The cytoskeletal protein profilin is an important allergen in saltwort (Salsola kali).

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.

Cytotoxic Effects of Doxorubicin on Cancer Cells and Macrophages Depend Differently on the Microcarrier Structure.

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.

Treatment of atopic dermatitis with upadacitinib: adcare single center experience.

Introduction: The role of upadacitinib in the management of moderate to severe atopic dermatitis seems promising, but more data on its efficacy and safety are needed. This study endeavors to assess the practical impact and safety of upadacitinib in patients with moderate to severe atopic dermatitis. The study aims to evaluate the efficacy and safety of upadacitinib in the treatment of moderate to severe atopic dermatitis, focusing on analyzing patient responses to the treatment. Methods: In this study, adult patients diagnosed with moderate to severe atopic dermatitis received upadacitinib at daily doses of 15 mg or 30 mg, as prescribed by their attending physicians. The therapeutic efficacy of upadacitinib was meticulously assessed using established clinical metrics. Simultaneously, a comprehensive safety assessment was conducted through monthly monitoring, including the evaluation of potential effects of upadacitinib intake on hepatic function, lipid profile, and hematopoiesis using the pertinent laboratory tests. Results: Sixteen participants were enrolled in the study. At 1month follow-up, there was a significant reduction in the mean Eczema Area and Severity Index (EASI) score to 18.8 points, which further increased to 24 points at the 4-month mark. Additionally, 9 participants (56%) demonstrated an EASI-50 response after 1 month of treatment, with this response increasing to 9 participants (90%) after 4 months. Furthermore, enhanced therapeutic responses were observed at 4 months, with 6 patients (38%) achieving an EASI-75 response at 1month and 8 patients (80%) achieving this milestone at the 4-month follow-up. This study highlights the potential of upadacitinib as an effective treatment option for moderate to severe atopic dermatitis. While it demonstrates improved symptom management, close monitoring for potential adverse events, particularly infections and the known risks of Janus kinase inhibitors, is essential. Further research is essential to determine the long-term safety and efficacy of upadacitinib.

Functionalized Calcium Carbonate-Based Microparticles as a Versatile Tool for Targeted Drug Delivery and Cancer Treatment.

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.

Photonic Crystal Surface Mode Real-Time Imaging of RAD51 DNA Repair Protein Interaction with the ssDNA Substrate.

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.

Allergenic Activity of Individual Cat Allergen Molecules.

More than 10% of the world's population suffers from an immunoglobulin E (IgE)-mediated allergy to cats which is accompanied mainly by respiratory symptoms such as rhinitis and asthma. Several cat allergen molecules have been identified, but their allergenic activity has not been investigated in depth. Purified cat allergen molecules (Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 6, Fel d 7 and Fel d 8) were characterized via mass spectrometry and circular dichroism spectroscopy regarding their molecular mass and fold, respectively. Cat-allergen-specific IgE levels were quantified via ImmunoCAP measurements in IgE-sensitized subjects with (n = 37) and without (n = 20) respiratory symptoms related to cat exposure. The allergenic activity of the cat allergens was investigated by loading patients' IgE onto rat basophils expressing the human FcεRI receptor and studying the ability of different allergen concentrations to induce ß-hexosaminidase release. Purified and folded cat allergens with correct masses were obtained. Cat-allergen-specific IgE levels were much higher in patients with a respiratory allergy than in patients without a respiratory allergy. Fel d 1, Fel d 2, Fel d 4 and Fel d 7 bound the highest levels of specific IgE and already-induced basophil degranulation at hundred-fold-lower concentrations than the other allergens. Fel d 1, Fel d 4 and Fel d 7 were recognized by more than 65% of patients with a respiratory allergy, whereas Fel d 2 was recognized by only 30%. Therefore, in addition to the major cat allergen Fel d 1, Fel d 4 and Fel d 7 should also be considered to be important allergens for the diagnosis and specific immunotherapy of cat allergy.

Albumins represent highly cross-reactive animal allergens.

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.

Real-world clinical effectiveness of Tixagevimab/Cilgavimab and Regdanvimab monoclonal antibodies for COVID-19 treatment in Omicron variant-dominant period.

