Quantification Of Cytokine Gene Expression Research Articles

Nanogold morphologies with the same surface chemistry provoke a different innate immune response: An in-vitro and in-vivo study

Gold nanomaterials (GNMs) have unique optical properties with less antigenicity, and their physicochemical properties have strong relation with an immunological response at bio-interface including antigenicity. An interpretation of this correlation would significantly impact on the clinical and theranostic applications of GNMs. Herein, we studied the effect of GNMs morphology on the cytotoxicity (in-vitro), innate immune responses, hepatotoxicity, and nephrotoxicity (in-vivo studies) using gold nano-cups (GNCs), porous gold nanospheres (PGNSs) and solid gold nano particles (SGNPs) coated with the same ligand to ensure similar surface chemistry. The cytotoxicity was assessed via sulfo-rhodamine B (SRB) assay, and the cytotoxicity data showed that morphological features at nanoscale dimensions like surface roughness and hollowness etc. have a significant impact on cellular viability. The biochemical and histopathological study of liver and kidney tissues also showed that all GNMs did not show any toxicity even at high concentration (100 μL). The relative quantification of cytokine gene expression of TNF-α, IFN-γ, IL-4, 1L-6, and 1L-17 (against each morphology) was checked after in-vivo activation in mice. Among the different nanogold morphologies, PVP stabilized GNCs (PVP-GNCs) showed the highest release of pro-inflammatory cytokines, which might be due to their high surface energy and large surface area for exposure as compared to other nanogold morphologies studied. The pro-inflammatory cytokine release could be suppressed by coating with some anti-inflammatory polymer, i.e., inulin. The in-vitro results of pro-inflammatory (TNF-α, IL-1) cytokines also suggested that all GNMs may induce activation of macrophages and Th1 immune response. The in-vivo activation results showed a decrease in mRNA expression of the cytokines (TNF-α, IFN-γ, IL-4, 1L-6 and 1L-17). Based on these findings, we proposed that the shape and morphology of GNMs control their immune response at nano-bio interface, and it must be considered while designing their role for different biomedical applications like immuno-stimulation and bio-imaging.

Immunological characterization of chitosan adjuvanted outer membrane proteins of Salmonella enterica serovar Typhi as multi-epitope typhoid vaccine candidate.

Outer membrane proteins (OMPs) of Gram-negative bacteria have been known as potential vaccine targets due to their antigenic properties and host specificity. Here, we focused on the exploration of the immunogenic potential and protective efficacy of total OMPs of Salmonella enterica serovar Typhi due to their multi epitope properties, adjuvanted with nanoporous chitosan particles (NPCPs). The study was designed to extrapolate an effective, low cost prophylactic approach for typhoid fever being getting uncontrolled in Pakistan due to emergence of extensively drug resistant (XDR) strains. The OMPs of two S. Typhi variants (with and without Vi capsule) alone and with nanoporous chitosan particles as adjuvant were comparatively analyzed for immunogenic potential in mice. Adaptive immunity was evaluated by ELISA and relative quantification of cytokine gene expression (IL4, IL6, IL9, IL17, IL10, TNF, INF and PPIA as house keeping gene) using RT-qPCR. Statistical analysis was done using Welch's test. The protection was recorded by challenging the immunized mice with 50% ×LD50 of S. Typhi. The Vi + ve-OMPs of S. Typhi showed the most promising results by ELISA and significantly high expression of IL-6, IL-10 and IL-17 and 92.5% protective efficacy with no detectable side effects. We can conclude that the OMPs of Vi + ve S. Typhi are the most promising candidates for future typhoid vaccines because of cost effective preparation without expensive purification steps and multi-epitope properties. Chitosan adjuvant may have applications for oral protein based vaccines but found less effective in injectable preparations.

Restricted Infection and Cytokine Expression in Primary Murine Astrocytes Induced by the H5N1 Influenza Virus

Central nervous system (CNS) dysfunction caused by infection with neurovirulent viruses is of interest, and may play an important role in many neurodegenerative diseases. Since neuronal functions are believed to be partly regulated by cytokines produced by astrocytes, neuroinflammation and neurodegeneration caused by H5N1 influenza virus infection could be due to abnormal cytokine production of those infected astrocytes. In the present study, cytokine responses of murine astrocytes following H5N1 virus infection were investigated. Primary astrocytes from neonatal outbred ICR mice were isolated and used to investigate cytopathology upon infection with the H5N1 virus (A/Chicken/Thailand/CUK2/04). Thereafter, cell lysates at 6, 24, 48, and 72 hours-post infection (hpi) were collected and subjected to quantification of cytokine gene expression, including TNF-α, IL-1β, IL-2, IL-6 and IL-12β, by quantitative RT-PCR. The results revealed that infection with the H5N1 virus in primary murine astrocytes was restricted, and resulted in abortive virus infection. However, this abortive infection in the astrocytes was found to result in significant upregulation of IL-1β mRNA expression at 72 hpi compared with the mock-infected group, while the mRNA expression of IL-2 and IL-12β was observed to have undergone significant down-regulation at 6-72 hpi and 24-48 hpi, respectively. The results of the present study could support the role of astrocytes in neuroinflammation and neurodegeneration.

Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells

Cytokines are global mediators of cellular communications that are involved in broad array of biological processes, including the immunological and inflammatory mechanisms of virus–host interactions. Measuring the gene expression of simultaneously expressed cytokines is necessary for understanding the pathogenesis of many viral infections, including influenza. We developed a multiplex quantitative real-time PCR (qPCR) method for the detection of the following human cytokines: IL-1B, IL-2, IL-4, IL-6, IL-10, IL-12B, IL-18, IFN-γ and TNF. The assay consisted of three sets of multiple qPCRs; in each qPCR, three target cytokines and reference GAPDH genes were amplified. The assay provided a precise and sensitive quantification of cytokine gene expression with a 20fmol limit of detection and a 1.5% coefficient of variation. This method was successfully applied to cytokine profiling in epithelial A549 cells that were infected with A/California/07/09 (H1N1pdm2009) virus.

127: Possible role of IL-10 in the natural resistance of mouse to leptospirosis

Leptospirosis is a widespread zoonosis with more than 1 million cases of human contaminations estimated annually. The disease is polymorphic with asymptomatic forms or clinical manifestations ranging from mild to severe forms possibly leading to death. Using a comparative study between susceptible (hamster) and resistant (OF1 mouse) animals infected with virulent leptospires, we previously showed that the anti-inflammatory interleukin-10 (IL-10), although upregulated in both animals, was rapidly and massively overexpressed in mice. We aimed at better understanding the role of IL-10 in the disease pathophysiology, and its possible involvement in the resistance of mouse to leptospirosis. Using anti-IL-10 antibody (Ab) as a pre-infection treatment, we studied the effect of IL-10 neutralization on the resistance of the OF1 mice infected with virulent leptospires ( Leptospira interrogans serovar Icterohaemorrhagiae). Growth variation was evaluated as a clinical sign after infection. The presence of leptospires was investigated in kidneys at D7; IL-1β and TNF-α gene expression in blood was assessed at D3 using RT-qPCR techniques. While either infection with virulent leptospires or Ab treatment did not affect the growth curve of mice, IL-10 neutralization significantly influenced the mouse growth during the early days (D1 and D2) postinfection suggesting a susceptibility of animals. Leptospires were detected in the kidneys of 5/7 infected mice (71.5%) whereas only 2/8 of Ab-injected animals (25%) were found positive for renal carriage. Quantification of cytokine gene expression did not show regulation of neither IL-1β nor TNF-α in blood of leptospiremic mice at D3. However, both cytokines were significantly up-regulated in infected animals pre-treated with anti-IL-10 ab. Results suggest that IL-10 neutralization might impact on the resistance of mouse to Leptospira infection, possibly related to an up-regulation of IL-1β and TNF-α, illustrating the possible key role of this cytokine in the immune response to leptospirosis.

Quantitative gene expression of cytokines in peripheral blood leukocytes stimulated in vitro: modulation by the anti-tumor nerosis factor-alpha antibody infliximab and comparison with the mucosal cytokine expression in patients with ulcerative colitis

Emerging data indicate that alterations in cytokine synthesis play a role in inflammatory bowel disease (IBD) pathogenesis. In this study, we quantified mRNA expression of the main acute-phase cytokines and T-cell cytokines in biopsies from patients with established ulcerative colitis (UC) and compared it with that obtained in biopsies from normal controls. Quantification of cytokine gene expression was also evaluated in in vitro phytohemagglutinin (PHA)-treated peripheral blood leukocytes (PBLs) at the RNA and protein levels. The in vitro influence of the anti-tumor necrosis factor-alpha (TNF-alpha) antibody infliximab (INFL) on PHA-treated PBLs was also evaluated. Analyzing inflamed specimens from UC patients compared with control samples, interleukin (IL)-6 was sharply the most induced cytokine. Interestingly, similar results were found in activated PBLs, where acute-phase cytokines were more abundantly expressed compared with T-cell cytokines. IL-6 was confirmed to be the most induced with a maximum increase of 1110-fold after 4 h of PHA stimulation, followed by TNF-alpha and IL-1beta as well as interferon-gamma (IFN-gamma). Surprisingly, analyzing cytokine-mRNA expression from activated PBLs, the time kinetics and quantity of IFN-gamma was more similar to that of the acute-phase proteins than to that of the T-cell cytokines, which were upregulated after 1 h. The upregulation of cytokine-mRNA was translated into protein as demonstrated by enzyme-linked immunosorbent assay. IFN-gamma was also strongly expressed in the RNA from UC biopsies. TNF-alpha protein was not detectable at all in INFL-treated cultures. INFL did not induce a reduction of TNF-alpha-mRNA nor of IL-1beta-mRNA, but it reduced IFN-gamma- mRNA and, to a lesser extent, IL-6-mRNA; it also reduced the T-cell-derived cytokine IL-2. The in vitro model of PHA-stimulated PBLs may mimic inflammation processes observed in vivo. INFL may reduce inflammation in vivo through inhibition of both monocyte and T-cell activation.

Automated RNA extraction by MagNA Pure followed by rapid quantification of cytokine and chemokine gene expression with use of fluorescence resonance energy transfer.

Cytokines and chemokines, a superfamily of small cytokine-like molecules, are key mediators of immunity and inflammation. Detection of cytokines and chemokines in disease states provides useful diagnostic tools for investigating host responses to invading organisms, tumors, and trauma. Different reverse transcription-PCR (RT-PCR) protocols have been described in the literature, including semiquantitative, quantitative, and competitive PCR techniques, but most of them were labor-intensive and time-consuming (1)(2). Recently, real-time PCR assays have become a major tool for quantifying the number of DNA or cDNA copies in different settings and clinical materials. These assays offer a standardized, rapid, accurate, and reproducible method that combines rapid in vitro amplification with real-time quantification of the DNA or cDNA load. However, to date, only limited data are available for quantification of cytokine gene expression (3)(4). In addition, there is a need for rapid, sensitive, reliable, and standardized methods for RNA extraction. Here we present an automated standardized protocol for the extraction of RNA from human peripheral blood mononuclear cells (PBMCs), using the MagNA Pure LC system (Roche Diagnostics), combined with a rapid and easy-to-perform quantitative PCR assay, using the LightCycler (Roche Diagnostics) amplification and detection system. We applied this protocol to quantification of mRNA expression of the following immunorelevant genes: interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-18, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), macrophage-inducing protein-3α (MIP-3α), SCYB-11 (IFN-inducible T-cell-α chemoattractant; H174; CXCL11), intracellular adhesion molecule-1 (ICAM-1; CD54), and nuclear factor-κB (NFκB), the transcription factor and central mediator of immune response. PBMCs were prepared from 50 mL of heparinized fresh venous blood obtained from healthy donors (n = 30). PBMCs were separated by centrifugation over Ficoll and resuspended in 1 mL of phosphate-buffered saline. The PBMC concentration was adjusted to 1 × 106/mL in RPMI 1640 containing 100 …

Rapid and Sensitive Closed-Tube Quantification of Human Interferon-γ mRNA by Reverse Transcription-PCR Utilizing Energy-Transfer Labeled Primers

Quantification of cytokine gene expression is essential for understanding the biological roles of cytokines in immune responses. Quantitative reverse transcription-PCR has become popular for the sensitive detection and quantification of mRNA (1). However, the necessity of post-PCR sample processing with the potential for carryover contamination of the amplicon makes it a difficult methodology for routine clinical testing. Using AmplifluorTM technology, we have developed a rapid and extremely sensitive assay for cytokine mRNA quantification. The high signal-to-noise ratio of the assay that is achieved by the unique structure of Amplifluor primers allows for closed-tube fluorescence detection and quantification during PCR amplification (real time) or at endpoint. Elimination of laborious post-PCR sample processing enables high-throughput analysis and greatly reduces the risk of carryover contamination. The Amplifluor primer system is a molecular switch for detecting DNA amplification by energy transfer between fluorophore and quencher (2)(3). The OFF-to-ON transition occurs when the conformation of the Amplifluor primer changes from “closed” intramolecular stem-loop structure to an “open” extended structure. This structural change is achieved when the Amplifluor primers …

Quantification of Cytokine Gene Expression in Lamina Propria Lymphocytes of Cattle Following Infection with Ostertagia ostertagi

Changes in cell surface markers and cytokine transcription were analyzed in lamina propria lymphocytes from control animals (noninfected calves) and calves after a single high but nonprotective primary infection with Ostertagia ostertagi. Flow cytometry of cells recovered from the lamina propria showed an increase in the percentages of IgM+, WC1+, and IL-2R+-bearing cells 10 days after infection; however, 2 mo after infection, cell staining was comparable to preinfection levels. Transcription levels of interleukins IL-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA were measured using a competitive reverse transcription-polymerase chain reaction. Results indicated elevated levels of IL-4 and IFN-gamma in the infected animals at 10 days and at 60 days after infection. Transcription of IL-10 also increased; however, this change was not observed until 60 days postinfection.