Marcelo Nociari

A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity

In this study, a fluorometric method using alamarBlue has been developed for detecting cell-mediated cytotoxicity in vitro. AlamarBlue is a non-toxic metabolic indicator of viable cells that becomes fluorescent upon mitochondrial reduction. Specific lysis of targets by effector cells is quantified by comparing the total number of viable cells in wells containing effector and targets together, with wells where target and effector cells were separately seeded. Cell-mediated cytotoxic activity by alloreactive T cells and natural killer cells has been detected using a novel application of the alamarBlue technique. The assay that we have developed to detect cell-mediated cytotoxicity is extremely sensitive and specific and requires a significant lower number of effector cells than the standard 51Cr assay. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile conditions, effector cells may be recovered at the end for further analysis or cell expansion, if desired. Direct comparison of cell-mediated cytotoxicity measured by the alamarBlue method with the standard 51Cr release assay revealed that the former method is as specific and more sensitive than the conventional assay. Moreover, very small inter and intra-assay variations have been observed for alamarBlue cytotoxicity assays. In conclusion, this study shows that the alamarBlue assay is an extremely sensitive, economical, simple and non-toxic procedure to evaluate cell-mediated cytotoxicity that yields accurate results using a limited number of effector cells. Furthermore, since this assay is a one-step procedure, and does not involve any risk for the personnel, it may be useful to analyze automatically cell-mediated cytotoxicity in a large number of samples.

Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator

ABSTRACT Infection with adenovirus vectors (AdV) results in rapid activation of innate immunity, which serves the dual purpose of stimulating inflammatory antiviral host defenses and the adaptive immune system. Viral recognition by macrophages, dendritic cells, and other cell types requires an ability to sense the presence of a foreign molecular pattern by “pattern recognition receptors.” The nature of the adenoviral sensor, the target ligand of the sensor, and the downstream antiviral signaling response triggered by virus infection have not been defined for this nonenveloped double-stranded DNA (dsDNA) virus. We have identified four critical links involved in AdV recognition by murine antigen-presenting cells (APC) and primary lung fibroblasts: (i) viral recognition occurs chiefly via a Toll-like receptor (TLR)-independent nucleic acid-sensing mechanism recognizing the viral dsDNA genome, (ii) the intact viral particle and capsid proteins are required for efficient intracellular delivery of the viral genome, (iii) delivery of the viral genome triggers interferon regulatory factor 3 (IRF3) phosphorylation, and (iv) IRF3 activation is the required dominant antiviral signaling pathway used by APC, whereas the “primary” involvement of NF-κB, mitogen-activated protein kinase, or Akt pathways is less prominent. In this study we provide the first direct evidence that infection by a dsDNA virus stimulates an IRF3-mediated interferon and proinflammatory response through a TLR-independent DNA-sensing mechanism.

Tuberculosis Is Associated with a Down-Modulatory Lung Immune Response That Impairs Th1-Type Immunity

Immune mediators associated with human tuberculosis (TB) remain poorly defined. This study quantified levels of lung immune mediator gene expression at the time of diagnosis and during anti-TB treatment using cells obtained by induced sputum. Upon comparison to patients with other infectious lung diseases and volunteers, active pulmonary TB cases expressed significantly higher levels of mediators that counteract Th1-type and innate immunity. Despite the concomitant heightened levels of Th1-type mediators, immune activation may be rendered ineffectual by high levels of intracellular (SOCS and IRAK-M) and extracellular (IL-10 and TGF-βRII, IL-1Rn, and IDO) immune suppressive mediators. These modulators are a direct response to Mycobacterium tuberculosis as, by day 30 of anti-TB treatment, many suppressive factors declined to that of controls whereas most Th1-type and innate immune mediators rose above pretreatment levels. Challenge of human immune cells with M. tuberculosis in vitro up-regulated these immune modulators as well. The observed low levels of NO synthase-2 produced by alveolar macrophages at TB diagnosis, along with the heightened amounts of suppressive mediators, support the conclusion that M. tuberculosis actively promotes down-modulatory mediators to counteract Th1-type and innate immunity as an immunopathological strategy. Our data highlight the potential application of immune mediators as surrogate markers for TB diagnosis or treatment response.

Antibodies to Streptococcal Surface Enolase React with Human α-Enolase: Implications in Poststreptococcal Sequelae

The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.

Influence of Fiber Detargeting on Adenovirus-Mediated Innate and Adaptive Immune Activation

ABSTRACT The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1β) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium

Significance Lipofuscin accumulation in the retinal pigment epithelium (RPE) is a hallmark of aging. High lipofuscin levels in the RPE have been associated with retinal degeneration and blindness in Stargardt disease patients and animal models. Currently, there is no treatment to prevent and/or revert lipofuscin-driven retinal degenerative changes. In this study, we report that beta cyclodextrins, cyclic sugars composed of seven glucose units, can bind retinal lipofuscin, prevent its oxidation and remove it from RPE. This study opens an avenue to develop small drugs against, currently untreatable, lipofuscin-associated blinding disorders. Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (β-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of β-CD. Importantly, β-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of β-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of β-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Adenovirus Induction of IRF3 Occurs through a Binary Trigger Targeting Jun N-Terminal Kinase and TBK1 Kinase Cascades and Type I Interferon Autocrine Signaling

ABSTRACT Pathogen recognition is a critical function of immune sentinel cells. Naïve macrophages or dendritic cells (DCs) undergo pathogen-directed activation and maturation, and as mature antigen-presenting cells (APCs), they contribute essential functions to both innate and adaptive immunity. Using recombinant adenovirus (rAdV) as a model for murine APC activation by DNA viruses, we demonstrate a critical role for stress kinase activation in cell intrinsic and extrinsic antiviral signaling cascades. We propose two viral triggers, viral capsid and viral DNA, are required for APC activation. Endosomal escape and presentation of cytosolic rAdV DNA induces phosphorylation of TANK-binding kinase 1 (TBK1) at serine 172 but does not induce IκB kinase ε activity as determined by in vitro kinase assays. However, induction of TBK1 alone is not sufficient for interferon regulatory factor 3 (IRF3) phosphorylation. We show that capsid-dependent activation of Jun N-terminal kinase (JNK) stress kinase is a necessary step, licensing TBK1 phosphorylation of IRF3 at Ser 396. A second later phase of JNK activity is required to coordinate phosphorylation of JNK-dependent transcription factors (c-Jun/ATF2) with activated IRF3 in the induction of primary IRF3-responsive transcripts. Finally, we demonstrate that maximal JNK/TBK1/IRF3 stimulation by rAdV depends on an intact type I interferon (IFN) signaling cascade. By requiring multiple viral triggers and type I IFN autocrine regulation, APCs have an inherent fail-safe mechanism against inappropriate activation and maturation.

Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes

Background and Objectives Dendritic cells (DCs) are promising adjuvants for clinical immunotherapy, but they are scantily distributed. Therefore, numerous in vitro methods have been developed to expand these cells while maintaining their normal functions. Current culture systems generally require the use of fetal bovine serum (FBS)‐supplemented media in order to attain DCs with high immunostimulatory activity. However, the presence of exogenous animal proteins sets limits for their use in clinical trials. The purpose of this study was to establish a simple, efficient and FBS‐free method for the generation of human DCs for clinical application.

Tumor Necrosis Factor Alpha‐Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

Especially when exposed to inflammatory stimuli, endothelial cells (EC) have been shown to promote the maturation of monocytes into dendritic cells (DC) and the long‐term proliferation of CD34+ cells by constitutive cytokine production and direct cellular contact. We therefore hypothesized that cytokine‐stimulated EC would induce hematopoietic progenitor cells to develop into mature dendritic cells. To test this theory, human CD34+ cells derived from cord blood or leukapheresis products were cultured with a monolayer of either interleukin (IL)‐1β, IL‐4, or tumor necrosis factor (TNF)‐α‐stimulated human umbilical cord EC. The cells in suspension were analyzed weekly over a period of 6 weeks. IL‐1β supported cell expansion, whereas IL‐4 had no effect on cell expansion or DC differentiation. Only TNF‐α‐stimulated EC induced the development of mature, allostimulatory DC with a high expression of CD83, HLA‐DR, CD1a, and costimulatory molecules like CD80 and CD86. Acute myeloid leukemia cells from the cell line Kasumi‐1 also developed DC‐like features when cocultured with TNF‐α‐stimulated EC. Direct contact between endothelial and progenitor cells increased the number of developing DC. Cell cycle analysis and apoptosis studies demonstrated a reduced G2M fraction, an increased S fraction, and a decrease in TNF‐α‐dependent apoptosis of DC developing in the presence of endothelial cells. As shown by electron and confocal microscopic studies, intimate interactions between EC and DC occurred, resulting in the internalization of the developing DC within the EC monolayer and a bidirectional exchange of proteins. We conclude that, via the action of TNF‐α, inflamed human endothelium can induce CD34+ and leukemic cells to differentiate into dendritic cells.