Search

Pollen from Salsola kali, i.e., saltwort, Russian thistle, is a major allergen source in the coastal regions of southern Europe, in Turkey, Central Asia, and Iran. S. kali-allergic patients mainly suffer from hay-fever (i.e., rhinitis and conjunctivitis), asthma, and allergic skin symptoms. The aim of this study was to investigate the importance of individual S. kali allergen molecules. Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5, and Sal k 6 were expressed in Escherichia coli as recombinant proteins containing a C-terminal hexahistidine tag and purified by nickel affinity chromatography. The purity of the recombinant allergens was analyzed by SDS-PAGE. Their molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and their fold and secondary structure were studied by circular dichroism (CD) spectroscopy. Sera from clinically well-characterized S. kali-allergic patients were used for IgE reactivity and basophil activation experiments. S. kali allergen-specific IgE levels and IgE levels specific for the highly IgE cross-reactive profilin and the calcium-binding allergen from timothy grass pollen, Phl p 12 and Phl p 7, respectively, were measured by ImmunoCAP. The allergenic activity of natural S. kali pollen allergens was studied in basophil activation experiments. Recombinant S. kali allergens were folded when studied by CD analysis. The sum of recombinant allergen-specific IgE levels and allergen-extract-specific IgE levels was highly correlated. Sal k 1 and profilin, reactive with IgE from 64% and 49% of patients, respectively, were the most important allergens, whereas the other S. kali allergens were less frequently recognized. Specific IgE levels were highest for profilin. Of note, 37% of patients who were negative for Sal k 1 showed IgE reactivity to Phl p 12, emphasizing the importance of the ubiquitous cytoskeletal actin-binding protein, profilin, for the diagnosis of IgE sensitization in S. kali-allergic patients. rPhl p 12 and rSal k 4 showed equivalent IgE reactivity, and the clinical importance of profilin was underlined by the fact that profilin-monosensitized patients suffered from symptoms of respiratory allergy to saltwort. Accordingly, profilin should be included in the panel of allergen molecules for diagnosis and in molecular allergy vaccines for the treatment and prevention of S. kali allergy.

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

The prevalence of allergic diseases has increased tremendously in recent decades, which can be attributed to growing exposure to environmental triggers, changes in dietary habits, comorbidity, and the increased use of medications. In this context, the multiplexed diagnosis of sensitization to various allergens and the monitoring of the effectiveness of treatments for allergic diseases become particularly urgent issues. The detection of allergen-specific antibodies, in particular, sIgE and sIgG, is a modern alternative to skin tests due to the safety and efficiency of this method. The use of allergen microarrays to detect tens to hundreds of allergen-specific antibodies in less than 0.1 mL of blood serum enables the transition to a deeply personalized approach in the diagnosis of these diseases while reducing the invasiveness and increasing the informativeness of analysis. This review discusses the technological approaches underlying the development of allergen microarrays and other protein microarrays, including the methods of selection of the microarray substrates and matrices for protein molecule immobilization, the obtainment of allergens, and the use of different types of optical labels for increasing the sensitivity and specificity of the detection of allergen-specific antibodies.

Microparticles are versatile carriers for controlled drug delivery in personalized, targeted therapy of various diseases, including cancer. The tumor microenvironment contains different infiltrating cells, including immune cells, which can affect the efficacy of antitumor drugs. Here, prototype microparticle-based systems for the delivery of the antitumor drug doxorubicin (DOX) were developed, and their cytotoxic effects on human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells were compared in vitro. DOX-containing calcium carbonate microparticles with or without a protective polyelectrolyte shell and polyelectrolyte microcapsules of about 2.4–2.5 μm in size were obtained through coprecipitation and spontaneous loading. All the microstructures exhibited a prolonged release of DOX. An estimation of the cytotoxicity of the DOX-containing microstructures showed that the encapsulation of DOX decreased its toxicity to macrophages and delayed the cytotoxic effect against tumor cells. The DOX-containing calcium carbonate microparticles with a protective polyelectrolyte shell were more toxic to the cancer cells than DOX-containing polyelectrolyte microcapsules, whereas, for the macrophages, the microcapsules were most toxic. It is concluded that DOX-containing core/shell microparticles with an eight-layer polyelectrolyte shell are optimal drug microcarriers due to their low toxicity to immune cells, even upon prolonged incubation, and strong delayed cytotoxicity against tumor cells.

Nano- and microparticles are increasingly widely used in biomedical research and applications, particularly as specific labels and targeted delivery vehicles. Silica has long been considered the best material for such vehicles, but it has some disadvantages limiting its potential, such as the proneness of silica-based carriers to spontaneous drug release. Calcium carbonate (CaCO3) is an emerging alternative, being an easily available, cost-effective, and biocompatible material with high porosity and surface reactivity, which makes it an attractive choice for targeted drug delivery. CaCO3 particles are used in this field in the form of either bare CaCO3 microbeads or core/shell microparticles representing polymer-coated CaCO3 cores. In addition, they serve as removable templates for obtaining hollow polymer microcapsules. Each of these types of particles has its specific advantages in terms of biomedical applications. CaCO3 microbeads are primarily used due to their capacity for carrying pharmaceutics, whereas core/shell systems ensure better protection of the drug-loaded core from the environment. Hollow polymer capsules are particularly attractive because they can encapsulate large amounts of pharmaceutical agents and can be so designed as to release their contents in the target site in response to specific stimuli. This review focuses first on the chemistry of the CaCO3 cores, core/shell microbeads, and polymer microcapsules. Then, systems using these structures for the delivery of therapeutic agents, including drugs, proteins, and DNA, are outlined. The results of the systematic analysis of available data are presented. They show that the encapsulation of various therapeutic agents in CaCO3-based microbeads or polymer microcapsules is a promising technique of drug delivery, especially in cancer therapy, enhancing drug bioavailability and specific targeting of cancer cells while reducing side effects. To date, research in CaCO3-based microparticles and polymer microcapsules assembled on CaCO3 templates has mainly dealt with their properties in vitro, whereas their in vivo behavior still remains poorly studied. However, the enormous potential of these highly biocompatible carriers for in vivo applications is undoubted. This last issue is addressed in depth in the Conclusions and Outlook sections of the review.

Albumins from animals are highly cross-reactive allergens for patients suffering from immunoglobulin E (IgE)-mediated allergy. Approximately 20-30% of cat and dog allergic patients show IgE reactivity and mount IgE-mediated allergic reactions to cat and dog albumin. It is astonishing that allergic patients can develop specific IgE responses against animal albumins because these proteins exhibit a more than 70% sequence identity to human serum albumin (HSA) which is the most abundant protein in the blood of the human body. The sequence identity of cat albumin (Fel d 2) and dog albumin (Can f 3) and HSA are 82% and 80%, respectively. Given the high degree of sequence identity between the latter two allergens and HSA one would expect that immunological tolerance would prohibit IgE sensitization to Fel d 2 and Can f 3. Here we discuss two possibilities for how IgE sensitization to Fel d 2 and Can f 3 may develop. One possibility is the failed development of immune tolerance in albumin-allergic patients whereas the other possibility is highly selective immune tolerance to HSA but not to Fel d 2 and Can f 3. If the first assumption is correct it should be possible to detect HSA-specific T cell responses and HSA-containing immune complexes in sensitized patients. In the latter scenario few differences in the sequences of Fel d 2 and Can f 3 as compared to HSA would be responsible for the development of selective T cell and B cell responses towards Fel d 2 as well as Can f 3. However, the immunological mechanisms of albumin sensitization have not yet been investigated in detail although this will be important for the development of allergen-specific prevention and allergen-specific immunotherapy (AIT) strategies for allergy to albumin.

Photonic crystals (PCs) are promising tools for label-free sensing in drug discovery screening, diagnostics, and analysis of ligand–receptor interactions. Imaging of PC surface modes has emerged as a novel approach to the detection of multiple binding events at the sensor surface. PC surface modification and decoration with recognition units yield an interface providing the highly sensitive detection of cancer biomarkers, antibodies, and oligonucleotides. The RAD51 protein plays a central role in DNA repair via the homologous recombination pathway. This recombinase is essential for the genome stability and its overexpression is often correlated with aggressive cancer. RAD51 is therefore a potential target in the therapeutic strategy for cancer. Here, we report the designing of a PC-based array sensor for real-time monitoring of oligonucleotide–RAD51 recruitment by means of surface mode imaging and validation of the concept of this approach. Our data demonstrate that the designed biosensor ensures the highly sensitive multiplexed analysis of association–dissociation events and detection of the biomarker of DNA damage using a microfluidic PC array. The obtained results highlight the potential of the developed technique for testing the functionality of candidate drugs, discovering new molecular targets and drug entities. This paves the way to further adaption and bioanalytical use of the biosensor for high-content screening to identify new DNA repair inhibitor drugs targeting the RAD51 nucleoprotein filament or to discover new molecular targets.

Molecular therapies, including anti-IgE, biologicals and small molecules are increasingly used for treatment of asthma. The effectiveness of these therapies may be increased with biomarkers. Aim of this study was to assess the value of measuring cumulative IgE levels specific for respiratory allergens to increase the efficacy of anti-IgE therapy for severe bronchial asthma. One hundred and thirty seven patients with severe asthma were recruited from 2016 to 2022. Standard empirical allergy diagnosis (i.e., anamnesis, skin testing, allergen-specific IgE measurement), blood eosinophil counting, measurement of total IgE and of cumulative IgE-specific for respiratory allergens by Phadiatop™ were performed. Thirty four patients with severe allergic asthma, for whom all three diagnostic methods were performed, were then used to analyze the efficacy of anti-IgE treatment in patients stratified in two groups according to cumulative IgE levels specific for respiratory allergens determined by Phadiatop™. Group #1 patients (n = 8) had cumulative specific IgE values ≥ 0.35 and < 1.53 PAU/l while in group #2 patients (n = 26) they were ≥ 1.53 PAU/l. Treatment with Omalizumab was performed for at least 12 months. The level of asthma control (ACT questionnaire), the number of asthma exacerbations, the quality of life (AQLQ questionnaire), the need for systemic corticosteroids, and the respiratory function (FEV1) was determined by “before-after” analysis for each group, followed by a comparison of the dynamics between groups. In group 2 patients with an initial allergen-specific IgE level ≥ 1.53 kUA/L, the efficacy of Omalizumab treatment was better regarding asthma control, number of exacerbations, and quality of life than in group 1 patients. Our study provides evidence that measuring cumulative levels of IgE specific for respiratory allergens could be a useful screening method for detecting an allergic phenotype of severe asthma and may serve as biomarker to enhance the success of IgE-targeted therapy.

Neutrophils and eosinophils are granulocytes which are characterized by the presence of granules in the cytoplasm. Granules provide a safe storage site for granule proteins that play important roles in the immune function of granulocytes. Upon granulocytes activation, diverse proteins are released from the granules into the extracellular space and contribute to the fight against infections. In this article, we describe granule proteins of both neutrophils and eosinophils able to kill pathogens and review their anticipated mechanism of antimicrobial toxicity. It should be noted that an excess of granules protein release can lead to tissue damage of the host resulting in chronic inflammation and organ dysfunction.

Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.

Autophagy is a highly conserved lysosomal degradation system that involves the creation of autophagosomes, which eventually fuse with lysosomes and breakdown misfolded proteins and damaged organelles with their enzymes. Autophagy is widely known for its function in cellular homeostasis under physiological and pathological settings. Defects in autophagy have been implicated in the pathophysiology of a variety of human diseases. The new line of evidence suggests that autophagy is inextricably linked to skin disorders. This review summarizes the principles behind autophagy and highlights current findings of autophagy’s role in skin disorders and strategies for therapeutic modulation.

Multiplexed fluorescent immunohistochemical analysis of breast cancer (BC) markers and high-resolution 3D immunofluorescence imaging of the tumor and its microenvironment not only facilitate making the disease prognosis and selecting effective anticancer therapy (including photodynamic therapy), but also provides information on signaling and metabolic mechanisms of carcinogenesis and helps in the search for new therapeutic targets and drugs. The characteristics of imaging nanoprobe efficiency, such as sensitivity, target affinity, depth of tissue penetration, and photostability, are determined by the properties of their components, fluorophores and capture molecules, and by the method of their conjugation. Regarding individual nanoprobe components, fluorescent nanocrystals (NCs) are widely used for optical imaging in vitro and in vivo, and single-domain antibodies (sdAbs) are well established as highly specific capture molecules in diagnostic and therapeutic applications. Moreover, the technologies of obtaining functionally active sdAb–NC conjugates with the highest possible avidity, with all sdAb molecules bound to the NC in a strictly oriented manner, provide 3D-imaging nanoprobes with strong comparative advantages. This review is aimed at highlighting the importance of an integrated approach to BC diagnosis, including the detection of biomarkers of the tumor and its microenvironment, as well as the need for their quantitative profiling and imaging of their mutual location, using advanced approaches to 3D detection in thick tissue sections. The existing approaches to 3D imaging of tumors and their microenvironment using fluorescent NCs are described, and the main comparative advantages and disadvantages of nontoxic fluorescent sdAb–NC conjugates as nanoprobes for multiplexed detection and 3D imaging of BC markers are discussed.

Semiconductor nanocrystals known as quantum dots (QDs) are of great interest for researchers and have potential use in various applications in biomedicine, such as in vitro diagnostics, molecular tracking, in vivo imaging, and drug delivery. Systematic analysis of potential hazardous effects of QDs is necessary to ensure their safe use. In this study, we obtained water-soluble core/shell QDs differing in size, surface charge, and chemical composition of the core. All the synthesized QDs were modified with polyethylene glycol derivatives to obtain outer organic shells protecting them from degradation. The physical and chemical parameters were fully characterized. In vitro cytotoxicity of the QDs was estimated in both normal and tumor cell lines. We demonstrated that QDs with the smallest size had the highest in vitro cytotoxicity. The most toxic QDs were characterized by a low negative surface charge, while positively charged QDs were less cytotoxic, and QDs with a greater negative charge were the least toxic. In contrast, the chemical composition of the QD core did not noticeably affect the cytotoxicity in vitro. This study provides a better understanding of the influence of the QD parameters on their cytotoxicity and can be used to improve the design of QDs.

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

Surface-enhanced Raman scattering (SERS) spectroscopy is a surface- or cavity-enhanced variant of Raman scattering spectroscopy that allows the detection of analytes with a sensitivity down to single molecules. This method involves the use of SERS-active surfaces or cavities capable of concentrating incident radiation into small mode volumes containing the analyte. Here, we have engineered an ultranarrow metal–dielectric nano-cavity out of a film of the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) glycoprotein and a silver surface, held together by interaction between reduced protein sulfhydryl groups and silver. The concentration of light in this nano-cavity allows the label-free recording of the characteristic Raman spectra of protein samples smaller than 1 pg. This is sufficient for the ultrasensitive detection of viral protein antigens at physiologically relevant levels. Moreover, the protein SERS signal can be increased by several orders of magnitude by coating the RBD film with a nanometer-thick silver shell, thereby raising the cavity Q-factor. This ensures a sub-femtogram sensitivity of the viral antigen detection. A simple theoretical model explaining the observed additional enhancement of the SERS signal from the silver-coated protein is proposed. Our study is the first to obtain the characteristic Raman and SERS spectra of the RBD of S glycoprotein, the key SARS-CoV-2 viral antigen, directly, without the use of Raman-reporter molecules. Thus, our approach allows label-free recording of the characteristic spectra of viral antigens at concentrations orders of magnitude lower than those required for detecting the whole virus in biological media. This makes it possible to develop a high-performance optical detection method and conformational analysis of the pathogen and its variants.

Background: The co-expression patterns of type 1 and 2 tumor necrosis factor (TNF)-α membrane receptors (TNFR1/TNFR2) are associated with the presence, stage, and activity of allergic diseases. The aim of this study was to assess the expression levels and dynamics of TNFRs on immune cells and to assess associations between their expression and severity of bronchial asthma (BA). Methods: Patients with severe (n = 8), moderate (n = 10), and mild (n = 4) BA were enrolled. As a comparison group, data from 46 healthy volunteers (HV) were accessed. Co-expression of TNFR1/2 was evaluated as a percentage of cells and the number of receptors of each type per cell. Multivariate logistic regression analysis was used to identify diagnostic biomarkers of BA. Results: More than 90% of the monocytes in patients with mild BA were TNFR1+TNFR2+ but had significantly lower TNFR1 expression density compared with HV (7.82- to 14.08-fold, depending on disease severity). Lower percentages of the TNFR+ B-lymphocytes were observed in combination with significantly lower receptors density in BA compared with HV (2.59- to 11.64-fold for TNFR1 and 1.72- to 3.4-fold for TNFR2, depending on disease severity). The final multivariate model for predicting the presence of BA included the percentage of double-positive CD5+ B-lymphocytes and average number of TNFR1 molecules expressed on cytotoxic naive T-lymphocytes and T-helper cells (R2 = 0.87). Conclusions: The co-expression patterns of TNFRs on immune cells in BA differed significantly compared with HV. The expression differences were associated with disease severity. TNFR1 expression changes were key parameters that discriminated patients with BA from those with HV.

