Comparison of Efficacy of the Disease-Specific LOX1- and Constitutive Cytomegalovirus-Promoters in Expressing Interleukin 10 through Adeno-Associated Virus 2/8 Delivery in Atherosclerotic Mice

The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of “disease-specific promoters” has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2) using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.     

Biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling

Protein microarrays represent an important new tool in proteomic systems biology. This review focuses on the contributions of protein microarrays to the discovery of novel disease biomarkers through antibody-based assays. Of particular interest is the use of protein microarrays for immune response profiling, through which a disease-specific antibody repertoire may be defined. The antigens and antibodies revealed by these studies are useful for clinical assay development, with enormous potential to aid in diagnosis, prognosis, disease staging and treatment selection. The discovery and characterization of novel biomarkers specifically tailored to disease type and stage are expected to enable personalized medicine by facilitating preventative medicine, predictive diagnostics and individualized curative therapies.     

Biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling

Protein microarrays represent an important new tool in proteomic systems biology. This review focuses on the contributions of protein microarrays to the discovery of novel disease biomarkers through antibody-based assays. Of particular interest is the use of protein microarrays for immune response profiling, through which a disease-specific antibody repertoire may be defined. The antigens and antibodies revealed by these studies are useful for clinical assay development, with enormous potential to aid in diagnosis, prognosis, disease staging and treatment selection. The discovery and characterization of novel biomarkers specifically tailored to disease type and stage are expected to enable personalized medicine by facilitating preventative medicine, predictive diagnostics and individualized curative therapies.     

Interaction between mesenchymal stromal cell-derived extracellular vesicles and immune cells by distinct protein content

Mesenchymal stromal cells (MSCs) can effectively contribute to tissue regeneration inside the inflammatory microenvironment mostly through modulating immune responses. MSC-derived extracellular vesicles (MSC-EVs) display immunoregulatory functions similar to parent cells. Interactions between MSC-EVs and immune cells make them an ideal therapeutic candidate for infectious, inflammatory, and autoimmune diseases. These properties of MSC-EVs have encouraged researchers to perform extensive studies on multiple factors that mediate MSC-EVs immunomodulatory effects. Investigation of proteins involved in the complex interplay of MSC-EVs and immune cells may help us to better understand their functions. Here, we performed a comprehensive proteomic analysis of MSC-EVs that was previously reported by ExoCarta database. A total of 938 proteins were identified as MSC-EV proteome using quantitative proteomics techniques. Kyoto Encyclopedia of Genes and Genomes analysis demonstrates that ECM–receptor interaction, focal adhesion, and disease-specific pathways are enriched in MSC-EVs. By detail analysis of proteins presence in immune system process, we found that expression of some cytokines, chemokines, and chemokine receptors such as IL10, HGF, LIF, CCL2, VEGFC, and CCL20, which leads to migration of MSC-EVs to injured sites, suppression of inflammation and promotion of regeneration in inflammatory and autoimmune diseases. Also, some chemoattractant proteins such as CXCL2, CXCL8, CXCL16, DEFA1, HERC5, and IFITM2 were found in MSC-EV proteome. They may actively recruit immune cells to the proximity of MSC or MSC-EVs, may result in boosting immune response under specific circumstances, and may have protective role in infectious diseases. In this review, we summarize available information about immunomodulation of MSC-EVs with particular emphasis on their proteomics analysis.     

Construction of disease-specific cytokine profiles by associating disease genes with immune responses

The pathogenesis of many inflammatory diseases is a coordinated process involving metabolic dysfunctions and immune response—usually modulated by the production of cytokines and associated inflammatory molecules. In this work, we seek to understand how genes involved in pathogenesis which are often not associated with the immune system in an obvious way communicate with the immune system. We have embedded a network of human protein-protein interactions (PPI) from the STRING database with 14,707 human genes using feature learning that captures high confidence edges. We have found that our predicted Association Scores derived from the features extracted from STRING’s high confidence edges are useful for predicting novel connections between genes, thus enabling the construction of a full map of predicted associations for all possible pairs between 14,707 human genes. In particular, we analyzed the pattern of associations for 126 cytokines and found that the six patterns of cytokine interaction with human genes are consistent with their functional classifications. To define the disease-specific roles of cytokines we have collected gene sets for 11,944 diseases from DisGeNET. We used these gene sets to predict disease-specific gene associations with cytokines by calculating the normalized average Association Scores between disease-associated gene sets and the 126 cytokines; this creates a unique profile of inflammatory genes (both known and predicted) for each disease. We validated our predicted cytokine associations by comparing them to known associations for 171 diseases. The predicted cytokine profiles correlate (p-value<0.0003) with the known ones in 95 diseases. We further characterized the profiles of each disease by calculating an “Inflammation Score” that summarizes different modes of immune responses. Finally, by analyzing subnetworks formed between disease-specific pathogenesis genes, hormones, receptors, and cytokines, we identified the key genes responsible for interactions between pathogenesis and inflammatory responses. These genes and the corresponding cytokines used by different immune disorders suggest unique targets for drug discovery.     

Potentialities of induced pluripotent stem (iPS) cells for treatment of diseases

Induced pluripotent stem (iPS) cell research has been growing a new height throughout the world due to its potentialities in medical applications. We can explore several therapeutic applications through the iPS cell research. In this review, we have first discussed the development of iPS cells, reprogramming factors, and effectiveness of iPS cells. Then we have emphasized the potential applications of iPS cells in pharmaceutical and medical sectors, such as, study of cellular mechanisms for spectrum of disease entities, disease-specific iPS cell lines for drugs discovery and development, toxicological studies of drugs development, personalized medicine, and regenerative medicine.     

Identification of pancreatic glycoprotein 2 as an endogenous immunomodulator of innate and adaptive immune responses

Pancreatic autoantibodies are Crohn disease-specific serologic markers. The function and immunological role of their recently identified autoantigen, glycoprotein 2 (GP2), are unknown. We therefore investigated the impact of GP2 on modulation of innate and adaptive immune responses to evaluate its potential therapeutic use in mucosal inflammation. Our data indicate a previously unknown function for GP2 as an immunomodulator. GP2 was ubiquitously expressed on cells vital to mucosal immune responses. The expression of GP2 was upregulated on activated human T cells, and it was further influenced by pharmaceutical TNF-α inhibitors. Recombinant GP2 significantly decreased human intestinal epithelial cells, mucosal and peripheral T cell proliferation, apoptosis, and activation, and it distinctly modulated cytokine secretion. Furthermore, intestinal epithelial cells stimulated with GP2 potently attracted T cells. In conclusion, we demonstrate a novel role for GP2 in immune regulation that could provide a platform for new therapeutic interventions in the treatment of Crohn disease.     

Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune‐boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340 nm resulted in fluorescence with an emission maximum of around 410 nm, which is fairly specific for NADH and NADPH. Reverse‐phase‐HPLC and electro‐spray ionization‐mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90 ± 0.01 µM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune‐stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.     

Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change

We herein report the development of a recombinant bacterial biosensor for the rapid and easy detection of phenolic compounds in the field. A plasmid was designed to encode a beta-galactosidase reporter gene under the control of capR, an activator involved in phenolic compound degradation. The construct was transformed into Escherichia coli, and transformed cells were stored after being freeze-dried in the presence of sucrose. For detection of phenolic compounds, the cells were rehydrated, and used instantly, without any growth step. In the presence of 0.1 microM-10mM phenol, we observed a red color from hydrolysis of chlorophenol red beta-D-galactopyranoside (CPRG) or an indigo color from hydrolysis of X-galactopyranoside (X-gal). Other phenolic compounds could be detected by this system, including catechol, 2-methylphenol, 2-chlorophenol, 3-methylphenol, 2-nitrophenol, and 4-chlorophenol. These results suggest that this novel bacteria biosensor may be useful for easy, on-site detection of phenolic compounds without the need for unwieldy equipment or sample pretreatment. Indeed, biosensor systems involving beta-galactosidase-expressing freeze-dried recombinant bacteria could prove useful for the in situ detection of many more compounds in the future.     

Re-purposing cancer therapeutics for breast cancer immunotherapy

After decades of work to develop immune-based therapies for cancer, the first drugs designed specifically to engage the host anti-tumor immune response for therapeutic benefit were recently approved for clinical use. Sipuleucel-T, a vaccine for advanced prostate cancer, and ipilimumab, a monoclonal antibody that mitigates the negative impact of cytotoxic T lymphocyte antigen-4 signaling on tumor immunity, provide a modest clinical benefit in some patients. The arrival of these drugs in the clinic is a significant advance that we can capitalize on for even better clinical outcomes. The strategic and scientifically rational integration of vaccines and other direct immunomodulators with standard cancer therapeutics should lead to therapeutic synergy and high rates of tumor rejection. This review focuses on the use of cyclophosphamide, doxorubicin, and HER-2-specific monoclonal antibodies to dissect mechanisms of immune tolerance relevant to breast cancer patients and illustrates how appropriate preclinical models can powerfully inform clinical translation. The immune-modulating activity of targeted, pathway-specific, small molecule therapeutics is also discussed. Fully understanding how cancer drugs impact the immune system should lead to the ultimate personalized cancer medicine: effective combinatorial immunotherapy strategies that simultaneously target signaling pathways essential for tumor growth and progression, and systematically break multiple, distinct immune tolerance pathways to maximize tumor rejection and effect cure.     

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.     

Recent progress on developing exogenous monocyte/macrophage-based therapies for inflammatory and degenerative diseases

Cell-based therapies are a rapidly developing area of regenerative medicine as dynamic treatments that execute therapeutic functions multimodally. Monocytes and macrophages, as innate immune cells that control inflammation and tissue repair, are increasing popular clinical candidates due to their spectrum of functionality. In this article, we review the role of monocytes and macrophages specifically in inflammatory and degenerative disease pathology and the evidence supporting the use of these cells as an effective therapeutic strategy. We compare current strategies of exogenously polarized monocyte/macrophage therapies regarding dosage, delivery and processing to identify outcomes, advances and challenges to their clinical use. Monocytes/macrophages hold the potential to be a promising therapeutic avenue but understanding and optimization of disease-specific efficacy is needed to accelerate their clinical use.     

Thermal biosensor for detecting nonylphenol in the environment

The main goal of the research was the development of thermal immune biosensor for highly sensitive and specific determination of nonylphenol (NPh), based on measuring the heat released as a result of the interaction between hapten and specific antibodies. As it was shown previously, in case of SPR based immune biosensor a number of algorithms of analysis was realized, including "competitive" (with the sensitivity on the level of about 7-10 ng/ml), "direct" (10 ng/ml) ways, and the so called algorithm "to saturation" (about 2-5 ng/ml). The time of analysis by immune SPR biosensor is about 10 min (on the previously prepared transducer surface, including immobilization of sensitive structures). The developed thermal biosensor provides direct detection of NPh with the sensitivity of about 1 microg/ml and the overall time of analysis of about 20-30 min. In spite of a lower sensitivity of the thermal biosensor, it is less sensitive to admixtures in real samples and simpler in use than the biosensor based on SPR and, consequently, the thermal biosensor is more applicable in the field conditions.     

Correlation between a novel calpastatin biosensor and traditional calpastatin assay techniques

An optical fiber biosensor to detect calpastatin has been investigated as a preliminary step in developing tenderness detection instrumentation. Longissimus dorsi samples were taken from beef carcasses (n=21) at 0, 24, 36 and 48h postmortem. Muscle homogenates were assayed for calpastatin activity using traditional methods and an optical fiber biosensor. Warner-Bratzler shear force was also performed on a steak from each carcass at 14d postmortem. Results demonstrated that the measurements with highest correlation between traditional calpastatin assays and optical biosensor readings were taken at 48h postmortem (r=0.597, P< or =0.01), suggesting that this is the best time for use of this biosensor in an on-line grading system. This research further advances the development of a calpastatin biosensor and would be useful in laboratory determination of the presence of biologically active calpastatin concentrations.     

Personalized health care: from theory to practice

The practice of medicine stands at the threshold of a transformation from its current focus on the treatment of disease events to an emphasis on enhancing health, preventing disease and personalizing care to meet each individual's specific health needs. Personalized health care is a new and strategic approach that is driven by personalized health planning empowered by personalized medicine tools, which are facilitated by advances in science and technology. These tools improve the capability to predict health risks, to determine and quantify the dynamics of disease development, and to target therapeutic approaches to the needs of the individual. Personalized health care can be implemented today using currently available technologies and know-how and thereby provide a market for the rational introduction of new personalized medicine tools. The need for early adoption of personalized health care stems from the necessity to reduce the egregious and wasteful burden of preventable chronic diseases, which is not effectively addressed by our current approach to care.     

Biosensors for determination of total and natural antioxidant capacity of red and white wines: comparison with other spectrophotometric and fluorimetric methods

Research was carried out to experimentally evaluate the antioxidant capacity of several red and white wines using a superoxide dismutase (SOD) biosensor recently developed by the present authors. Measurements were performed by comparing the biosensor response to increasing concentration of the superoxide radical produced in solution by the xanthine/xanthine oxidase system, both in the presence and absence of the test sample.The results were compared with those of two traditional spectrophotometric methods and of a spectrofluorimetric method described in literature.Lastly, also the polyphenol, sulfite and ascorbic acid contents of the different wine samples examined were measured using a tyrosinase biosensor, a sulfite oxidase biosensor and an ascorbate oxidase biosensor, respectively.     

Characterization of a self-assembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-GAD antibody

A biosensor chip utilizing surface plasmon resonance (SPR) was fabricated for detecting anti-glutamic acid decarboxylase (GAD) antibody, which is an indicator of the presence of type I diabetes mellitus. The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 3-mercaptopropionic acid (3-MPA) to 11-mercaptoundecanoic acid (11-MUA). To determine the surface characteristics of the different alkanethiol monolayers, several quantitative and kinetic measurements were carried out. The extent of immobilization of streptavidin (SA) and biotin-GAD (the anti-GAD receptor) and the immune response of anti-GAD antibody against GAD were measured using the SPR biosensor. The terminal functional group of a thiol has different effects on the adsorption and covalent binding of protein depending on the steric hindrance. The protein chip described herein permits simple, rapid detection of anti-GAD antibody.     

Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes

The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.     

Innovative analytical system for screening on lectins

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., those located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest. In a new approach, biomolecular interaction analysis (BIA) was utilized for a screening program on lectins. The BIA has been done by a new interferometric biosensor based on spectral-phase interference (SPI). The system can be used either for characterisation of lectin binding domains or for a screening on lectins obtained from natural sources. Several lectin binding surfaces on the basis of SPI have been established.     

Antitumour immune responses

The role of the immune system in combating tumour progression has been studied extensively. The two branches of the immune response - humoral and cell-mediated - act both independently and in concert to combat tumour progression, the success of which depends on the immunogenicity of the tumour cells. The immune system discriminates between transformed cells and normal cells by virtue of the presence of unique antigens on tumour cells. Despite this, the immune system is not always able to detect and kill cancerous cells because neoplasms have also evolved various strategies to escape immune surveillance. Attempts are being made to trigger the immune system into an early and efficient response against malignant cells, and various therapeutic modalities are being developed to enhance the strength of the immune response against tumours. This review aims to elucidate the tumoricidal role of various components of the immune system, including macrophages, lymphocytes, dendritic cells and complement.