Recombinant PreS-fusion protein vaccine for birch pollen and apple allergy.

BACKGROUND: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. METHODS: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA. RESULTS: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV. CONCLUSION: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

Role and Molecular Mechanisms of Alternative Splicing of Th2-Cytokines IL-4 and IL-5 in Atopic Bronchial Asthma.

Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the respiratory tract. Allergic (atopic) asthma is the most common (up to 80% of cases) phenotype developing through the Th2-dependent mechanisms involving cytokines: IL-4, IL-5, IL-9, and IL-13. The genes encoding Th2-cytokines have a mosaic structure (encode exons and introns). Therefore, several mature mRNA transcripts and protein isoforms can be derived from a single mRNA precursor through alternative splicing, and they may contribute to BA pathogenesis. Analysis of the published studies and databases revealed existence of the alternative mRNA transcripts for IL-4, IL-5, and IL-13. The alternative transcripts of IL-4 and IL-5 carry open reading frames and therefore can encode functional proteins. It was shown that not only alternative mRNA transcripts exist for IL-4, but alternative protein isoforms, as well. Natural protein isoform (IL-4δ2) lacking the part encoded by exon-2 was identified. Similarly, alternative mRNA transcript with deleted exon-2 (IL-5δ2) was also identified for IL-5. In this review, we summarize current knowledge about the identified alternative mRNA transcripts and protein isoforms of Th2-cytokinins, first of all IL-4 and IL-5. We have analyzed biological properties of the alternative variants of these cytokines, their possible role in the allergic asthma pathogenesis, and considered their diagnostic and therapeutic potential.

Cell-Penetrating Peptides as Vehicles for Delivery of Therapeutic Nucleic Acids. Mechanisms and Application in Medicine.

Currently, nucleic acid therapeutics are actively developed for the treatment and prophylactic of metabolic disorders and oncological, inflammatory, and infectious diseases. A growing number of approved nucleic acid-based drugs evidences a high potential of gene therapy in medicine. Therapeutic nucleic acids act in the cytoplasm, which makes the plasma membrane the main barrier for the penetration of nucleic acid-based drugs into the cell and requires development of special vehicles for their intracellular delivery. The optimal carrier should not only facilitate internalization of nucleic acids, but also exhibit no toxic effects, ensure stabilization of the cargo molecules, and be suitable for a large-scale and low-cost production. Cell-penetrating peptides (CPPs), which match all these requirements, were found to be efficient and low-toxic carriers of nucleic acids. CPPs are typically basic peptides with a positive charge at physiological pH that can form nanostructures with negatively charged nucleic acids. The prospects of CPPs as vehicles for the delivery of therapeutic nucleic acids have been demonstrated in numerous preclinical studies. Some CPP-based drugs had successfully passed clinical trials and were implemented into medical practice. In this review, we described different types of therapeutic nucleic acids and summarized the data on the use of CPPs for their intracellular delivery, as well as discussed, the mechanisms of CPP uptake by the cells, as understanding of these mechanisms can significantly accelerate the development of new gene therapy approaches.

Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

BACKGROUND: The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. OBJECTIVE: To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. METHODS: Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. RESULTS: IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. CONCLUSION: IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Antiviral Therapy of COVID-19.

Since the beginning of the COVID-19 pandemic, the scientific community has focused on prophylactic vaccine development. In parallel, the experience of the pharmacotherapy of this disease has increased. Due to the declining protective capacity of vaccines against new strains, as well as increased knowledge about the structure and biology of the pathogen, control of the disease has shifted to the focus of antiviral drug development over the past year. Clinical data on safety and efficacy of antivirals acting at various stages of the virus life cycle has been published. In this review, we summarize mechanisms and clinical efficacy of antiviral therapy of COVID-19 with drugs based on plasma of convalescents, monoclonal antibodies, interferons, fusion inhibitors, nucleoside analogs, and protease inhibitors. The current status of the drugs described is also summarized in relation to the official clinical guidelines for the treatment of COVID-19. In addition, here we describe innovative drugs whose antiviral effect is provided by antisense oligonucleotides targeting the SARS-CoV-2 genome. Analysis of laboratory and clinical data suggests that current antivirals successfully combat broad spectra of emerging strains of SARS-CoV-2 providing reliable defense against COVID-19.

Treatment of COVID-19 patients with a SARS-CoV-2-specific siRNA-peptide dendrimer formulation.

BACKGROUND: Severe acute respiratory syndrome corona virus (SARS-CoV-2) infection frequently causes severe and prolonged disease but only few specific treatments are available. We aimed to investigate safety and efficacy of a SARS-CoV-2-specific siRNA-peptide dendrimer formulation MIR 19® (siR-7-EM/KK-46) targeting a conserved sequence in known SARS-CoV-2 variants for treatment of COVID-19. METHODS: We conducted an open-label, randomized, controlled multicenter phase II trial (NCT05184127) evaluating safety and efficacy of inhaled siR-7-EM/KK-46 (3.7 mg and 11.1 mg/day: low and high dose, respectively) in comparison with standard etiotropic drug treatment (control group) in patients hospitalized with moderate COVID-19 (N = 52 for each group). The primary endpoint was the time to clinical improvement according to predefined criteria within 14 days of randomization. RESULTS: Patients from the low-dose group achieved the primary endpoint defined by simultaneous achievement of relief of fever, normalization of respiratory rate, reduction of coughing, and oxygen saturation of >95% for 48 h significantly earlier (median 6 days; 95% confidence interval [CI]: 5-7, HR 1.75, p = .0005) than patients from the control group (8 days; 95% CI: 7-10). No significant clinical efficacy was observed for the high-dose group. Adverse events were reported in 26 (50.00%), 25 (48.08%), and 28 (53.85%) patients from the low-, high-dose and control group, respectively. None of them were associated with siR-7-EM/KK-46. CONCLUSIONS: siR-7-EM/KK-46, a SARS-CoV-2-specific siRNA-peptide dendrimer formulation is safe, well tolerated and significantly reduces time to clinical improvement in patients hospitalized with moderate COVID-19 compared to standard therapy in a randomized controlled trial.

Murine model of steroid-resistant neutrophilic bronchial asthma as an attempt to simulate human pathology.

Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the airways. The majority of patients with mild to moderate BA develop Th2-biased eosinophilic pulmonary inflammation and respond well to corticosteroid treatment. However up to 10% of BA patients develop severe pathology, which is associated with neutrophilic inflammation and resistant to conventional corticosteroid therapy. Contrary to eosinophil-predominant airway inflammation neutrophilic BA is developed through Th1- and Th17-immune responses. However, the etiology of corticoid insensitive neutrophilic BA is still remains unclear. Therefore, in the current study we developed a mouse model of BA with predominant neutrophilic rather than eosinophilic pulmonary inflammation. BALB/c mice were immunized with the mixture of the ovalbumin allergen and Freund's adjuvant, followed by aerosol challenge with the same allergen mixed with E. coli lipopolysaccharide. As a result, mice developed the main BA manifestations: production of allergen specific IgE, development of airway hyperreactivity, airway remodeling and pulmonary neutrophilic inflammation. Moreover, this pathology developed through Th1- and Th17-dependent mechanisms and mice with induced neutrophilic BA phenotype responded poorly to dexamethasone treatment, that coincide to clinical observations. The established mouse model could be useful both for studying the pathogenesis and for testing novel approaches to control neutrophilic BA.

Role of STAT3 Transcription Factor in Pathogenesis of Bronchial Asthma.

Bronchial asthma is a heterogeneous chronic inflammatory disease of airways. The studies of molecular and cellular mechanisms of bronchial asthma have established that a wide range of immune (T and B cells, eosinophils, neutrophils, macrophages, etc.) and structural (epithelial and endothelial) cells are involved in its pathogenesis. These cells are activated in response to external stimuli (bacteria, viruses, allergens, and other pollutants) and produce pro-inflammatory factors (cytokines, chemokines, metalloproteinases, etc.), which ultimately leads to the initiation of pathological processes in the lungs. Genes encoding transcription factors of the STAT family (signal transducer and activator of transcription), that includes seven representatives, are involved in the cell activation. Recent studies have shown that the transcription factor STAT3 plays an important role in the activation of the abovementioned cells, thus contributing to the development of asthma. In animal studies, selective inhibition of STAT3 significantly reduces the severity of lung inflammation, which indicates its potential as a therapeutic target. In this review, we describe the mechanisms of STAT3 activation and its role in polarization of Th2/Th17 cells and M2 macrophages, as well as in the dysfunction of endothelial cells, which ultimately leads to development of bronchial asthma symptoms, such as infiltration of neutrophils and eosinophils into the lungs, bronchial hyperreactivity, and the respiratory tract remodeling.

From Allergen Molecules to Molecular Immunotherapy of Nut Allergy: A Hard Nut to Crack.

Peanuts and tree nuts are two of the most common elicitors of immunoglobulin E (IgE)-mediated food allergy. Nut allergy is frequently associated with systemic reactions and can lead to potentially life-threatening respiratory and circulatory symptoms. Furthermore, nut allergy usually persists throughout life. Whether sensitized patients exhibit severe and life-threatening reactions (e.g., anaphylaxis), mild and/or local reactions (e.g., pollen-food allergy syndrome) or no relevant symptoms depends much on IgE recognition of digestion-resistant class I food allergens, IgE cross-reactivity of class II food allergens with respiratory allergens and clinically not relevant plant-derived carbohydrate epitopes, respectively. Accordingly, molecular allergy diagnosis based on the measurement of allergen-specific IgE levels to allergen molecules provides important information in addition to provocation testing in the diagnosis of food allergy. Molecular allergy diagnosis helps identifying the genuinely sensitizing nuts, it determines IgE sensitization to class I and II food allergen molecules and hence provides a basis for personalized forms of treatment such as precise prescription of diet and allergen-specific immunotherapy (AIT). Currently available forms of nut-specific AIT are based only on allergen extracts, have been mainly developed for peanut but not for other nuts and, unlike AIT for respiratory allergies which utilize often subcutaneous administration, are given preferentially by the oral route. Here we review prevalence of allergy to peanut and tree nuts in different populations of the world, summarize knowledge regarding the involved nut allergen molecules and current AIT approaches for nut allergy. We argue that nut-specific AIT may benefit from molecular subcutaneous AIT (SCIT) approaches but identify also possible hurdles for such an approach and explain why molecular SCIT may be a hard nut to crack.

Molecular and Cellular Mechanisms of Respiratory Syncytial Viral Infection: Using Murine Models to Understand Human Pathology.

Respiratory syncytial virus (RSV) causes severe pathology of the lower respiratory tract in infants, immunocompromised people, and elderly. Despite decades of research, there is no licensed vaccine against RSV, and many therapeutic drugs are still under development. Detailed understanding of molecular and cellular mechanisms of the RSV infection pathology can accelerate the development of efficacious treatment. Current studies on the RSV pathogenesis are based on the analysis of biopsies from the infected patients; however deeper understanding of molecular and cellular mechanisms of the RSV pathology could be achieved using animal models. Mice are the most often used model for RSV infection because they exhibit manifestations similar to those observed in humans (bronchial obstruction, mucous hypersecretion, and pulmonary inflammation mediated by lymphocytes, macrophages, and neutrophils). Additionally, the use of mice is economically feasible, and many molecular tools are available for studying RSV infection pathogenesis at the molecular and cellular levels. This review summarizes new data on the pathogenesis of RSV infection obtained in mouse models, which demonstrated the role of T cells in both the antiviral defense and the development of lung immunopathology. T cells not only eliminate the infected cells, but also produce significant amounts of the proinflammatory cytokines TNFα and IFNγ. Recently, a new subset of tissue-resident memory T cells (TRM) was identified that provide a strong antiviral defense without induction of lung immunopathology. These cells accumulate in the lungs after local rather than systemic administration of RSV antigens, which suggests new approaches to vaccination. The studies in mouse models have revealed a minor role of interferons in the anti-RSV protection, as RSV possesses mechanisms to escape the antiviral action of type I and III interferons, which may explain the low efficacy of interferon-containing drugs. Using knockout mice, a significant breakthrough has been achieved in understanding the role of many pro-inflammatory cytokines in lung immunopathology. It was found that in addition to TNFα and IFNγ, the cytokines IL-4, IL-5, IL-13, IL-17A, IL-33, and TSLP mediate the major manifestations of the RSV pathogenesis, such as bronchial obstruction, mucus hyperproduction, and lung infiltration by pro-inflammatory cells, while IL-6, IL-10, and IL-27 exhibit the anti-inflammatory effect. Despite significant differences between the mouse and human immune systems, mouse models have made a significant contribution to the understanding of molecular and cellular mechanisms of the pathology of human RSV infection.

Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

IgE-reactivity profiles to allergen molecules in Russian children with and without symptoms of allergy revealed by micro-array analysis.

BACKGROUND: The analysis of longitudinal birth cohorts with micro-arrayed allergen molecules has provided interesting information about the evolution of IgE sensitization in children. However, so far no cross-sectional study has been performed comparing IgE sensitization profiles in children with and without symptoms of allergy. Furthermore, no data are available regarding molecular IgE sensitization profiles in children from Russia. METHODS: We recruited two groups of age- and gender-matched children, one (Group 1: n = 103; 12.24 ± 2.23 years; male/female: 58/45) with symptoms and a second (Group 2: n = 97; 12.78 ± 2.23 years; male/female: 53/44), without symptoms of allergy according to international ISAAC questionnaire. Children were further studied regarding symptoms of allergy (rhinitis, asthma, atopic dermatitis) according to international guidelines, and skin prick testing with a panel of aeroallergen extracts was performed before sera were analyzed in an investigator-blinded manner for IgE specific to more than 160 micro-arrayed allergen molecules using ImmunoCAP ISAC technology. RESULTS: IgE sensitization = or >0.3 ISU to at least one of the micro-arrayed allergen molecules was found in 100% of the symptomatic children and in 36% of the asymptomatic children. Symptomatic and asymptomatic children showed a comparable IgE sensitization profile; however, frequencies of IgE sensitization and IgE levels to the individual allergen molecules were higher in the symptomatic children. Aeroallergen sensitization was dominated by sensitization to major birch pollen allergen, Bet v 1, and major cat allergen, Fel d 1. Food allergen sensitization was due to cross-sensitization to PR10 pollen and food allergens whereas genuine peanut sensitization was absent. CONCLUSION: This is the first study analyzing molecular IgE sensitization profiles to more than 160 allergen molecules in children with and without symptoms of allergy. It detects similar molecular IgE sensitization profiles in symptomatic and asymptomatic children and identifies Bet v 1 and Fel d 1 as the predominant respiratory allergen molecules and PR10 proteins as the major food allergens and absence of genuine peanut allergy in Moscow region (Russia).

Past, present, and future of allergen immunotherapy vaccines.

Allergen-specific immunotherapy (AIT) is an allergen-specific form of treatment for patients suffering from immunoglobulin E (IgE)-associated allergy; the most common and important immunologically mediated hypersensitivity disease. AIT is based on the administration of the disease-causing allergen with the goal to induce a protective immunity consisting of allergen-specific blocking IgG antibodies and alterations of the cellular immune response so that the patient can tolerate allergen contact. Major advantages of AIT over all other existing treatments for allergy are that AIT induces a long-lasting protection and prevents the progression of disease to severe manifestations. AIT is cost effective because it uses the patient´s own immune system for protection and potentially can be used as a preventive treatment. However, broad application of AIT is limited by mainly technical issues such as the quality of allergen preparations and the risk of inducing side effects which results in extremely cumbersome treatment schedules reducing patient´s compliance. In this article we review progress in AIT made from its beginning and provide an overview of the state of the art, the needs for further development, and possible technical solutions available through molecular allergology. Finally, we consider visions for AIT development towards prophylactic application.

Respiratory syncytial virus upregulates IL-33 expression in mouse model of virus-induced inflammation exacerbation in OVA-sensitized mice and in asthmatic subjects.

BACKGROUND: Bronchial asthma (BA) is a chronic disease of the airways. The great majority of BA exacerbations are associated with respiratory viral infections. Recent findings point out a possible role of proinflammatory cytokine interleukin-33 (IL-33) in the development of atopic diseases. Although, little is known about the role of IL-33 in virus-induced BA exacerbations. METHODS: We used mouse models of RSV (respiratory syncytial virus)-induced inflammation exacerbation in OVA-sensitized mice and RSV infection alone in adult animals to characterize expression of il33 in the mouse lungs. Moreover, we studied the influence of il33 knockdown with intranasally administrated siRNA on the development of RSV-induced inflammation exacerbation. In addition, we evaluated the expression of IL33 in the ex vivo stimulated PBMCs from allergic asthma patients and healthy subjects with and without confirmed acute respiratory viral infection. RESULTS: Using mouse models, we found that infection with RSV drives enhanced il33 mRNA expression in the mouse lung. Treatment with anti-il33 siRNA diminishes airway inflammation in the lungs (we found a decrease in the number of inflammatory cells in the lungs and in the severity of histopathological alterations) of mice with RSV-induced inflammation exacerbation, but do not influence viral load. Elevated level of the IL33 mRNA was detected in ex vivo stimulated blood lymphocytes of allergic asthmatics infected with respiratory viruses. RSV and rhinovirus were the most detected viruses in volunteers with symptoms of respiratory infection. CONCLUSION: The present study provides additional evidence of the crucial role of the IL-33 in pathogenesis of RSV infection and virus-induced allergic bronchial asthma exacerbations.

Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases.

Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6-50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.

Genotoxicity of cationic lipopeptide nanoparticles.

The genotoxicity of cationic lipopeptide nanoparticles (cLPNPs) was evaluated in vivo and in vitro comet assay and the in vivo chromosome aberrations test. In vitro comet assay, human blood cells were exposed to cLPNPs at the concentration of 2.5, 5, 10, 20, 40 and 100 µg/mL. Significant DNA damage was observed after 1 h exposure, but no effects were detected after 3 h. In vivo, cLPNPs were administered in single or five daily injection doses at 8, 20 and 40 mg/kg of body weight by subcutaneous injection to male mice. The cLPNPs caused DNA damage in the liver, lung and kidney, but not in the spleen. The kidney was more prone to genotoxic effects that persisted from 24 h to 14d after a single injection of cLPNPs. No statistically significant increase in the percentage of cells with chromosomal aberrations above the vehicle control was observed in mice bone marrow after a single or repeated injection of cLPNPs. In summary, cLPNPs shown to be genotoxic both in vivo and in vitro. The results suggest the importance of the use of highly sensitive methods, such as the comet assay, in order to determine the full genotoxic potential of nanoparticles.

Linear and dendrimeric antiviral peptides: design, chemical synthesis and activity against human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the most common viral pathogens. It is especially dangerous for newborns and young children. In some cases it could lead to severe bronchiolitis, pneumonia with hospitalization or even a lethal outcome. Despite decades of investigation of RSV biology, effective and safe therapeutics are still under development. Certain natural peptides have been found to exhibit antiviral activity against respiratory viruses, but their implementation is limited by low stability in biological media. One of the current approaches to enhance the peptide therapeutic opportunities is chemical synthesis of peptide dendrimers with hyperbranched structures. Taking into account the recent data of bioactive cationic and helical regions of natural peptides and the structure features of nucleolin identified as an RSV cellular receptor, the main goal of this study was to design relatively short linear and dendrimeric cationic peptides and to test their antiviral activity against RSV. As a result 3 linear cationic peptides and 4 peptide dendrimers were synthesized and compared with known LL-37 (cathelicidin family) and anti-F0 monoclonal antibodies in terms of cytotoxicity and antiviral activity. Their affinity to the supposed molecular target - nucleolin (C23) - was estimated in silico by molecular docking analysis. Four synthesized peptides demonstrated a cytotoxic effect, two of them were even more cytotoxic than LL-37, which could be explained by a combination of a high amount of positive charge and amphipathicity. Contrariwise, non-hydrophobic dendrimer peptides did not exhibit cytotoxicity in mammalian cells in the studied concentration range. Two of the seven synthesized peptides, LTP (dendrimer) and SA-35 (linear), used in this study had a stronger antiviral effect than natural peptide LL-37, and three others showed slightly lower activity than anti-F0 monoclonal antibodies. The data obtained in this study suggest that evenly distributed positive charge, and low or medium amphipathicity play a key role in the antiviral activity of the studied peptides. Moreover, the calculated free energy values of the peptide/nucleolin complex for the most active peptides supported the idea that the peptide ability of nucleolin interaction promotes the anti-RSV properties of the molecules.

Identification of a novel splice variant for mouse and human interleukin-5.

Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30-40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

Cell-Type-Specific Responses to Interleukin-1 Control Microbial Invasion and Tumor-Elicited Inflammation in Colorectal Cancer.

Chronic inflammation drives the progression of colorectal cancer (CRC). Increased expression of interleukin (IL)-17A is associated with poor prognosis, and IL-17A blockade curbs tumor progression in preclinical models of CRC. Here we examined the impact of IL-1 signaling, a key regulator of the IL-17 pathway, in different cell types within the CRC microenvironment. Genetic deletion of the IL-1 receptor (IL-1R1) in epithelial cells alleviated tumorigenesis in the APC model of CRC, demonstrating a cell-autonomous role for IL-1 signaling in early tumor seed outgrowth. T cell specific ablation of IL-1R1 decreased tumor-elicited inflammation dependent on IL-17 and IL-22, thereby reducing CRC progression. The pro-tumorigenic roles of IL-1 were counteracted by its effects on myeloid cells, particularly neutrophils, where IL-1R1 ablation resulted in bacterial invasion into tumors, heightened inflammation and aggressive CRC progression. Thus, IL-1 signaling elicits cell-type-specific responses, which, in aggregate, set the inflammatory tone of the tumor microenvironment and determine the propensity for disease progression.