IL-4 regulates neutrophilic pulmonary inflammation in a mouse model of bronchial asthma.

Neutrophilic pulmonary inflammation in asthmatics substantially exacerbates the severity of the disease leading to resistance to conventional corticosteroid therapy. Many studies established the involvement of Th1- and Th17-cells and cytokines produced by them (IFNg, IL-17A, IL-17F etc.) in neutrophilic pulmonary inflammation. Recent studies revealed that IL-4 - a Th2-cytokine regulates neutrophil effector functions and migration. It was showed that IL-4 substantially reduces neutrophilic inflammation of the skin in a mouse model of cutaneous bacterial infection and blood neutrophilia in a mouse model systemic bacterial infection. However, there are no data available regarding the influence of IL-4 on non-infectious pulmonary inflammation. In the current study we investigated the effects of IL-4 in a previously developed mouse model of neutrophilic bronchial asthma. We showed that systemic administration of IL-4 significantly restricts neutrophilic inflammation of the respiratory tract probably through the suppression of Th1-/Th17-immune responses and downregulation of CXCR2. Additionally, pulmonary neutrophilic inflammation could be alleviated by IL-4-dependant polarization of N2 neutrophils and M2 macrophages, expressing anti-inflammatory TGFß. Considering these, IL-4 might be used for reduction of exaggerated pulmonary neutrophilic inflammation and overcoming corticosteroid insensitivity of asthma patients.

Wound healing activity of aqueous dispersion of fullerene C60 produced by "green technology".

In addition to exhibited antioxidant and anti-inflammatory activity, fullerene C60 is a promising wound healing agent. An important stage in the production of fullerene-based ointments is the stability of the aqueous fullerene dispersion (AFD) with minimum size of colloidal fullerene aggregates and sufficiently high concentration. To achieve these parameters tangential flow filtration of fullerene C60 was used ("green technology"). As estimated by small-angle neutron scattering and dynamic light scattering purified AFDs with narrow-size distribution nanoclusters have a size of 6 nm and are assembled into agglomerates which reach a size of 150 nm. The ability of the AFD to exhibit regenerative activity was studied using the animal wound model. This study shows for the first time that the fullerene-based composition stimulates the healing of wounds of various origins. We assume that the mechanism of the AFD wound-healing activity is associated with the aryl hydrocarbon receptor and macrophages activity.

Modern View of Neutrophilic Asthma Molecular Mechanisms and Therapy.

For a long time asthma was commonly considered as a homogeneous disease. However, recent studies provide increasing evidence of its heterogeneity and existence of different phenotypes of the disease. Currently, classification of asthma into several phenotypes is based on clinical and physiological features, anamnesis, and response to therapy. This review describes five most frequently identified asthma phenotypes. Neutrophilic asthma (NA) deserves special attention, since neutrophilic inflammation of the lungs is closely associated with severity of the disease and with the resistance to conventional corticosteroid therapy. This review focuses on molecular mechanisms of neutrophilic asthma pathogenesis and on the role of Th1- and Th17-cells in the development of this type of asthma. In addition, this review presents current knowledge of neutrophil biology. It has been established that human neutrophils are represented by at least three subpopulations with different biological functions. Therefore, total elimination of neutrophils from the lungs can result in negative consequences. Based on the new knowledge of NA pathogenesis and biology of neutrophils, the review summarizes current approaches for treatment of neutrophilic asthma and suggests new promising ways to treat this type of asthma that could be developed in future.

[Prospects For the Use of Peptides against Respiratory Syncytial Virus].

The human respiratory syncytial virus (RSV) is one of the most common viral pathogens that affects the lower respiratory tract and could be a reason of bronchiolitis and/or pneumonia. Currently, there are no available effective ways of treating the RSV infection. Attempts to develop preventive vaccine have been unsuccessful. The only therapeutic agent used for RSV treatment is virazole (ribavirin); however, it induces adverse effects. Medications based on neutralizing monoclonal antibodies, such as IGIV (Respigam), palivizumab (Synagis), and MEDI-524 (Numab), are under clinical trials; however, their use will be limited by their high cost. One of the promising approaches for antiviral therapy is the use of natural peptides (defensins and cathelicidins), or their synthetic analogs. The majority of currently described antiviral peptides are developed against the human immunodeficiency virus, the herpes simplex virus, and the influenza virus. At the same time, a body of experimental data evidencing anti-RSV activity of peptides has been accumulated. The main advantages of peptide drugs are their wide spectrum of antiviral activity and low toxicity. However, there are obstacles in implementing peptide-based drugs in clinical practice. Due to their low resistance to the action of serum proteases, most authors consider peptides promising only for local application. Given that RSV affects the epithelium of the respiratory tract, where the protease activity is lower than in the systemic circulation, it is possible to develop locally active peptide drugs, for example, as inhalation forms. Their stability could also be increased by the synthesis of dendrimer peptides and by the development of recombinant peptides as precursor proteins. Anti-RSV peptides can be divided into several groups: (1) attachment and/or fusion blockers; (2) peptides displaying direct virucidal activity, disrupting the viral envelope. Such peptides, which suppress early stages of the viral life cycle, are considered prophylactic agents. However, for several peptides, their immunoregulatory properties have been described, which opens the possibility for therapeutic use. This review summarizes the information on the antiviral properties of such peptides and mechanisms of their action and describes the prospects of the future development of antiviral peptides.

[Gene expression levels of cytokines in different phenotypes of CRSwNP]. / Uroven' ékspressii genov tsitokinov pri raznykh fenotipakh polipoznogo rinosinusita.

INTRODUCTION: Rhinosinusitis with nasal polyps (CRSwNP) is often followed by a range of comorbid states, influence of which on the course of the main pathology process remains insufficiently studied. PURPOSE: To study the gene expression level of cytokines potentially talking part in the development of inflammation in nasal polyps with different phenotypes of CRSwNP. MATERIAL AND METHODS: All the patients with CRSwNP were divided into 4 equal groups, 36 patients in each subgroup: group 1 - CRSwNP without comorbid pathology; group 2 - CRSwNP+atopy; group 3 - CRSwNP + non-allergic bronchial asthma (BA); control group 4 - 36 patients diagnosed with hypertrophic rhinitis without atopy and without bronchial asthma. Using the real-time polymerase chain reaction (Real-Time PCR) method, the study of expression level of mRNA genes coding proteins IL-1ß, IL-4, IL-5, IL-13, IL-17F, IL-25, IFN-y, TSLP in polyp tissue was conducted. RESULTS: The statistically proved difference of expression level of cytokines depending on the CRSwNP phenotype was educed. If CRSwNP and atopy were combined, the gene expression level of all studied cytokines was statistically higher than that of CRSwNP without comorbid pathology; and the expression level of IL-17F, IL-25 and TSLP was more intense that in the group of CRSwNP + BA. There was no difference between the patients with comorbid allergy and comorbid BA regarding the gene expression of IFN-y, IL-5 and IL-13 cytokines. Among different phenotypes of CRSwNP no difference in IL-1ß expression level was detected, which evidences of persisting inflammatory process, and the IL-4 gene expression level was lower than the detection level in all the groups. CONCLUSION: With different CRSwNP phenotypes different inflammatory patterns are detected, which indicates different character of the pathology process course among these groups of patients. Higher expression level of cytokine genes, which are a marker of epithelial damage of IL-25 and TSLP, is found only among the patients with CRSwNP and atopy. It suggests that forming of CRSwNP without comorbid pathology is connected with other pathologic mechanisms, not with the damage to epithelial barrier. If CRSwNP + BA and CRSwNP + atopy were combined, the expression level of IFN-y, IL-5, IL-13 and IL-17F genes was higher than the one in the group of patients with CRSwNP without comorbid pathology. In view of obtained data, all the patients with CRSwNP shall be screened for bronchial asthma and the allergy diagnostic shall be conducted.

The potential of anti-infectives and immunomodulators as therapies for asthma and asthma exacerbations.

Asthma is responsible for approximately 25,000 deaths annually in Europe despite available medicines that maintain asthma control and reduce asthma exacerbations. Better treatments are urgently needed for the control of chronic asthma and reduction in asthma exacerbations, the major cause of asthma mortality. Much research spanning >20 years shows a strong association between microorganisms including pathogens in asthma onset, severity and exacerbation, yet with the exception of antibiotics, few treatments are available that specifically target the offending pathogens. Recent insights into the microbiome suggest that modulating commensal organisms within the gut or lung may also be a possible way to treat/prevent asthma. The European Academy of Allergy & Clinical Immunology Task Force on Anti-infectives in Asthma was initiated to investigate the potential of anti-infectives and immunomodulators in asthma. This review provides a concise summary of the current literature and aimed to identify and address key questions that concern the use of anti-infectives and both microbe- and host-based immunomodulators and their feasibility for use in asthma.

The Role of Interleukin-33 in Pathogenesis of Bronchial Asthma. New Experimental Data.

Interleukin-33 (IL-33) belongs to the IL-1 cytokine family and plays an important role in modulating immune system by inducing Th2 immune response via the ST2 membrane receptor. Epithelial cells are the major producers of IL-33. However, IL-33 is also secreted by other cells, e.g., bone marrow cells, dendritic cells, macrophages, and mast cells. IL-33 targets a broad range of cell types bearing the ST2 surface receptor. Many ST2-positive cells, such as Th2 cells, mast cells, basophils, and eosinophils, are involved in the development of allergic bronchial asthma (BA). This suggests that IL-33 directly participates in BA pathogenesis. Currently, the role of IL-33 in pathogenesis of inflammatory disorders, including BA, has been extensively investigated using clinical samples collected from patients, as well as asthma animal models. In particular, numerous studies on blocking IL-33 and its receptor by monoclonal antibodies in asthma mouse model have been performed over the last several years; IL-33- and ST2-deficient transgenic mice have also been generated. In this review, we summarized and analyzed the data on the role of IL-33 in BA pathogenesis and the prospects for creating new treatments for BA.

[Anticytokine therapy of allergic asthma].

Bronchial asthma is a chronic inflammatory disease of the airways that is characterized by episodes of shortness of breath, expiratory dyspnea, cough, wheezing, and pulmonary emphysema. At the present time, asthma is a global public health problem and affects about 5% of the worldwide population. Although a wide range of anti-inflammatory drugs is available, uncontrolled or poorly controlled asthma is still a problem, requiring the development of novel therapeutic approaches. Intense studies of the molecular mechanisms of asthma in transgenic animals performed since the 1990s implicated cytokines, such as IL-4, IL-5, and IL-13, and their receptors in the initiation and maintenance of asthma. These findings led to anticytokine therapy as a novel approach for bronchial asthma treatment. To date, many preclinical and clinical studies have been performed in this field especially with drugs based on humanized monoclonal antibodies, soluble receptors, peptide inhibitors, etc. The review summarizes the data from preclinical and clinical studies of anti-cytokine therapeutics in humans.

Effects of Release-Active Antibodies to CD4 Receptor on the Level of lck-Kinase in Cultured Mononuclear Cells from Human Peripheral Blood.

For evaluation of effects of release-active antibodies to CD4 on cultured lymphocytes from human peripheral blood, we measured intracellular content of lck-kinase cell-based ELISA. In cells treated with release-active antibodies to CD4, the content of intracellular lck-kinase significantly (p<0.01) decreased in comparison with the control (purified water processed in a similar way). Phytohemagglutinin had no effect on the concentration of lck-kinase in cells. The decrease in the content of CD4-associated lck protein suggests that the preparation enhanced intracellular coupling of lck-kinase with T-cell receptor and potentiated T-cell immune response.

Interleukins 1 and 6 as Main Mediators of Inflammation and Cancer.

The idea of a potential link between cancer and inflammation was first proposed by R. Virchow in the nineteenth century. However, clear evidence regarding a key role of inflammation in oncogenesis appeared only during the last decade. Now the tumor microenvironment is commonly considered as an obligatory and significant component of almost all types of cancer, and the cells infiltrating such microenvironment are a source of inflammatory cytokines. Such cytokines play a key role in regulating inflammation during both normal immune response and developing cancer. In this review, we explore the role of two inflammatory cytokines interleukin 1 and interleukin 6 in cancer development. These cytokines have pleiotropic effects on various cell types in the tumor microenvironment, particularly being able to regulate pro-oncogenic transcription factors NF-κB and STAT3. For this reason, such cytokines influence key parameters of oncogenesis, increasing cell resistance to apoptosis, proliferation of cancer cells, angiogenesis, invasion and malignancy as well as the ability of tumor cells to respond to anticancer therapy. Here we summarize novel experimental data regarding mechanisms underlying the interaction between chronic inflammation and malignant neoplasms.

Antiviral Activity of Ergoferon against Group A Rotavirus.

Antiviral activity of Ergoferon was studied in vitro on an experimental model of rotavirus infection in MA-104 cell line. In infected cells treated with Ergoferon, rotavirus titer was shown to decrease by 83 and 90% in comparison with cells treated with solvent used for Ergoferon preparation (p<0.05) and distilled water (p<0.05), respectively. These findings demonstrate high anti-rotavirus activity of Ergoferon.

RNA INTERFERENCE: NEW APPROACH TO THE TREATMENT OF ALLERGIC ASTHMA (A REVIEW).

Asthma is among the most common chronic disorders of airways, which affects both children and adults. Asthma being a common disease among different segments of population, it has a high mortality rate and, in the absence of appropriate care, affects the quality of life and leads to economics losses. In a view of continuing growth in the incidence of asthma, it is important to find relevant biological targets for developing new approaches to astma therapy. Recent advances in molecular immunology, genetics, and bioinformatics allowed genes involved in the pathogenesis of asthma to be identified, which provided prerequisites for the development of new types of drugs that can regulate the activity of pathogenically significant genes. To date, a number of technologies for sequence-specific gene regulation (ASO, ribozymes, DNAzymes, EGS, DNA-decoys, U 1-adapters) are available, but RNA interference is the most promising approach in both terms of efficacy and financial cost. This review focuses on the generalization and analysis of experimental data regarding the use of RNA interference technology for the treatment of astma.

CXC chemokines and antimicrobial peptides in rhinovirus-induced experimental asthma exacerbations.

RATIONALE: Rhinoviruses (RVs) are the major triggers of asthma exacerbations. We have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental RV-induced asthma exacerbations. OBJECTIVES: We hypothesized that neutrophil-related CXC chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in RV-induced exacerbations of asthma. METHODS: Protein levels of antimicrobial peptides (SLPI, HNP 1-3, elafin, and LL-37) and neutrophil chemokines (CXCL1/GRO-α, CXCL2/GRO-ß, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, and CXCL8/IL-8) were determined in bronchoalveolar lavage (BAL) fluid of 10 asthmatics and 15 normal controls taken before, at day four during and 6 weeks post-experimental infection. RESULTS: BAL HNP 1-3 and Elafin were higher, CXCL7/NAP-2 was lower in asthmatics compared with controls at day 4 (P = 0.035, P = 0.048, and P = 0.025, respectively). BAL HNP 1-3 and CXCL8/IL-8 were increased during infection (P = 0.003 and P = 0.011, respectively). There was a trend to increased BAL neutrophils at day 4 compared with baseline (P = 0.076). BAL HNP 1-3 was positively correlated with BAL neutrophil numbers at day 4. There were no correlations between clinical parameters and HNP1-3 or IL-8 levels. CONCLUSIONS: We propose that RV infection in asthma leads to increased release of CXCL8/IL-8, attracting neutrophils into the airways where they release HNP 1-3, which further enhances airway neutrophilia. Strategies to inhibit CXCL8/IL-8 may be useful in treatment of virus-induced asthma exacerbations.

[Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein].

INTRODUCTION: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid. MATERIALS AND METHODS: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated. RESULTS: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60. CONCLUSIONS: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

RSV infection modulates IL-15 production and MICA levels in respiratory epithelial cells.

The cytokine interleukin (IL)-15, major histocompatibility complex (MHC) class I molecules and MHC class I chain-related proteins (MIC) A and B are involved in cellular immune responses to virus infections but their role in respiratory syncytial virus (RSV) infection has not been studied. We aimed to determine how RSV infection modulates IL-15 production, MHC class I and MICA expression in respiratory epithelial cells, the molecular pathways implicated in virus-induced IL-15 production and how interferon (IFN)-γ alters RSV-induced IL-15 production and MHC class I and MICA expression. We infected respiratory epithelial cell lines (A549 and BEAS-2B cells) and primary bronchial epithelial cells with RSV and measured production of IL-15, expression of MHC I and MICA and the role of the transcription factor nuclear factor (NF)-κB. We report here that RSV increases IL-15 in respiratory epithelial cells via virus replication and NF-κB-dependent mechanisms. Furthermore, RSV infection of epithelial cells upregulated cell surface expression of MICA and levels of soluble MICA. IFN-γ upregulated RSV induction of soluble IL-15 but inhibited induction of MICA. Upregulation of IL-15, MHC I and MICA are likely to be important mechanisms in activating immune responses to RSV by epithelial cells.

[Anti-peptide antibodies specifically recognize the L1 protein of human papilloma virus type 31].

Antibodies that specifically recognize the capsid protein (L1) of human papillomavirus (HPV) are an important tool necessary for designing vaccines against HPV infection. In this work, we have predicted and synthesized peptide fragments mimicking B cell epitopes of L1 HPV type 31 (sequences 49-65, 131-145, 172-189, 349-362 and 402-414), and conjugated their to KLH and BSA to generate the L1-31-specific anti-peptide antibodies in mice. Variants of recombinant L1-31, including full-size and mutants with C-terminal single amino acid changes and deletions and full-size L1-16 were produced in the yeast using monitoring with L1 HPV16-specific monoclonal antibody. Testing of anti-peptide antisera in ELISA showed that antibodies to peptides 49-65 and 172-189 were capable to recognize specifically L1-31 protein, but not L1-16 one. Such antibodies may be used for assay of L1-31 production in various expression systems.

[Biological models of IgE-dependent bronchial asthma: methodology and prospects for application].

The present work was aimed to review modern approaches to simulation of bronchial asthma using laboratory animals. Various modes of immunization and challenge for the induction of typical pathological features in mice are described along with the methods for detection of these markers (including ones for studying murine lung function). Advantages and drawbacks of the models, difficulties and prospects of their application for the study of pathogenesis and preliminary estimation of the safety and efficacy of antiasthmatic therapy (especially antigen-specific immunotherapy) are analyzed. Moreover, the paper presents concise characteristic of short-term adjuvant-free murine models of timothy pollen extract- and ovalbumin-induced bronchial asthma developed in this country.

[The development of vector constructions for respiratory syncitial virus (RSV) P-gene silencing].

Interfering RNA (RNAi) is a powerful tool to silence gene expression on the level of mRNA. To knock-down gene expression by using RNAi two major methods of mRNA silencing exist. First method utilizes siRNA (small interfering RNA), a readily processed dsRNA, that enters RISC complex and destroy target mRNA after transfection into the cells. The second method based on the construction of plasmid DNA that expresses shRNA (short harpin RNA) from U6 or CMV promoter. shRNA gets processed by Drosha and Dicer RNAses inside the cell before it translocates to the cell cytoplasm and affects the level of target RNA. In this study we modified lentiviral vector pGIPZ expressing tFP-IRES-Puro-shRNA(mir30) cassette by introducing BamH I restriction site downstream of this cassette. This modification makes possible to clone specific shRNA sequences in pGIPZ vector using XhoI/BamHI restriction sites instead of the original recombination. Three shRNAs against phosphoprotein P of respiratory sinthitial virus (RSV) and shRNA against human CD43 as a control were generated and cloned into modified so-called pCIPD vector. Monkey kidney cells MA-104 were stably transduced with four shRNA constructs. In conclusion, the generated lentiviral vector pCIPD can be successfully used for efficient gene silencing and virus replication in a broad variety of cells.

Respiratory virus induction of alpha-, beta- and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells.

BACKGROUND: Respiratory viruses, predominantly rhinoviruses are the major cause of asthma exacerbations. Impaired production of interferon-beta in rhinovirus infected bronchial epithelial cells (BECs) and of the newly discovered interferon-lambdas in both BECs and bronchoalveolar lavage cells, is implicated in asthma exacerbation pathogenesis. Thus replacement of deficient interferon is a candidate new therapy for asthma exacerbations. Rhinoviruses and other respiratory viruses infect both BECs and macrophages, but their relative capacities for alpha-, beta- and lambda-interferon production are unknown. METHODS: To provide guidance regarding which interferon type is the best candidate for development for treatment/prevention of asthma exacerbations we investigated respiratory virus induction of alpha-, beta- and lambda-interferons in BECs and peripheral blood mononuclear cells (PBMCs) by reverse transferase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Rhinovirus infection of BEAS-2B BECs induced interferon-alpha mRNA expression transiently at 8 h and interferon-beta later at 24 h while induction of interferon-lambda was strongly induced at both time points. At 24 h, interferon-alpha protein was not detected, interferon-beta was weakly induced while interferon-lambda was strongly induced. Similar patterns of mRNA induction were observed in primary BECs, in response to both rhinovirus and influenza A virus infection, though protein levels were below assay detection limits. In PBMCs interferon-alpha, interferon-beta and interferon-lambda mRNAs were all strongly induced by rhinovirus at both 8 and 24 h and proteins were induced: interferon-alpha>-beta>-lambda. Thus respiratory viruses induced expression of alpha-, beta- and lambda-interferons in BECs and PBMCs. In PBMCs interferon-alpha>-beta>-lambda while in BECs, interferon-lambda>-beta>-alpha. CONCLUSIONS: We conclude that interferon-lambdas are likely the principal interferons produced during innate responses to respiratory viruses in BECs and interferon-alphas in PBMCs, while interferon-beta is produced by both cell types.

Diversity of PEGylation methods of liposomes and their influence on RNA delivery.

Gene therapy is a promising approach for personalized medicine, but its application in humans requires development of efficient and safe vehicles. PEGylated liposomes are some of the most suitable delivery systems for nucleic acids because of their stability under physiological conditions and prolonged circulation time, compared to conventional and other types of "stealth" liposomes. In vitro/in vivo activity of PEGylated liposomes is highly dependent on PEG motif abundance. The process of "stealth" coverage formation is a very important parameter for efficient transfection assays and further fate determination of the PEG layer after tissue penetration. In this review, we discuss the latest methods of PEGylated liposome preparation.