Several virus-neutralizing monoclonal antibodies (mAbs) have become new tools in the treatment of the coronavirus disease (COVID-19), but their effectiveness against the rapidly mutating virus is questionable. The present study investigated the effectiveness of Tixagevimab/Cilgavimab and Regdanvimab for mild and moderate COVID-19 treatment in real-world clinical practice during the Omicron variant-dominant period. Patients with known risk factors for disease progression and increasing disease severity were enrolled in the study within the first 7 days of symptom onset. Seventy-seven patients were divided into four groups: first 15 patients received 300 mg Tixagevimab/Cilgavimab intravenously (IV) and 23 patients got the same drug 300 mg intramuscularly (IM), the next 15 patients was on the same combination in dose of 600 mg IV, and 24 patients were on Regdanvimab at a dose of 40 mg/kg IV. By Day 4, 100% of Tixagevimab/Cilgavimab IV patients showed negative polymerase chain reaction results for SARS-CoV-2 Ribonucleic acid (RNA) regardless of the mAbs dose while in the Regdanvimab group 29% of the patients were positive for SARS-CoV-2 virus RNA. The testing for virus neutralizing antibodies (nAbs) to various Omicron sublineages (BA.1, BA.2, and BA.5) showed that an increase in nAb levels was detected in blood serum immediately after the drug administration only in Tixagevimab/Cilgavimab 300 mg and 600 mg IV groups. In the group of intravenous Regdanvimab, a significant increase in the level of nAbs to the Wuhan variant was detected immediately after the drug administration, while no increase in nAbs to different Omicron sublineages was observed. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05982704.

Impact of Macrophages on the Interaction of Cetuximab-Functionalized Polyelectrolyte Capsules with EGFR-Expressing Cancer Cells.

Polyelectrolyte capsules (PCs) are a promising tool for anticancer drug delivery and tumor targeting. Surface functionalization of PCs with antibodies is widely used for providing their specific interactions with cancer cells. The efficiency of PC-based targeted delivery systems can be affected by the cellular heterogeneity of the tumor, particularly by the presence of tumor-associated macrophages. We used human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells in either monoculture or coculture to analyze the targeting capacity and internalization efficiency of PCs with a mean size of 1.03 ± 0.11 µm. The PCs were functionalized with the monoclonal antibody cetuximab targeting the human epidermal growth factor receptor (EGFR). We have shown that surface functionalization of the PCs with cetuximab ensures a specific interaction with EGFR-expressing cancer cells and promotes capsule internalization. In monoculture, the macrophages derived from human leukemia monocytic cells have been found to internalize both nonfunctionalized PCs and cetuximab-functionalized PCs (Cet-PCs) more intensely compared to epidermoid carcinoma cells. The internalization of Cet-PCs by cancer cells is mediated by lipid rafts of the cell membrane, whereas the PC internalization by macrophages is only slightly influenced by lipid rafts. Experiments with a coculture of human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells have shown that Cet-PCs preferentially interact with cancer cells, which are subsequently attacked by macrophages. These data can be used to further improve the strategy of PC functionalization for targeted delivery, with the cellular heterogeneity of the tumor microenvironment taken into consideration.

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.

Exploring the potential of photodynamic therapy in the treatment of non-melanoma skin cancer: Analysis of two clinical cases.

This article presents two cases of non-melanoma skin cancer treated with photodynamic therapy (PDT). The first case involved a 74-year-old woman with a 4 cm basal cell carcinoma on her right zygomatic region, while the second case involved a 83-year-old woman with a 6 cm squamous cell carcinoma on her left peri­auricular area. Both patients underwent two sessions of systemic PDT, with chlorin E6 (Photoran) as the photosensitizer, followed by red light illumination. The treatment was well-tolerated with no significant adverse effects. Each three months, the patients showed clinical improvement with partial to complete regression of the tumors. Fluorescence diagnostics and photobleaching control were performed during the PDT sessions to monitor the treatment response. Regular follow-up examinations were conducted, including visual inspections, CT scans, and cytology investigations, which revealed no evidence of any neoplastic processes. These two cases demonstrate that PDT can be a safe and effective treatment option for non-melanoma skin cancer, with good cosmetic outcomes and minimal scarring.

Positive experience with TNF-α inhibitor in toxic epidermal necrolysis resistant to high-dose systemic corticosteroids.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare, potentially life-threatening syndromes characterized by the development of necrotic epidermal and mucosal lesions. The most common etiologic cause of SJS/TEN is drug-induced mechanisms. The group of drugs with high potential risk includes sulfonamides, anticonvulsants, non-steroidal anti-inflammatory drugs (NSAIDs), allopurinol, phenobarbital, etc. There is no gold standard treatment algorithm for SJS/TEN. In medical practice, systemic glucocorticosteroids (sGCS), intravenous immunoglobulin (IVIG), plasmapheresis, and cyclosporine are used empirically and in various combinations. Recently published studies have demonstrated the efficacy of TNF-α inhibitors as a promising approach in SJS/TEN, including cases resistant to high-dose sGCS, with etanercept and infliximab being the most commonly used drugs. In a large multicenter study by Zhang J et al. (XXXX), 242 patients treated with etanercept, sGCS, or a combination of both had lower mortality compared to the control group. A shorter skin healing time was documented compared to sGCS monotherapy, thus reducing the risk of secondary infections. The published data show a high efficacy with THF-α inhibitor blockade, but the safety of TNF-α inhibitors in patients with SJS/TEN is still questionable due to the paucity of available information. As all clinical research data should be accumulated to provide reliable evidence that the use of TNF-α inhibitors may be beneficial in SJS/TEN, we report a case of etoricoxib-associated SJS with progression to TEN in a 50-year-old woman who was refractory to high-dose sGCS therapy.

Impact of local human microbiota on the allergic diseases: Organ-organ interaction.

The homogeneous impact of local dysbiosis on the development of allergic diseases in the same organ has been thoroughly studied. However, much less is known about the heterogeneous influence of dysbiosis within one organ on allergic diseases in other organs. A comprehensive analysis of the current scientific literature revealed that most of the relevant publications focus on only three organs: gut, airways, and skin. Moreover, the interactions appear to be mainly unidirectional, that is, dysbiotic conditions of the gut being associated with allergic diseases of the airways and the skin. Similar to homogeneous interactions, early life appears to be not only a crucial period for the formation of the microbiota in one organ but also for the later development of allergic diseases in other organs. In particular, we were able to identify a number of specific bacterial and fungal species/genera in the intestine that were repeatedly associated in the literature with either increased or decreased allergic diseases of the skin, like atopic dermatitis, or the airways, like allergic rhinitis and asthma. The reported studies indicate that in addition to the composition of the microbiome, also the relative abundance of certain microbial species and the overall diversity are associated with allergic diseases of the corresponding organs. As anticipated for human association studies, the underlying mechanisms of the organ-organ crosstalk could not be clearly resolved yet. Thus, further work, in particular experimental animal studies are required to elucidate the mechanisms linking dysbiotic conditions of one organ to allergic diseases in other organs.

Milk Allergen Micro-Array (MAMA) for Refined Detection of Cow's-Milk-Specific IgE Sensitization.

BACKGROUND: Immunoglobulin-E(IgE)-mediated hypersensitivity to cow's milk allergens is a frequent cause of severe and life-threatening anaphylactic reactions. Besides case histories and controlled food challenges, the detection of the IgE antibodies specific to cow's milk allergens is important for the diagnosis of cow-milk-specific IgE sensitization. Cow´s milk allergen molecules provide useful information for the refined detection of cow-milk-specific IgE sensitization. METHODS: A micro-array based on ImmunoCAP ISAC technology was developed and designated milk allergen micro-array (MAMA), containing a complete panel of purified natural and recombinant cow's milk allergens (caseins, α-lactalbumin, ß-lactoglobulin, bovine serum albumin-BSA and lactoferrin), recombinant BSA fragments, and α-casein-, α-lactalbumin- and ß-lactoglobulin-derived synthetic peptides. Sera from 80 children with confirmed symptoms related to cow's milk intake (without anaphylaxis: n = 39; anaphylaxis with a Sampson grade of 1-3: n = 21; and anaphylaxis with a Sampson grade of 4-5: n = 20) were studied. The alterations in the specific IgE levels were analyzed in a subgroup of eleven patients, i.e., five who did not and six who did acquire natural tolerance. RESULTS: The use of MAMA allowed a component-resolved diagnosis of IgE sensitization in each of the children suffering from cow's-milk-related anaphylaxis according to Sampson grades 1-5 requiring only 20-30 microliters of serum. IgE sensitization to caseins and casein-derived peptides was found in each of the children with Sampson grades of 4-5. Among the grade 1-3 patients, nine patients showed negative reactivity to caseins but showed IgE reactivity to alpha-lactalbumin (n = 7) or beta-lactoglobulin (n = 2). For certain children, an IgE sensitization to cryptic peptide epitopes without detectable allergen-specific IgE was found. Twenty-four children with cow-milk-specific anaphylaxis showed additional IgE sensitizations to BSA, but they were all sensitized to either caseins, alpha-lactalbumin, or beta-lactoglobulin. A total of 17 of the 39 children without anaphylaxis lacked specific IgE reactivity to any of the tested components. The children developing tolerance showed a reduction in allergen and/or peptide-specific IgE levels, whereas those remaining sensitive did not. CONCLUSIONS: The use of MAMA allows for the detection, using only a few microliters of serum, of IgE sensitization to multiple cow's milk allergens and allergen-derived peptides in cow-milk-allergic children with cow-milk-related anaphylaxis.

Nontoxic Fluorescent Nanoprobes for Multiplexed Detection and 3D Imaging of Tumor Markers in Breast Cancer.

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.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Comorbidity defines asthmatic patients' risk of COVID-19 hospitalization: A global perspective.

BACKGROUND: The global epidemiology of asthma among patients with coronavirus disease 2019 (COVID-19) presents striking geographic differences, defining prevalence zones of high and low co-occurrence of asthma and COVID-19. OBJECTIVE: We aimed to compare asthma prevalence among hospitalized patients with COVID-19 in major global hubs across the world by applying common inclusion criteria and definitions. METHODS: We built a network of 6 academic hospitals in Stanford (Stanford University)/the United States; Frankfurt (Goethe University), Giessen (Justus Liebig University), and Marburg (Philipps University)/Germany; and Moscow (Clinical Hospital 52 in collaboration with Sechenov University)/Russia. We collected clinical and laboratory data for patients hospitalized due to COVID-19. RESULTS: Asthmatic individuals were overrepresented among hospitalized patients with COVID-19 in Stanford and underrepresented in Moscow and Germany as compared with their prevalence among adults in the local community. Asthma prevalence was similar among patients hospitalized in an intensive care unit and patients hospitalized in other than an intensive care unit, which implied that the risk for development of severe COVID-19 was not higher among asthmatic patients. The numbers of males and comorbidities were higher among patients with COVID-19 in the Stanford cohort, and the most frequent comorbidities among these patients with asthma were other chronic inflammatory airway disorders such as chronic obstructive pulmonary disease. CONCLUSION: The observed disparity in COVID-19-associated risk among asthmatic patients across countries and continents is connected to the varying prevalence of underlying comorbidities, particularly chronic obstructive pulmonary disease.

Cumulative IgE-levels specific for respiratory allergens as biomarker to predict efficacy of anti-IgE-based treatment of severe asthma.

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.

Dependence of Quantum Dot Toxicity In Vitro on Their Size, Chemical Composition, and Surface Charge.

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.

Structure-function relationships in polymeric multilayer capsules designed for cancer drug delivery.

The targeted delivery of cancer drugs to tumor-specific molecular targets represents a major challenge in modern personalized cancer medicine. Engineering of micron and submicron polymeric multilayer capsules allows the obtaining of multifunctional theranostic systems serving as controllable stimulus-responsive tools with a high clinical potential to be used in cancer therapy and detection. The functionalities of such theranostic systems are determined by the design and structural properties of the capsules. This review (1) describes the current issues in designing cancer cell-targeting polymeric multilayer capsules, (2) analyzes the effects of the interactions of the capsules with the cellular and molecular constituents of biological fluids, and (3) presents the key structural parameters determining the effectiveness of capsule targeting. The influence of the morphological and physicochemical parameters and the origin of the structural components and surface ligands on the functional activity of polymeric multilayer capsules at the molecular, cellular, and whole-body levels are summarized. The basic structural and functional principles determining the future trends of theranostic capsule development are established and discussed.