ELISpot Assay for the Detection of ASFV-Specific Interferon-Gamma (IFN-γ)-Producing Cells.

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.

Immunoprotective effect of an in silico designed multiepitope cancer vaccine with BORIS cancer-testis antigen target in a murine mammary carcinoma model.

In our previous study, immunoinformatic tools were used to design a novel multiepitope cancer vaccine based on the most immunodominant regions of BORIS cancer-testis antigen. The final vaccine construct was an immunogenic, non-allergenic, and stable protein consisted of multiple cytotoxic T lymphocytes epitopes, IFN-γ inducing epitopes, and B cell epitopes according to bioinformatic analyzes. Herein, the DNA sequence of the final vaccine construct was placed into the pcDNA3.1 vector as a DNA vaccine (pcDNA3.1-VAC). Also, the recombinant multiepitope peptide vaccine (MPV) was produced by a transfected BL21 E. coli strain using a recombinant pET-28a vector and then, purified and screened by Fast protein liquid chromatography technique (FPLC) and Western blot, respectively. The anti-tumor effects of prophylactic co-immunization with these DNA and protein cancer vaccines were evaluated in the metastatic non-immunogenic 4T1 mammary carcinoma in BALB/c mice. Co-immunization with the pcDNA3.1-VAC and MPV significantly (P < 0.001) increased the serum levels of the MPV-specific IgG total, IgG2a, and IgG1. The splenocytes of co-immunized mice exhibited a significantly higher efficacy to produce interleukin-4 and interferon-γ and proliferation in response to MPV in comparison with the control. The prophylactic co-immunization regime caused significant breast tumors' growth inhibition, tumors' weight decrease, inhibition of metastasis formation, and enlarging tumor-bearing mice survival time, without any considerable side effects. Taking together, this cancer vaccine can evoke strong immune response against breast tumor and inhibits its growth and metastasis.

IFNγ-stimulated dendritic cell extracellular vesicles can be nasally administered to the brain and enter oligodendrocytes.

Extracellular vesicles secreted from IFNγ-stimulated rat dendritic cells (referred to here as IFNγ-DC-EVs) contain miRNAs which promote myelination (including but not limited to miR-219), and preferentially enter oligodendrocytes in brain slice cultures. IFNγ-DC-EVs also increase myelination when nasally administered to naïve rats. While we can infer that these extracellular vesicles enter the CNS from functional studies, here we demonstrate biodistribution throughout the brain after nasal delivery by way of imaging studies. After nasal administration, Xenolight DiR-labelled IFNγ-DC-EVs were detected 30 minutes later throughout the brain and the cervical spinal cord. We next examined cellular uptake of IFNγ-DC-EVs by transfecting IFNγ-DC-EVs with mCherry mRNA prior to nasal administration. mCherry-positive cells were found along the rostrocaudal axis of the brain to the brainstem. These cells morphologically resembled oligodendrocytes, and indeed cell-specific co-staining for neurons, astrocytes, microglia and oligodendrocytes showed that mcherry positive cells were predominantly oligodendrocytes. This is in keeping with our prior in vitro results showing that IFNγ-DC-EVs are preferentially taken up by oligodendrocytes, and to a lesser extent, microglia. To confirm that IFNγ-DC-EVs delivered cargo to oligodendrocytes, we quantified protein levels of miR-219 mRNA targets expressed in oligodendrocyte lineage cells, and found significantly reduced expression. Finally, we compared intranasal versus intravenous delivery of Xenolight DiR-labelled IFNγ-DC-EVs. Though labelled IFNγ-DC-EVs entered the CNS via both routes, we found that nasal delivery more specifically targeted the CNS with less accumulation in the liver. Taken together, these data show that intranasal administration is an effective route for delivery of IFNγ-DC-EVs to the CNS, and provides additional support for their development as an EV-based neurotherapeutic that, for the first time, targets oligodendrocytes.

Near-Infrared-Light Remote-Controlled Activation of Cancer Immunotherapy Using Photothermal Conjugated Polymer Nanoparticles.

Remote control of the therapeutic process is an ideal strategy for maximizing efficacy and avoiding side effects, especially for cancer immunotherapy. Herein, a conjugated polymer nanoparticles (CPNs)-mediated optogenetic system for in situ activation of immunotherapy under near-infrared laser irradiation is reported. This system is composed of photothermal CPNs and interferon-gamma (IFN-γ) plasmid driven by heat shock promoter HSP70. The photothermally responsive CPNs serve as a photo-heat nanotransducer to trigger the gene transcription of IFN-γ cytokine. The secreted IFN-γ from cancer cells can sufficiently elicit surrounding tumor-associated macrophages activation through IFN-γ-JAK-STAT1 transcription-factor signaling pathway and finally induce cancer cell killing by immunotherapy. Therefore, this synergetic optogenetic system provides a promising approach to remotely control the process of cancer immunotherapy.

Inflammation protein quantification by multiple reaction monitoring mass spectrometry in lipopolysaccharide-stimulated THP-1 cells.

RATIONALE: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. METHODS: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. RESULTS: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. CONCLUSIONS: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis.

Enhancement of interferon gamma stability as an anticancer therapeutic protein against hepatocellular carcinoma upon interaction with calycosin.

The stability of IFN-γ as a therapeutic protein can play a key role on its anticancer effects. Herein, we explored the thermodynamic parameters and conformational stability of IFN-γ in the presence of calycosin, the main active compound of Radix astragali, by different biophysical and theoretical analysis. Afterwards, the improved anticancer effects of IFN-γ-calycosin interaction relative to IFN-γ alone were assessed on hepatocellular carcinoma (HepG2) cell line by MTT and caspase assays. ITC data indicated that upon interaction of calycosin with IFN-γ the binding and thermodynamic parameters were as follows: Kd = 1.9 µM, ΔG° = -32.45 kJ/mol, ΔH° = -11.91 kJ/mol, and TΔS° = 20.54 kJ/mol. ANS/synchronous fluorescence, CD and UV-Vis spectroscopy studies indicated that the interaction between calycosin and IFN-γ caused the folding of the IFN-γ backbone in to a more packed structure with enhanced α-helix content and higher melting temperature (Tm) value. The spectroscopic outcomes were then verified by molecular docking and molecular dynamic analysis. It was also shown that after incubation of the IFN-γ samples at 50 °C for 60 min in the presence of calycosin (5 µM), the IFN-γ-calycosin system showed a significant antiproliferative effects against hepatocellular carcinoma (HepG2) cells through caspase-9/3 activation and this anticancer effect was more pronounced than free IFN-γ. This data may provide useful information about the development of IFN-γ-based therapeutic platforms.

Exploring the Potential of Interferon Gamma Gene as Major Immune Responder for Bovine Tuberculosis in River Buffalo.

Bovine tuberculosis (bTB) is a widespread zoonotic infection targeting the livestock sector, especially in developing countries, and posing a risk to humans and animal populations. Its recent prevalence in river buffaloes has been estimated as higher as 33.7%. In emergent countries like Pakistan, there is likeliness of human-livestock interfaces extensively and lacking of effective preventive measures that illustrate the risk of spreading the infection at a remarkable rate. The river buffalo (Bubalus bubalis) is an upkeep host of Mycobacterium bovis and is responsible for disease transmission among buffaloes and other livestock species. In this study, potential molecular biomarkers in the Interferon-gamma gene (IFNg) were identified after genomic screening of river buffaloes. Unique genomic loci in river buffalo proved the novelty of the genomic structure of this phenomenal animal but also highlighted its significance in natural immunity against the Mycobacterium. A total of eight single nucleotide polymorphisms were identified in the coding region of IFNg. The SNPs in the exonic region were all transitions, i.e., the conversion of purines to purines. These SNPs were analyzed for Hardy Weinberg Equilibrium, chi2 test, gene diversity, and protein structural conformation. Pathway analysis in tuberculosis revealed that IFNg inhibits the antigen-presenting cells (APC) through JAK and STAT pathways. Network analysis of IFNg proteins in both species showed strong associations among the immunity-related proteins (interleukins, tissue necrosis factors) and receptors of interferons. The identified polymorphic sites might be novel-potentiated markers for the selection of animals with superior immune response against bTB and can be exploited as promising genomic sites for breeding the resistant animal herds to combat Mycobacterium infection in a long run.

IFN-γ/SrBG composite scaffolds promote osteogenesis by sequential regulation of macrophages from M1 to M2.

The macrophage-dominated bone immune response plays an important role in osteogenesis of bone defects. Generally, M2 macrophages are beneficial to promote osteogenesis. However, recent studies have confirmed that M1 also plays an important role in early angiogenesis, osteointegration. Therefore, achieving sequential polarization of macrophages from M1 to M2 may be more beneficial for osteogenesis. In this study, a new type of bone immunomodulatory IFN-γ/Sr-dropped bioactive glass composite scaffold (IFN-γ/SrBG) was successfully prepared which integrates multiple biological functions at different stages of the bone healing process. The scaffold can polarize macrophages into pro-inflammatory M1 type at the early stage of implantation by releasing IFN-γ within the first day, and then polarize macrophages into anti-inflammatory M2 type at a later stage by releasing Sr2+ from SrBG, which promoted mature bone formation in bone defects to a greater extent. Therefore, IFN-γ/SrBG scaffolds are expected to become excellent bone tissue engineering materials by sequential regulation of macrophage polarization.

Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle.

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

Total Synthesis of Glycosylated Human Interferon-γ.

Interferon-γ (IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical synthesis of homogeneously glycosylated variants of human IFN-γ using a tandem diselenide-selenoester ligation-deselenization strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral functions were assessed.

Immunoinformatics design of multivalent chimeric vaccine for modulation of the immune system in Pseudomonas aeruginosa infection.

Increasing in drug-resistant Pseudomonas aeruginosa and high mortality and morbidity rate have become a health challenge worldwide; therefore, developing the novel therapeutic strategies such as immunogenic vaccine candidate are required. Despite a substantial research effort, the future of immunization against P. aeruginosa due to failure in covering two separate stages of infection, and furthermore, inducing ineffective type of immune response, still remains controversial. In this study, immunoinformatics approach was utilized to design multivalent chimeric vaccine from both stages of infection containing Lectin, HIV TAT peptide, N-terminal fragment of exotoxin A and Epi8 of outer membrane protein F (OprF) with hydrophobic linkers which have a high density of B-cell, T Lymphocytes (HTL), T Lymphocytes (CTL), and IFN-γ epitopes. The physicochemical properties, antigenicity, and allergenicity for designed vaccine were analyzed. 3D model generation and refinement further validation of the final vaccine were followed by computational docking with molecular dynamics analyses that demonstrated high- affinity interaction between vaccine and TLR-4. Finally, designed vaccine was in silico cloned in pET22b. We have expected that the designed vaccine able to elucidate innate, humoral and cellular innate immune responses and control the interaction of P. aeruginosa with host and maybe overcome to P. aeruginosa vaccines drawback.

The role of interferon-γ in cardiovascular disease: an update.

PURPOSE: Cardiovascular disease (CVD) is the leading cause of death, globally, and its prevalence is only expected to rise due to the increasing incidence of co-morbidities such as obesity and diabetes. Medical treatment of CVD is directed primarily at slowing or reversing the underlying atherosclerotic process by managing circulating lipids with an emphasis on control of low-density lipoprotein (LDL) cholesterol. However, over the past several decades, there has been increasing recognition that chronic inflammation and immune system activation are important contributors to atherosclerosis. This shift in focus has led to the elucidation of the complex interplay between cholesterol and cellular secretion of cytokines involved in CVD pathogenesis. Of the vast array of cytokine promoting atherosclerosis, interferon (IFN)-γ is highly implicated and, therefore, of great interest. METHODS: Literature review was performed to further understand the effect of IFN-γ on the development of atherosclerotic CVD. RESULTS: IFN-γ, the sole member of the type II IFN family, is produced by T cells and macrophages, and has been found to induce production of other cytokines and to have multiple effects on all stages of atherogenesis. IFN-γ activates a variety of signaling pathways, most commonly the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, to induce oxidative stress, promote foam cell accumulation, stimulate smooth muscle cell proliferation and migration into the arterial intima, enhance platelet-derived growth factor expression, and destabilize plaque. These are just a few of the contributions of IFN-γ to the initiation and progression of atherosclerotic CVD. CONCLUSION: Given the pivotal role of IFN-γ in the advancement of CVD, activation of its signaling pathways is being explored as a driver of atherosclerosis. Manipulation of this key cytokine may lead to novel therapeutic avenues for CVD prevention and treatment. A number of therapies are being explored with IFN-γ as the potential target.

Accuracy of PCR, mycobacterial culture and interferon-γ assays for detection of Mycobacterium bovis in blood and milk samples from Egyptian dairy cows using Bayesian modelling.

The number of bovine tuberculosis (bTB) infected dairy herds in Egypt is growing and this calls for accurate and reliable diagnostic methods at cow level for cost-effective bTB eradication as culling of the whole herd is not economically sustainable. The present study aimed to estimate the sensitivity (Se) and specificity (Sp) of PCR, mycobacterial culture and interferon-γ (IFN-γ) assays for Mycobacterium bovis (M. bovis) detection in blood and milk samples from dairy cows in Egyptian dairy herds within a Bayesian framework. As a secondary objective, the distribution of true within-herd prevalence of M. bovis infection was estimated. Blood and milk samples were collected from 245 Holstein dairy cows in 11 Egyptian dairy herds and subjected to PCR, mycobacterial culture and IFN-γ testing. With respect to the detection of M. bovis in blood, IFN-γ recorded higher Se [0.97 (95% Posterior Credible Interval (PCI): 0.87-1.00)] than PCR [0.68 (95% PCI: 0.53-0.95)] and culture [0.22 (95% PCI: 0.13-0.37)]. However, Sp estimates of PCR [0.98 (95% PCI: 0.95-1.00)], culture [0.99 (95% PCI: 0.98-1.00)] and IFN-γ [0.97 (95% PCI: 0.88-1.00)] were comparable. As for milk samples, Se estimate of PCR [0.29 (95% PCI: 0.01-0.60)] was higher than that of culture [0.08 (95% PCI: 0.001-0.23)]. However, the Sp estimates of both tests were statistically similar. The estimated true within-herd prevalences of M. bovis varied across the tested bovine subpopulations and ranged between 0.06 and 0.66. In conclusion, IFN-γ registered a similar overall performance to PCR but was superior to mycobacterial culture. With its good accuracy and wide applicability, IFN-γ lends itself to use in the Egyptian bTB eradication program.

Functional characterization of a group II interferon, IFNc in the perciform fish, Nile tilapia (Oreochromis niloticus).

Interferons are a family of class II α-helical cytokines playing vital roles in antiviral immune response, and little information is available to date regarding the interferon system of tilapia. In this study, a type I IFN gene, named On-IFNc, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-IFNc contains several structural features known in type I IFNs, and On-IFNc was clustered together with the known IFNc in fish into a separated clade in the phylogenetic tree. On-IFNc gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, recombinant On-IFNc has been proven to markedly induce the expression of the antiviral effectors, Mx and viperin, the signalling components, STAT1, STAT2, and IRF9, and the transcription factors, IRF3 and IRF7, as well as the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. Furthermore, recombinant On-IFNc has been proven to possess antiviral activity against ISKNV. The present study thus contributes to a better understanding of the functional properties of the type I IFN system in tilapia.

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Covalently Immobilizing Interferon-γ Drives Filopodia Production through Specific Receptor-Ligand Interactions Independently of Canonical Downstream Signaling.

Immobilizing a signaling protein to guide cell behavior has been employed in a wide variety of studies. This approach draws inspiration from biology, where specific, affinity-based interactions between membrane receptors and immobilized proteins in the extracellular matrix guide many developmental and homeostatic processes. Synthetic immobilization approaches, however, do not necessarily recapitulate the in vivo signaling system and potentially lead to artificial receptor-ligand interactions. To investigate the effects of one example of engineered receptor-ligand interactions, we focus on the immobilization of interferon-γ (IFN-γ), which has been used to drive differentiation of neural stem cells (NSCs). To isolate the effect of ligand immobilization, we transfected Cos-7 cells with only interferon-γ receptor 1 (IFNγR1), not IFNγR2, so that the cells could bind IFN-γ but were incapable of canonical signal transduction. We then exposed the cells to surfaces containing covalently immobilized IFN-γ and studied membrane morphology, receptor-ligand dynamics, and receptor activation. We found that exposing cells to immobilized but not soluble IFN-γ drove the formation of filopodia in both NSCs and Cos-7, showing that covalently immobilizing IFN-γ is enough to affect cell behavior, independently of canonical downstream signaling. Overall, this work suggests that synthetic growth factor immobilization can influence cell morphology beyond enhancing canonical cell responses through the prolonged signaling duration or spatial patterning enabled by protein immobilization. This suggests that differentiation of NSCs could be driven by canonical and non-canonical pathways when IFN-γ is covalently immobilized. This finding has broad implications for bioengineering approaches to guide cell behavior, as one ligand has the potential to impact multiple pathways even when cells lack the canonical signal transduction machinery.

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles.

Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.

The role of heparin/heparan sulphate in the IFN-γ-led Arena.

IFN-γ (Interferon-gamma) is a pleiotropic cytokine. It is often involved in a variety of physiological processes by binding to the cell surface transmembrane receptor (IFN-γR) to initiate a series of signalling pathways that transmit external signals from cell surface receptors to the cell nucleus. Heparan sulphate (HS), a highly sulphated linear polysaccharide, is ubiquitous on the mammalian cell surface and extracellular matrix. Electrostatic interactions can be generated between the highly sulphated HS region and specific basic amino acid residues in the IFN-γ structure, thereby detaining IFN-γ on the cell surface, and the concentration of IFN-γ on the cell surface is thus, changed. IFN-γ retained on the cell surface will optimize the binding of IFN-γ to the transmembrane receptor resulting in high efficiency signalling. Heparin is a glycosaminoglycan with a structure similar to HS. The structural similarity provides a basis for modelling exogenous heparin dependence for interference with IFN-γ function. This model can be summarized as follows: First, the competitive binding effect; heparin bound to cytokines by competing with membrane-associated HS, causes a decrease in cytokine concentration on the cell surface. Second, the principle of priority occupancy; heparin can occupy the receptor binding site on cytokines, partially preventing the IFN-γ-IFN-γR interaction. These two models interfere with IFN-γ signal transmission. To decipher the mechanism by which heparin influences IFN-γ activity, studies of the structure-activity relationship are in progress. This paper summarizes research progress on the IFN-γ signalling pathway, heparin interference with IFN-γ activity and the structure-activity relationship between heparin and IFN-γ.

An amplified label-free electrochemical aptasensor of γ-interferon based on target-induced DNA strand transform of hairpin-to-linear conformation enabling simultaneous capture of redox probe and target.

In this work, a novel and signal-amplified label-free electrochemical aptasensor was developed and enabled efficient determination of γ-interferon (IFN-γ), based on target-induced DNA strand transform of hairpin-to-linear conformation combining with simultaneous capture of redox probe and target. Gold nanoparticles (AuNPs) were electrodeposited in the matrix of poly(amidoamine) dendrimer (PAMAM), followed by drop-casting addition on MoS2 nanosheets to prepare AuNPs- PAMAM/MoS2 composites. HS-terminated hairpin-DNA aptamer of IFN-γ was conjugated with AuNPs to prepare aptamer-AuNPs-PAMAM/MoS2 onto glassy carbon electrode (GCE), by using bovine serum albumin as the cross-linker and stabilizer. Methylene blue (MB) as a redox probe was absorbed on IFN-γ aptamer. In the presence of IFN-γ, MB electrochemical signal increased gradually. The preparation processes, mechanisms and optimal experiment conditions of aptamer- AuNPs-PAMAM/MoS2/MB/GCE sensing platform were studied by electron microscope imaging technologies, spectral curves and electrochemical measurements. There is a well plotting linear relationship between the peak current intensities of MB and IFN-γ contents in the range of 0.01-1000 pg mL-1, showing a low detection limit of 2 fg mL-1. Experimental results testified that the aptasensor had highly sensitive and selective responses toward IFN-γ, over potential interferents. In real biological samples, the aptasensor of IFN-γ had superior detection recoveries, indicating its high detection performance and feasibility for practicability.

Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA.

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10-9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers' applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.