its chemical constituents. EE and fEA were assayed for their anti-inflammatory potential, using formalin-induced licking behavior and carrageenan-induced inflammation into the subcutaneous air pouch (SAP) models. Reduction in polymorphonuclear cells (PMN) activation was performed in freshly isolated PMN. Chromatographic analysis of fEA was performed by HPLC-DAD. Hispiduloside was isolated as the main constituent in fEA, and its quantity was estimated to be 39.3% in fEA. EE (30 mg/kg) significantly reduced the second phase of formalin-induced licking. fEA demonstrated a reduction in leukocyte migration into the SAP. EE and fEA drastically reduced cytokines (TNF-α, IL-1ß, and IFN-γ), nitric oxide (NO) production, in vitro PMN migration induced by C5a and IL-8, and TNF-α and IL-1ß gene expression. Taken together, our data indicate that either ethanol extract or its fEA fraction from leaves of T. granulosa present an anti-inflammatory effect, contributing to the pharmacological and chemical knowledge of this species and confirming the rationale behind its traditional use.

Osteoblast retraction induced by adherent neutrophils promotes osteoclast bone resorption: implication for altered bone remodeling in chronic gout.

Bone destruction in chronic gout is correlated with deposits of monosodium urate (MSU) crystals. Bone with MSU tophi were histopathologically shown to have altered remodeling and cellular distribution. We investigated the impact of neutrophils in bone remodeling associated with MSU and demonstrated that neutrophils, through elastase localized at their surface, induced retraction of confluent osteoblasts (OBs) previously layered on calcified matrix. This OB retraction allowed osteoclasts to resorb cell-free areas of the matrix. This neutrophil effect was concentration dependent and time dependent and required direct contact with OBs. Exposure of OBs to MSU greatly promoted neutrophil adherence to OBs. Neutrophil membrane at the contact zone with OBs showed concentrated fluorescence of dye PKH-67, indicating a cellular contact. Neutrophil-OB interaction increased the survival of neutrophils, reduced their release of lactoferrin in presence of MSU and did not change OB-mediated mineralization. The adhesion of neutrophils to OBs was heterotypic through neutrophil CD29/CD49d and OB-fibronectin peptide CS1. Leukotriene B4 (LTB4) and platelet-activating factor (PAF) were also involved in neutrophil adherence to OBs, as shown by the blocking effect of selective LTB4 and PAF receptor antagonists, and a cytosolic phospholipase A(2α) (cPLA(2α)) inhibitor. Blockade of CD49d/CS1 and inhibition of the cPLA(2α) had subadditive effects, reducing by 60% the adherence of neutrophils to OBs. Taken together, these data showed that neutrophil adhesion to MSU-activated OBs was mediated by the ß1 integrin CD29/CD49d-fibronectin peptide CS1 receptors and cPLA(2α)-derived metabolites and impacts on OB and osteoclast functions. These interactions could be involved in the local bone remodeling process of gout.

Proteinase-activated receptor-2 up-regulation by Fcgamma-receptor activation in human neutrophils.

We shed new light on the expression and function of the proteinase-activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR-2 and PAR-3 mRNA but not PAR-1 or PAR-4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up-regulated cell surface expression of PAR-2 in a concentration-dependent and time-dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR-2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR-2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR-2 up-regulated cells. Increase of cell-surface PAR-2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)-binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig-receptor activation, up-regulates PAR-2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR-2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.

Platelets release mitochondrial antigens in systemic lupus erythematosus.

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bß3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

[Neutrophil: foe or friend?]. / Le neutrophile : ennemi ou ami ?

Neutrophils are well-recognized phagocytes in the first line of host defense, and are also a major source of pro-inflammatory cytokines, chemokines and lipid mediators, thereby contributing to the onset and early orchestration of the inflammatory response. In contrast, recent studies indicate that neutrophils have tools to limit the magnitude and length of an inflammatory response, and may take part in engaging the resolution process. This article describes endogenous signals that may transform the phenotype of a neutrophil: from a pro-inflammatory cell to one that promotes resolution. Adenosine, an autacoid which can be found at high concentrations in inflammatory sites, inhibits several inflammatory functions of the neutrophil via engagement of the A2A receptor and reshapes the profile of lipid mediators and cytokines released, causing cells to terminate the release of pro-inflammatory signals while progressing toward resolution. These endogenous resolution pathways may represent a key target for better treatments of inflammatory diseases.

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils.

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.

The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis.

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet-activating factor, and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha, promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.

Immunomodulatory impact of the A2A adenosine receptor on the profile of chemokines produced by neutrophils.

In LPS-stimulated human neutrophils, engagement of the adenosine A2A receptor selectively prevented the expression and release of TNF-alpha, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2alpha/CXCL2, and MIP-3alpha/CCL20. In mice lacking the A2A receptor, granulocytes that migrated into the air pouch 4 h after LPS injection expressed higher mRNA levels of TNF-alpha, MIP-1alpha, and MIP-1beta than PMNs from wild-type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL-1beta, TNF-alpha, IL-6, and MCP-2/CCL6 was higher in A2AR knockout mice. In addition to highlighting neutrophils as an early and pivotal target for mediating adenosine anti-inflammatory activities, these results identify TNF-alpha and the MIP chemokine family as gene products whose expression is pivotally affected by activation of A2AR in LPS-activated PMNs. Modulation by A2AR in the production of inflammatory signals by PMNs may thus influence the evolution of an inflammatory response by reducing the activation status of inflammatory cells.

Early tyrosine phosphorylation events following adenosine A2A receptor in human neutrophils: identification of regulated pathways.

Activation of the adenosine 2A receptor (A2AR) elevates intracellular levels of cAMP and acts as a physiologic inhibitor of inflammatory neutrophil functions. In this study, we looked into the impact of A2AR engagement on early phosphorylation events. Neutrophils were stimulated with well-characterized proinflammatory agonists in the absence or presence of an A2AR agonist {3-[4-[2-[ [6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl] phenyl] propanoic acid (CGS 21680)}, PGE2, or a mixture of the compounds RO 20-1724 and forskolin. As assessed by immunoblotting, several proteins were tyrosine phosphorylated; CGS 21680 markedly decreased tyrosine phosphorylation levels of 4 regions (37-45, 50-55, 60, and 70 kDa). Key signaling protein kinases-p38 MAPK, Erk-1/2, PI3K/Akt, Hck, and Syk-showed decreased phosphorylation, whereas Lyn, SHIP-1, or phosphatase and tensin homolog (PTEN) was spared. PGE2 or the intracellular cAMP-elevating combination of RO 20-1724 and forskolin mostly mimicked the effect of CGS 21680. Together, results unveil intracellular signaling pathways targeted by the A2AR, some of which might be key in modulating neutrophil functions.

Age-related decline of the acute local inflammation response: a mitigating role for the adenosine A2A receptor.

Aging is accompanied by an increase in markers of innate immunity. How aging affects neutrophil functions remains of debate.The adenosine A2A receptor (A2AR), essential to the resolution of inflammation, modulates neutrophil functions. We sought to determine whether or not A2AR protects against the effects of aging. We monitored neutrophil influx, viability, and activation as well as cytokine accumulation in wild-type (WT) and A2AR-knockout mice (KO) at three different ages.Several readouts decreased with aging: neutrophil counts in dorsal air pouches (by up to 55%), neutrophil viability (by up to 56%), elastase and total protein in exudates (by up to 80%), and local levels of cytokines (by up to 90%). Each of these parameters was significantly more affected in A2AR-KO mice. CXCL1-3 levels were largely unaffected. The effects of aging were not observed systemically. Preventing neutrophil influx into the air pouch caused a comparable cytokine pattern in young WT mice. Gene expression (mRNA) in leukocytes was affected, with CXCL1 and CCL4 increasing and with TNF and IL-1α decreasing.ConclusionAging has deleterious effects on the acute inflammatory response and neutrophil-related activities, and defective migration appears as an important factor. A functional A2AR signaling pathway delays some of these.

Rapid and simple comparison of messenger RNA levels using real-time PCR.

Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species.