Vaccines for infectious diseases have improved the life of the human species in a tremendous manner. The principle of vaccination is to establish de novo adaptive immune response consisting of antibody and T cell responses against pathogens which should defend the vaccinated person against future challenge with the culprit pathogen. The situation is completely different for immunoglobulin E (IgE)-associated allergy, an immunologically-mediated hypersensitivity which is already characterized by increased IgE antibody levels and T cell responses against per se innocuous antigens (i.e., allergens). Thus, allergic patients suffer from a deviated hyper-immunity against allergens leading to inflammation upon allergen contact. Paradoxically, vaccination with allergens, termed allergen-specific immunotherapy (AIT), induces a counter immune response based on the production of high levels of allergen-specific IgG antibodies and alterations of the adaptive cellular response, which reduce allergen-induced symptoms of allergic inflammation. AIT was even shown to prevent the progression of mild to severe forms of allergy. Consequently, AIT can be considered as a form of therapeutic vaccination. In this article we describe a strategy and possible road map for the use of an AIT approach for prophylactic vaccination against allergy which is based on new molecular allergy vaccines. This road map includes the use of AIT for secondary preventive vaccination to stop the progression of clinically silent allergic sensitization toward symptomatic allergy and ultimately the prevention of allergic sensitization by maternal vaccination and/or early primary preventive vaccination of children. Prophylactic allergy vaccination with molecular allergy vaccines may allow halting the allergy epidemics affecting almost 30% of the population as it has been achieved for vaccination against infectious diseases.

A large number of allergens have been discovered but we know little about their potential to induce inflammation (allergenic activity) and symptoms. Nowadays, the clinical importance of allergens is determined by the frequency and intensity of their IgE antibody binding (allergenicity). This is a rather limited parameter considering the development of experimental allergology in the last 20 years and the criteria that support personalized medicine. Now it is known that some allergens, in addition to their IgE antibody binding properties, can induce inflammation through non IgE mediated pathways, which can increase their allergenic activity. There are several ways to evaluate the allergenic activity, among them the provocation tests, the demonstration of non-IgE mediated pathways of inflammation, case control studies of IgE-binding frequencies, and animal models of respiratory allergy. In this review we have explored the current status of basic and clinical research on allergenic activity of indoor allergens and confirm that, for most of them, this important property has not been investigated. However, during recent years important advances have been made in the field, and we conclude that for at least the following, allergenic activity has been demonstrated: Der p 1, Der p 2, Der p 5 and Blo t 5 from HDMs; Per a 10 from P. americana; Asp f 1, Asp f 2, Asp f 3, Asp f 4 and Asp f 6 from A. fumigatus; Mala s 8 and Mala s 13 from M. sympodialis; Alt a 1 from A. alternata; Pen c 13 from P. chrysogenum; Fel d 1 from cats; Can f 1, Can f 2, Can f 3, Can f 4 and Can f 5 from dogs; Mus m 1 from mice and Bos d 2 from cows. Defining the allergenic activity of other indoor IgE antibody binding molecules is necessary for a precision-medicine-oriented management of allergic diseases.

Abstract Studies on the methods of nanoparticle (NP) synthesis, analysis of their characteristics, and exploration of new fields of their applications are at the forefront of modern nanotechnology. The possibility of engineering water-soluble NPs has paved the way to their use in various basic and applied biomedical researches. At present, NPs are used in diagnosis for imaging of numerous molecular markers of genetic and autoimmune diseases, malignant tumors, and many other disorders. NPs are also used for targeted delivery of drugs to tissues and organs, with controllable parameters of drug release and accumulation. In addition, there are examples of the use of NPs as active components, e.g., photosensitizers in photodynamic therapy and in hyperthermic tumor destruction through NP incorporation and heating. However, a high toxicity of NPs for living organisms is a strong limiting factor that hinders their use in vivo. Current studies on toxic effects of NPs aimed at identifying the targets and mechanisms of their harmful effects are carried out in cell culture models; studies on the patterns of NP transport, accumulation, degradation, and elimination, in animal models. This review systematizes and summarizes available data on how the mechanisms of NP toxicity for living systems are related to their physical and chemical properties.

The engineering of delivery systems for drugs and contrasting labels ensuring the simultaneous imaging and treatment of malignant tumors is an important hurdle in developing new tools for cancer therapy and diagnosis. Polyelectrolyte microcapsules (MCs), formed by nanosized interpolymer complexes, represent a promising platform for the designing of multipurpose agents, functionalized with various components, including high- and low-molecular-weight substances, metal nanoparticles, and organic fluorescent dyes. Here, we have developed size-homogenous MCs with different structures (core/shell and shell types) and microbeads containing doxorubicin (DOX) as a model anticancer drug, and fluorescent semiconductor nanocrystals (quantum dots, QDs) as fluorescent nanolabels. In this study, we suggest approaches to the encapsulation of DOX at different stages of the MC synthesis and describe the optimal conditions for the optical encoding of MCs with water-soluble QDs. The results of primary characterization of the designed microcarriers, including particle analysis, the efficacy of DOX and QDs encapsulation, and the drug release kinetics are reported. The polyelectrolyte MCs developed here ensure a modified (prolonged) release of DOX, under conditions close to normal and tumor tissues; they possess a bright fluorescence that paves the way to their exploitation for the delivery of antitumor drugs and fluorescence imaging.

Designing nanoprobes in which quantum dots (QDs) are used as photoluminescent labels is an especially promising line of research due to their possible medical applications ranging from disease diagnosis to drug delivery. In spite of the significant progress made in designing such nanoprobes, the properties of their individual components, i.e., photoluminescent QDs, vectorization moieties, and pharmacological agents, still require further optimization to enhance the efficiency of diagnostic or therapeutic procedures. Here, we have developed a method of engineering compact multifunctional nanoprobes based on functional components with optimized properties: bright photoluminescence of CdSe/ZnS (core/shell) QDs, a compact and effective antitumor agent (an acridine derivative), and direct conjugation of the components via electrostatic interaction, which provides a final hydrodynamic diameter of nanoprobes smaller than 15 nm. Due to the possibility of conjugating various biomolecules with hydroxyl and carboxyl moieties to QDs, the method represents a versatile approach to the biomarker-recognizing molecule imaging of the delivery of the active substance as part of compact nanoprobes.

Nanoparticles attract much interest as fluorescent labels for diagnostic and therapeutic tools, although their applications are often hindered by size- and shape-dependent cytotoxicity. This cytotoxicity is related not only to the leak of toxic metals from nanoparticles into a biological solution, but also to molecular cytotoxicity effects determined by the formation of a protein corona, appearance of an altered protein conformation leading to exposure of cryptic epitopes and cooperative effects involved in the interaction of proteins and peptides with nanoparticles. In the last case, nanoparticles may serve, depending on their nature, as centers of self-association or fibrillation of proteins and peptides, provoking amyloid-like proteinopathies, or as inhibitors of self-association of proteins, or they can self-assemble on biopolymers as on templates. In this study, human insulin protein was used to analyze nanoparticle-induced proteinopathy in physiological conditions. It is known that human insulin may form amyloid fibers, but only under extreme experimental conditions (very low pH and high temperatures). Here, we have shown that the quantum dots (QDs) may induce amyloid-like fibrillation of human insulin under physiological conditions through a complex process strongly dependent on the size and surface charge of QDs. The insulin molecular structure and fibril morphology have been shown to be modified at different stages of its fibrillation, which has been proved by comparative analysis of the data obtained using circular dichroism, dynamic light scattering, amyloid-specific thioflavin T (ThT) assay, transmission electron microscopy, and high-speed atomic force microscopy. We have found important roles of the QD size and surface charge in the destabilization of the insulin structure and the subsequent fibrillation. Remodeling of the insulin secondary structure accompanied by remarkable increase in the rate of formation of amyloid-like fibrils under physiologically normal conditions was observed when the protein was incubated with QDs of exact specific diameter coated with slightly negative specific polyethylene glycol (PEG) derivatives. Strongly negatively or slightly positively charged PEG-modified QDs of the same specific diameter or QDs of bigger or smaller diameters had no effect on insulin fibrillation. The observed effects pave the way to the control of amyloidosis proteinopathy by varying the nanoparticle size and surface charge.

Eosinophils are a subset of granulocytes characterized by a high abundance of specific granules in their cytoplasm. To act as effector cells, eosinophils degranulate and form eosinophil extracellular traps (EETs), which contain double-stranded DNA (dsDNA) co-localized with granule proteins. The exact molecular mechanism of EET formation remains unknown. Although the term “EET release” has been used in scientific reports, it is unclear whether EETs are pre-formed in eosinophils and subsequently released. Moreover, although eosinophil degranulation has been extensively studied, a precise time-course of granule protein release has not been reported until now. In this study, we investigated the time-dependent release of eosinophil peroxidase (EPX) and mitochondrial DNA (mtDNA) following activation of both human and mouse eosinophils. Unexpectedly, maximal degranulation was already observed within 1 min with no further change upon complement factor 5 (C5a) stimulation of interleukin-5 (IL-5) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-primed eosinophils. In contrast, bulk mtDNA release in the same eosinophil populations occurred much slower and reached maximal levels between 30 and 60 min. Although no single-cell analyses have been performed, these data suggest that the molecular pathways leading to degranulation and mtDNA release are at least partially different. Moreover, based on these data, it is likely that the association between the mtDNA scaffold and granule proteins in the process of EET formation occurs in the extracellular space.

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure (“inner body”), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations.

Fluorescent semiconductor nanocrystals or quantum dots (QDs) are characterized by unique optical properties, including a high photostability, wide absorption spectrum, and narrow, symmetric fluorescence spectrum. This makes them attractive fluorescent nanolabels for the optical encoding of microcarriers intended for targeted drug delivery, diagnosis, and imaging of transport processes on the body, cellular, and subcellular levels. Incorporation of QDs into carriers in the form of polyelectrolyte microcapsules through layer-by-layer adsorption of oppositely charged polyelectrolyte polymers yields microcapsules with a bright fluorescence signal and adaptable size, structure, and surface characteristics without using organic solvents. The easily modifiable surface of the microcapsules allows for its subsequent functionalization with capture molecules, such as antibodies, which ensures specific and selective interaction with cells, including tumor cells, with the use of the bioconjugation technique developed here. We obtained stable water-soluble nanolabels based on QDs whose surface was modified with polyethylene glycol (PEG) derivatives and determined their colloidal and optical characteristics. The obtained nanocrystals were used to encode polyelectrolyte microcapsules optically. The microcapsule surface was modified with humanized monoclonal antibodies (Abs) recognizing a cancer marker, epidermal growth factor receptor (EGFR). The possibility of effective, specific, and selective delivery of the microcapsules to tumor cells expressing EGFR has been demonstrated. The results show that the QD-encoded polyelectrolyte microcapsules functionalized with monoclonal Abs against EGFR can be used for targeted imaging and diagnosis.

Immunoglobulin E (IgE) is the key immunoglobulin in the pathogenesis of IgE associated allergic diseases affecting 30% of the world population. Recent data suggest that allergen-specific IgE levels in serum of allergic patients are sustained by two different mechanisms: inducible IgE production through allergen exposure, and continuous IgE production occurring even in the absence of allergen stimulus that maintains IgE levels. This assumption is supported by two observations. First, allergen exposure induces transient increases of systemic IgE production. Second, reduction in IgE levels upon depletion of IgE from the blood of allergic patients using immunoapheresis is only temporary and IgE levels quickly return to pre-treatment levels even in the absence of allergen exposure. Though IgE production has been observed in the peripheral blood and locally in various human tissues (e.g., nose, lung, spleen, bone marrow), the origin and main sites of IgE production in humans remain unknown. Furthermore, IgE-producing cells in humans have yet to be fully characterized. Capturing IgE-producing cells is challenging not only because current staining technologies are inadequate, but also because the cells are rare, they are difficult to discriminate from cells bearing IgE bound to IgE-receptors, and plasma cells express little IgE on their surface. However, due to the central role in mediating both the early and late phases of allergy, free IgE, IgE-bearing effector cells and IgE-producing cells are important therapeutic targets. Here, we discuss current knowledge and unanswered questions regarding IgE production in allergic patients as well as possible therapeutic approaches targeting IgE.

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S–23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the Gen-Bank electronic database has been demonstrated. Differences in the 16S–23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and nonplasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B-B, Ba, D, Da, E, L1, L2, L2a; intermediate group-F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life development cycle disorder, and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration