Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.

Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rß1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rß1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.

Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

An unconventional role of an ASB family protein in NF-κB activation and inflammatory response during microbial infection and colitis.

Nuclear factor κB (NF-κB)-mediated signaling pathway plays a crucial role in the regulation of inflammatory process, innate and adaptive immune responses. The hyperactivation of inflammatory response causes host cell death, tissue damage, and autoinflammatory disorders, such as sepsis and inflammatory bowel disease. However, how these processes are precisely controlled is still poorly understood. In this study, we demonstrated that ankyrin repeat and suppressor of cytokine signaling box containing 1 (ASB1) is involved in the positive regulation of inflammatory responses by enhancing the stability of TAB2 and its downstream signaling pathways, including NF-κB and mitogen-activated protein kinase pathways. Mechanistically, unlike other members of the ASB family that induce ubiquitination-mediated degradation of their target proteins, ASB1 associates with TAB2 to inhibit K48-linked polyubiquitination and thereby promote the stability of TAB2 upon stimulation of cytokines and lipopolysaccharide (LPS), which indicates that ASB1 plays a noncanonical role to further stabilize the target protein rather than induce its degradation. The deficiency of Asb1 protects mice from Salmonella typhimurium- or LPS-induced septic shock and increases the survival of mice. Moreover, Asb1-deficient mice exhibited less severe colitis and intestinal inflammation induced by dextran sodium sulfate. Given the crucial role of ASB proteins in inflammatory signaling pathways, our study offers insights into the immune regulation in pathogen infection and inflammatory disorders with therapeutic implications.

GL261 luciferase-expressing cells elicit an anti-tumor immune response: an evaluation of murine glioma models.

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 104 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 105 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 104 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.

GloBody Technology: Detecting Anti-Drug Antibody against VH/VL domains.

The occurrence of anti-drug antibodies following administration of therapeutic monoclonal antibody to patients is a growing problem that is attracting attention from frontline clinicians. Ideally, an initial indicative point of care test would provide guidance to seek testing approved by the regulatory authorities. Here we describe a platform for the detection of IgG anti-drug antibodies that may provide an initial screen for all therapeutic monoclonal antibodies. Synthetic genes encoding Nanoluciferase polypeptides were inserted between the variable heavy and light domain encoding region of known antibody drugs (alemtuzumab and adalimumab) to generate recombinant single chain GloBodies, which retain the drug antibody paratopes and Nanoluciferase activity. In the presence of anti-drug antibodies, the GloBody is bound by specific IgG in the sample. These complexes are captured on immobilised Protein G and the luciferase activity determined. The amount of light generated being indicative of the anti-drug IgG antibody levels in serum. It should be possible to assemble GloBody reagents for all therapeutic monoclonal antibodies and adapt the capture phase to include additional specific isotypes. The assay has the potential to be developed for use with a drop of blood allowing initial pre-screening in a point of care setting.

Engineered immune cells as highly sensitive cancer diagnostics.

Endogenous biomarkers remain at the forefront of early disease detection efforts, but many lack the sensitivities and specificities necessary to influence disease management. Here, we describe a cell-based in vivo sensor for highly sensitive early cancer detection. We engineer macrophages to produce a synthetic reporter on adopting an M2 tumor-associated metabolic profile by coupling luciferase expression to activation of the arginase-1 promoter. After adoptive transfer in colorectal and breast mouse tumor models, the engineered macrophages migrated to the tumors and activated arginase-1 so that they could be detected by bioluminescence imaging and luciferase measured in the blood. The macrophage sensor detected tumors as small as 25-50 mm3 by blood luciferase measurements, even in the presence of concomitant inflammation, and was more sensitive than clinically used protein and nucleic acid cancer biomarkers. Macrophage sensors also effectively tracked the immunological response in muscle and lung models of inflammation, suggesting the potential utility of this approach in disease states other than cancer.

Evaluation of LIPS (luciferase immunoprecipitation system) for serodiagnosis of Toxoplasmosis.

Development of reliable, quantitative technologies for serodiagnosis of Toxoplasma gondii infection remains desirable. The luciferase immunoprecipitation system (LIPS) is a relatively simple, highly sensitive, and rapid quantitative immunoassay. The major advantages of this assay over ELISA are a wider dynamic range, shorter overall assay time, and less sample volume. In this study, we aimed to use this method for the serodiagnosis of toxoplasmosis. Recombinant Toxoplasma antigens (dense granule antigens GRA6, GRA7, and GRA8 and bradyzoite antigen BAG1) fused with nanoluciferase (Nluc, a small luciferase enzyme) were expressed in Escherichia coli, purified, and tested in LIPS assays with sera from experimental mice infected with T. gondii and a WHO standard anti-Toxoplasma human immunoglobulin (TOXM). In the experimentally infected mice, LIPS assays detected antibodies against Nluc-GRA6, Nluc-GRA7, and Nluc-GRA8 as early as day 14, whereas antibodies against Nluc-BAG1 remained undetected until day 21 and then showed significant elevation on day 60. In TOXM sera, LIPS assays with each Nluc recombinant protein produced reliable standard curves with a coefficient of determination (R2) of 0.980-0.989 for GRA6, 0.986-0.990 for GRA7, 0.998-0.999 for GRA8, and 0.942-0.987 for BAG1. The detection limits were estimated to be 3.9, 2, 1, and 1 IU/ml for rGRA6, rGRA7, rGRA8, and rBAG1, respectively. The LIPS assay for toxoplasmosis could detect antibodies against T. gondii in the mouse and human sera with a reasonably high sensitivity. We consider the LIPS assay to be a promising alternative tool for screening, diagnosing, and monitoring toxoplasmosis. In particular, detection of antibodies against BAG1 may be useful for a longitudinal seroprevalence study in suspected high-risk areas on the basis of its elevated serum concentration in the chronic phase.

Measuring the bioactivity of anti-IL-6/anti-IL-6R therapeutic antibodies: presentation of a robust reporter gene assay.

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.

Phosphatase Cdc25A Negatively Regulates the Antiviral Immune Response by Inhibiting TBK1 Activity.

The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-ß) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-ß stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-ß. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-ß reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity.IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-ß transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.

Modulation of mRNA Translation and Cell Viability by Influenza A Virus Derived Nonstructural Protein 1.

Translation of in vitro transcribed messenger RNA (mRNA) is known to be compromised by cell's innate immune responses. Herein we show that when mRNA encoding nonstructural protein 1 (NS1), an immune evasion gene derived from influenza A virus, is co-delivered with mRNA encoding green fluorescent protein (GFP), higher GFP expression can be observed in four different interferon competent cell types within 6 h, indicating NS1's wide host range property and rapid counter response to the cells' innate immune response. Enhanced mRNA translation correlates with reduced interferon production in all tested cell types and substituting a small portion of luciferase mRNA with NS1 mRNA enhances luciferase production compared to the same dose composing of only luciferase mRNA although in a cell type specific manner. Toxicity caused by transfection of unmodified mRNA is mitigated with the delivery of NS1 mRNA and is observed only in NS1 without cleavage and polyadenylation specificity factor 30 kda (CPSF30) inhibition function. Conversely, delivery of mRNA encoding NS1 with CPSF30 inhibition function aggravated toxicity. Overall, we demonstrate that NS1 enhanced mRNA transfection through active evasion of innate immune responses and modulated cellular viability during mRNA transfection.

Recombinant Peptidomimetic-Nano Luciferase Tracers for Sensitive Single-Step Immunodetection of Small Molecules.

Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate, and biological nature of the phage particles create some limitations to their use and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as a fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E7 was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). NanoLuc-C2-15 showed better performance than C2-15-NanoLuc and was adopted to develop a bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA) for imidaclothiz. The luminescence signal of NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation, which enabled one to capture the BLLFIA readout by using a smartphone without an external light source. The IC50 of the BLEIA and BLLFIA were 3.3 ± 0.2 and 6.4 ± 0.4 ng mL-1, respectively. Both immunoassays exhibited good accuracy for the detection of imidaclothiz in environmental and agricultural samples.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells.

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

Interferon induces interleukin 8 and bone marrow stromal cell antigen 2 expression, inhibiting the production of hepatitis B virus surface antigen from human hepatocytes.

Hepatitis B virus (HBV) surface antigen (HBsAg) loss is one of the treatment goals of chronic HBV infection. Bone marrow stromal cell antigen 2 (BST2) is one of the interferon (IFN)-stimulated genes (ISGs) and inhibits the release of various enveloped viruses. Here we examined the effects of antiviral treatment on HBsAg levels and its intracellular mechanism in HBsAg-producing hepatocytes. In PLC/PRF/5 and Huh1, IFNα-2a treatment decreased HBsAg levels in their conditioned media. Upregulation of interleukin 8 (IL8), toll-like receptor 2 (TLR2) and interferon gamma-induced protein 10 (IP10) mRNAs was associated with the reduction of HBsAg in both PLC/PRF/5 and Huh1. The HBsAg level was upregulated by knockdown of IL8, TLR2 or IP10. Exogenous addition of IL8 enhanced BST2 promoter activity and BST2 mRNA expression. Additionally, knockdown of IL8 could lead to the downregulation of BST2 mRNA. Transfection of poly(I-C) enhanced IL8 and BST2 mRNA expression and inhibited HBsAg secretion from PLC/PRF/5 cells. In conclusion, IL8 might play an important role in the enhancement of BST2 and be involved in HBsAg eradication.

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy.

Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

Diosgenin effectively suppresses skin inflammation induced by phthalic anhydride in IL-4/Luc/CNS-1 transgenic mice.

To quantitatively evaluate the therapeutic effects of diosgenin (DG) and investigate the role of IL-4 on skin inflammation, alterations in luciferase-derived signal and general phenotype biomarkers were measured in IL-4/Luc/CNS-1 transgenic mice with phthalic anhydride (PA)-induced skin inflammation after treatment with DG for 4 weeks. High levels of luciferase-derived signal detected in the abdominal region and submandibular lymph node (SL) of the PA treated group was significantly decreased by 67-88% in the PA + DG cotreated group. Furthermore, the weight of the lymph node and spleen, IgE concentration, epidermis thickness, and number of infiltrated mast cells were lower in the PA + DG treated group than the PA + Vehicle treated group. Moreover, expression of IL-6 and vascular endothelial growth factor (VEGF) also decreased in the PA + DG cotreated group. These results suggest that PA-induced skin inflammation could be successfully suppressed by DG treatment in IL-4/Luc/CNS-1 Tg mice through attenuation of IL-4 and IL-6 expression, as well as decreased IgE concentration and mast cells infiltration.

Effects of transgene expression level per cell in mice livers on induction of transgene-specific immune responses after hydrodynamic gene transfer.

We previously showed that high and sustained transgene expression of antigenic proteins induced transgene-specific immune responses. In the present study, a detailed relationship between the level of transgene expression per cell and immune response after hydrodynamic gene transfer was investigated. Cypridina luciferase (cLuc), a secretory antigenic reporter protein, was selected as a model antigen, and pROSA-cLuc, a plasmid expressing cLuc, was constructed. A fixed dose (30 µg) of pROSA-cLuc was delivered to mice by a single hydrodynamic injection or three injections at 24-h intervals because the number of cells transfected with plasmids is dependent on the number of hydrodynamic injections. Serum cLuc activity, an indicator of the total amount of cLuc transgene expression, was almost equal between these two groups. In contrast, the high-dose single injection induced higher levels of cLuc-specific humoral and cellular immune responses than the three low-dose injections. Moreover, the serum cLuc activity of the high-dose single injection group began to decline ~10 days after injection, whereas the activity remained constant in the three low-dose injection group. These results indicate that it is preferable to reduce the level of transgene expression per cell to avoid induction of the transgene-specific immune response after hydrodynamic gene transfer.

Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome.

When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling.

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice.

Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study.

Comparative Functional Analysis of 12 Mammalian IFN-λ4 Orthologs.

IFN-λ4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals--up to 10% of Asians, 50% of Europeans, and 90% of Africans--carry the ΔG allele of a genetic variant rs368234815-TT/ΔG and are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-λ4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-λ4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-λ4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-λ4 proteins are comparable-they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-λ4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-λ4. Studies of IFN-λ4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions.