mRNA vaccines: The future of prevention of viral infections?

Messenger RNA (mRNA) vaccines against COVID-19 are the first authorized biological preparations developed using this platform. During the pandemic, their administration has been proven to be a life-saving intervention. Here, we review the main advantages of using mRNA vaccines, identify further technological challenges to be met during the development of the mRNA platform, and provide an update on the clinical progress on leading mRNA vaccine candidates against different viruses that include influenza viruses, human immunodeficiency virus 1, respiratory syncytial virus, Nipah virus, Zika virus, human cytomegalovirus, and Epstein-Barr virus. The prospects and challenges of manufacturing mRNA vaccines in low-income countries are also discussed. The ongoing interest and research in mRNA technology are likely to overcome some existing challenges for this technology (e.g., related to storage conditions and immunogenicity of some components of lipid nanoparticles) and enhance the portfolio of vaccines against diseases for which classical formulations are already authorized. It may also open novel pathways of protection against infections and their consequences for which no safe and efficient immunization methods are currently available.

Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

Evaluation of the ability of colistin, amoxicillin (components of Potencil® ), and fluoroquinolones to attenuate bacterial endotoxin- and Shiga exotoxin-mediated cytotoxicity-In vitro studies.

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS- and Stx-mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS-mediated cytotoxicity in an experiment mimicking "natural infection," (c) the effect of antibiotics to decrease Stx2e-mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS- and Stx2e-induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS- and Stx2e-mediated cytotoxicity.

Chemerin, retinol binding protein-4, cytokeratin-18 and transgelin-2 presence in sera of patients with non-alcoholic liver fatty disease.

 Background. Chemerin and retinol binding protein-4 (RBP-4) are adipokines which may play a role in the progression of NAFLD. It has been also suggested that cytokeratin-18 (CK-18) could be a marker of hepatocyte caspase-directed death while transgelin-2 production could reflect stage of liver fibrosis. The aim of this study was to evaluate the level of the above adipokines in sera of patients with NAFLD and determine the relation between the level of transgelin-2 and fibrosis-4 index (FIB-4). MATERIAL AND METHODS: Ninety-five subjects included initially to the study were divided into four groups: (I) prediabetics, obese with NAFLD and metabolic syndrome (MS), (II) lean with NAFLD and without MS, (III) obese without NAFLD and MS, and (IV) healthy individuals. We determined the levels of chemerin, RBP-4, transgelin-2 and CK-18 fragments in sera of patients with NAFLD. Moreover, we examined if the levels of CK-18 fragments and transgelin-2 correlates with FIB4 value. RESULTS: Chemerin and RBP-4 were highly expressed in sera of all NAFLD, especially in obese individuals. Chemerin level was also linked to MS. High level of serum CK-18 fragments and transgelin-2 did not correlate with obesity and MS, but seemed to correlate with progression of NAFLD to liver fibrosis. CONCLUSIONS: In conclusion, the production of the two adipokines, chemerin and RBP-4, is strongly associated with obesity in patients with NAFLD. Serum concentrations of CK-18 fragments and transgelin-2 correlate with the severity of NAFLD, but no with obesity.

Antidepressant-like activity of sildenafil following acute and subchronic treatment in the forced swim test in mice: effects of restraint stress and monoamine depletion.

Sildenafil is a highly effective oral agent for the treatment of erectile dysfunction of multiple etiologies. Although in clinical practice sildenafil is often used in depressed patients, its influence on the pathophysiology of depression remains unclear. The aim of the present study was to evaluate the antidepressant-like activity following acute and subchronic treatment with sildenafil in naïve mice as well as in mice with reserpine- and restraint stress-induced depressive-like behavior. Since corticosterone is released in response to acute stress, we also aimed to assess the influence of sildenafil on serum corticosterone level in non-stressed and stressed animals. The antidepressant activity of sildenafil was assessed in the forced swim test. Corticosterone serum level was determined by using ELISA method, while brain and serum sildenafil level via HPLC method. Sildenafil administered acutely exerted an antidepressant-like effect. Subchronic (14 days) administration of sildenafil resulted only in a weak antidepressant-like effect when evaluated 24 h after the last dose. Acute but not subchronic sildenafil administration reversed the reserpine- and stress-induced immobility in the forced swim test. The lack of effects of sildenafil after subchronic treatment could have been related to its complete elimination from the brain within 24 h from the last injection. Interestingly, acute administration of sildenafil produced a marked increase in serum corticosterone level in both non-stressed and stressed animals. Sildenafil exerts differential effects in the forced swim test after acute and subchronic administration. Further studies on the antidepressant activity of sildenafil are required.

Amphotericin B-silver hybrid nanoparticles: synthesis, properties and antifungal activity.

High antifungal activity is reported, in comparison with commercially available products, of a novel hybrid system based on silver nanoparticles synthesized using a popular antifungal macrocyclic polyene amphotericin B (AmB) acting both as a reducing and stabilizing/capping agent. The synthesis reaction proceeds in an alkaline environment which prevents aggregation of AmB itself and promotes nanoparticle formation. The innovative approach produces monodisperse (PDI=0.05), AmB-coated silver nanoparticles (AmB-AgNPs) with the diameter ~7nm. The products were characterized using imaging (electron microscopy) and spectroscopic (UV-vis and infrared absorption, dynamic light scattering and Raman scattering) methods. The nanoparticles were tested against Candida albicans, Aspergillus niger and Fusarium culmorum species. For cytotoxicity studies CCD-841CoTr and THP-1 cell lines were used. Particularly high antifungal activity of AmB-AgNPs is interpreted as the result of synergy between the antifungal activity of amphotericin B and silver antimicrobial properties (Ag(+) ions release). FROM THE CLINICAL EDITOR: Amphotericin B (AmB) is a common agent used for the treatment against severe fungal infections. In this article, the authors described a new approach in using a combination of AmB and silver nanoparticles, in which the silver nanoparticles were synthesized and stabilized by AmB. Experimental data confirmed synergistic antifungal effects between amphotericin B and silver. This novel synthesis process could potentially be important in future drug development and fabrication.

New Norcantharidin Analogs: Synthesis and Anticancer Activity.

The reaction of direct condensation between S-ethyl-N-(7-oxabicyclo-[2.2.1]heptane-2,3-dicarbonyl)isothiosemicarbazide (1) and primary amines was used for synthesizing new N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)-7-oxabicyclo-[2.2.1]heptane-2-carboxylic acid (2-12) as norcantharadin analogs. Moreover, the anticancer activity of the obtained compounds was studied. Among all compounds, the N-3-methylbutyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)-7-oxabicyclo-[2.2.1]heptane-2-carboxylic acid (4) presented selective in vitro toxic and antiproliferative effects against the human hepatoma cell line Hep3B, without affecting normal human liver stellate cells (LX-2 cell line).

Legionella dumoffii utilizes exogenous choline for phosphatidylcholine synthesis.

Phosphatidycholine (PC) is the major membrane-forming phospholipid in eukaryotes but it has been found in only a limited number of prokaryotes. Bacteria synthesize PC via the phospholipid N-methylation pathway (Pmt) or via the phosphatidylcholine synthase pathway (Pcs) or both. Here, we demonstrated that Legionella dumoffii has the ability to utilize exogenous choline for phosphatidylcholine (PC) synthesis when bacteria grow in the presence of choline. The Pcs seems to be a primary pathway for synthesis of this phospholipid in L. dumoffii. Structurally different PC species were distributed in the outer and inner membranes. As shown by the LC/ESI-MS analyses, PC15:0/15:0, PC16:0/15:0, and PC17:0/17:1 were identified in the outer membrane and PC14:0/16:0, PC16:0/17:1, and PC20:0/15:0 in the inner membrane. L. dumoffii pcsA gene encoding phosphatidylcholine synthase revealed the highest sequence identity to pcsA of L. bozemanae (82%) and L. longbeachae (81%) and lower identity to pcsA of L. drancourtii (78%) and L. pneumophila (71%). The level of TNF-α in THP1-differentiated cells induced by live and temperature-killed L. dumoffii cultured on a medium supplemented with choline was assessed. Live L. dumoffii bacteria cultured on the choline-supplemented medium induced TNF-α three-fold less efficiently than cells grown on the non-supplemented medium. There is an evident effect of PC modification, which impairs the macrophage inflammatory response.

Fungal Aeroallergens-The Impact of Climate Change.

The incidence of allergic diseases worldwide is rapidly increasing, making allergies a modern pandemic. This article intends to review published reports addressing the role of fungi as causative agents in the development of various overreactivity-related diseases, mainly affecting the respiratory tract. After presenting the basic information on the mechanisms of allergic reactions, we describe the impact of fungal allergens on the development of the allergic diseases. Human activity and climate change have an impact on the spread of fungi and their plant hosts. Particular attention should be paid to microfungi, i.e., plant parasites that may be an underestimated source of new allergens.

Effectiveness of the BNT162b2 vaccine in preventing COVID-19-associated deaths in Poland.

INTRODUCTION: Development of vaccines was a turning point of the COVID­19 pandemic. In this study, we describe the course of the vaccination program in Poland and the effectiveness of the BNT162b2 vaccine. OBJECTIVES: The aim of the study was to analyze the vaccination rates and effectiveness stratified by age groups in Poland. PATIENTS AND METHODS: This is a retrospective study based on the data on the vaccination rate and survival status among Polish citizens, obtained from the registries kept by the Polish Ministry of Health, the Statistics Poland, and the European Centre for Disease Prevention and Control. The data were collected between week 53 of 2020 and week 3 of 2022. The final analysis included patients who were either not vaccinated at all or fully vaccinated with the BNT162b2 vaccine. RESULTS: The database contained records of 36 362 777 individuals, of whom 14 441 506 (39.71%) were fully vaccinated with the BNT162b2 vaccine and 14 220 548 (39.11%) were not vaccinated at all. The weekly average effectiveness of the BNT162b2 vaccine in preventing death was 92.62% and varied from 89.08% for the citizens aged 80 years and older, to 100% for individuals aged 5 to 17 years. The estimated mortality rate was significantly higher in the unvaccinated group than in the fully vaccinated group in the entire cohort (447.9 per 100 000 vs 43.76 per 100 000; P <0.001) in all age categories. CONCLUSIONS: The study results confirm high effectiveness of the BNT162b2 vaccine in preventing COVID­19 deaths in all analyzed age groups.

Potential Proallergenic Activity of Phytopathogenic Erysiphe palczewskii and Erysiphe convolvuli in in vitro Studies.

Purpose: Allergic diseases have reached epidemic proportions globally, affecting nearly 30% of the world's population. One of the most prominent sources of allergens is fungi, causing up to 6% of respiratory diseases in the general population. However, the cause of respiratory allergies is not always identifiable. Therefore, we studied the ability of two representatives of common powdery mildew (Erysiphales), Erysiphe palczewskii and Erysiphe convolvuli, to induce a proinflammatory response in in vitro models of the upper and lower respiratory tract. Materials and Methods: Two cell lines, BEAS-2B and A549, were used to mimic upper and lower respiratory epithelial cells. The toxicity of fungal extracts was assessed with MTT and flow cytometry assay. The production of reactive oxygen species in the cells was measured with flow cytometry. ELISA tests were used to determine the production of proinflammatory cytokines. The presence of the cell integrity marker was assessed with the immunofluorescence method. Results: In both cell lines, the extract of E. palczewskii and E. convolvuli microfungi induced marked production of proinflammatory IL-1ß, TNF-α, and GM-CSF cytokines involved in developing allergic reactions. The higher levels of these cytokines with higher reactive oxygen species synthesis positively correlated with the disruption of epithelial cell junctions. Conclusion: We conclude that E. palczewskii and E. convolvuli microfungi have strong proinflammatory and proallergenic potential, but this finding needs in vivo confirmation.

The COVID-19 Vaccination Still Matters: Omicron Variant Is a Final Wake-Up Call for the Rich to Help the Poor.

By June 2022, COVID-19 vaccine coverage in low-income countries remained low, while the emergence of the highly-transmissible but less clinically-severe Omicron lineage of SARS-CoV-2 has led to the assumption expressed outside the academic realm that Omicron may offer a natural solution to the pandemic. The present paper argues that this assumption is based on the false premise that this variant could be the final evolutionary step of SARS-CoV-2. There remains a risk of the emergence of novel viral subvariants and recombinants, and entirely novel lineages, the clinical consequences of which are hard to predict. This is particularly important for regions with a high share of immunocompromised individuals, such as those living with HIV/AIDS, in whom SARS-CoV-2 can persist for months and undergo selection pressure. The vaccination of the least-vaccinated regions should remain the integral strategy to control viral evolution and its potential global consequences in developed countries, some of which have decided to ease sanitary and testing measures in response to the rise and dominance of the Omicron variant. We argue that low-income countries require help in improving COVID-19 vaccine availability, decreasing vaccine hesitancy, and increasing the understanding of long-term vaccination goals during the circulation of a viral variant that causes milder disease.

Prognostic Value of Tie2-Expressing Monocytes in Chronic Lymphocytic Leukemia Patients.

Tie2-expressing monocytes (TEMs) are associated with tumor progression and metastasis. This unique subset of monocytes has been identified as a potential prognostic marker in several solid tumors. However, TEMs remain poorly characterized in hematological cancers, including chronic lymphocytic leukemia (CLL). This study analyzed, for the first time, the clinical significance of TEM population in CLL patients. Flow cytometry analysis of TEMs (defined as CD14+CD16+Tie2+ cells) was performed at the time of diagnosis on peripheral blood mononuclear cells from 104 untreated CLL patients. Our results revealed an expansion of circulating TEM in CLL patients. These monocytes express high levels of VEGF and suppressive IL-10. A high percentage of TEM was associated closely with unfavorable prognostic markers (ZAP-70, CD38, 17p and 11q deletion, and IGHV mutational status). Moreover, increased percentages of circulating TEMs were significantly higher in patients not responding to the first-line therapy as compared to responding patients, suggesting its potential predictive value. High TEM percentage was also correlated with shorter overall survival (OS) and shorter time to treatment (TTT). Importantly, based on multivariate Cox regression analysis, TEM percentage was an independent predictor for TTT. Thus, we can suggest the adverse role of TEMs in CLL.

Isothiocyanate l-argininato copper(II) complexes - Solution structure, DNA interaction, anticancer and antimicrobial activity.

l-argininato copper(II) complexes have been intensively investigated in a variety of diseases due to their therapeutic potential. Here we report the results of comprehensive structural studies (ESI-MS, NIR-VIS-UV, EPR) on the complexes arising in aqueous solutions of two ternary copper(II) complexes with molecular formulas from crystal structures, [Cu(l-Arg)2(NCS)](NCS)·H2O (1) and [Cu(l-Arg)(NCS)2] (2) (l-Arg = l-arginine). Reference systems, the ternary Cu(II)/l-Arg/NCS- as well as binary Cu(II)/NCS- and Cu(II)/l-Arg, were studied in parallel in aqueous solutions by pH-potentiometric titration, EPR and VIS spectroscopy to characterize stability, structures and speciation of the formed species over the broad pH range. Comparative analysis of the obtained results showed that at a pH close to 7.0 mononuclear [Cu(l-Arg)2(NCS)]+ is the only species in water solution of 1, while equilibrium between [Cu(l-Arg)(SCN)]+ and binary [Cu(l-Arg)2]2+ was detected in water solution of 2. According to DNA binding studies, the [Cu(l-Arg)2(NCS)]+, [Cu(l-Arg)(SCN)]+ and [Cu(l-Arg)2]2+ species could be considered as strong minor groove binding agents causing, in the presence of H2O2, the involvement of ROS in plasmid damage. The human carcinoma cells (A549 cell line) were generally significantly more sensitive to cytotoxic and antiproliferative effect of compounds 1 and 2 than human normal cells. The studied compounds shown antimicrobial activity against bacteria belonging to Enterobacteriaceae family.

Transforming growth factor-beta1 modulates metalloproteinase-2 and -9, nitric oxide, RhoA and alpha-smooth muscle actin expression in colon adenocarcinoma cells.

Colon carcinoma invasiveness is a process involving cell-cell and cell-matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF-beta1 (transforming growth factor-beta1) and small G protein RhoA in tumour progression, the influence of TGF-beta1 treatment or RhoA-associated kinase inhibitor on the production of NO (nitric oxide) and MMP-2 and MMP-9 (metalloproteinases-2 and -9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP-2 and MMP-9. rhTGF-beta1 and the synthetic Rho kinase inhibitor (Y-27632) decreased MMP-2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF-beta1 decreased NO secretion by cells, while Y-27632 had no effect on it. Immunoblotting with anti-RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF-beta1. rhTGF-beta1 induced alpha-smooth muscle actin (alpha-SMA) expression, especially in high Duke's grade of colon cancer, while Y-27632 blocked it. Summing up, in colon carcinoma cells, TGF-beta1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and alpha-SMA expression or assayed using motility data and may be a good target for cancer therapy.

[Role of stellate cells in alcoholic liver fibrosis]. / Rola komórek gwiazdzistych w procesie alkoholowego wlóknienia watroby.

Many different diseases and toxins can cause liver damage, which is difficult to treat and often leads to the development of liver fibrosis or even cirrhosis. The key event in this process is the activation of hepatic stellate cells (HSCs). During such activation, HSCs undergo a dramatic transformation in morphology and behavior, changing from a neuronal-like to a fibroblast-like morphology. After activation, HSCs increase their proliferation rate and extracellular matrix (ECM)production. Overproduction of ECM, which contains mainly collagen type I, is a direct cause of liver disruption. HSCs also produce substances which inhibit protease activities, such as TIMPs,which enhance ECM deposition in the liver. On the molecular level, HSCs are activated by cytokines,growth factors, and oxidative stress, which are abundant in afflicted liver. These factors induce intracellular signals transmitted by many kinases, the most important of which are JNK,ERK1/2, p38, TAK-1, PKC, FAK, and P3IK. Signals transmitted via these pathways change the activities of transcription factors such as Smad, AP-1, and NF-kB. This in turn causes changes in gene transcription and ultimately alters the whole cell's behavior and morphology. The cell begins the production collagen type I, TIMP-1, and alphaSMA. Activated HSCs can sustain their own activation by producing growth factors such as PDGF and TGF-beta. Despite the vast knowledge about the mechanisms causing liver fibrosis and cirrhosis, there is still no effective cure. Further studies are therefore needed to solve this problem.

Metformin inhibits both oleic acid-induced and CB1R receptor agonist-induced lipid accumulation in Hep3B cells: The preliminary report.

Fatty liver is characterized by excessive accumulation of triglycerides within hepatocytes. Recent findings indicate that natural history of nonalcoholic fatty liver is regulated, in part, by endogenous cannabinoids. Metformin is an oral hypoglycemic medication which inhibits gluconeogenesis and glycogenolysis in hepatocytes and limits lipid storage in the liver through the inhibition of free fatty acid formation via induction of activated protein kinase activity (AMPK). Both endocannabinoids and metformin may modulate hepatosteatosis; therefore, it was interesting to examine whether metformin may affect lipid accumulation in hepatocytes by acting on cannabinoid receptors, CB1 and CB2, in in vitro study. Hep3B cells were incubated with or without metformin (Met), phosphatidylcholine (PC), and oleic acid (OA). Cells without any of the examined substances served as negative control. Cells treated only with OA served as positive control. The quantity of intracellular lipids was assessed using Oil-Red-O staining. Selective CB1R agonist, arachidonyl-2-chloromethylamide (ACEA), and CB2R agonist, AM1241 (2-iodo-5-nitrophenyl)-[1-(methylpiperidin-2-ylmethyl)-1 H-indol-3-yl]methanone, were also used to treat Hep3B cells. In some experiments, antagonist for CB1R, AM6545, or SR144528 as selective antagonist of CB2R were used. In the study, Met decreased lipid accumulation in cells treated with OA and inhibited CB1R agonist-induced lipid accumulation in hepatocytes. The CB2R agonist-induced hepatic lipid accumulation was not inhibited by metformin. The results indicate that metformin may interact with endocannabinoid system in the liver by inhibiting CB1R agonist-stimulated fat accumulation in hepatocytes.

Glycoconjugates of Gram-negative bacteria and parasitic protozoa - are they similar in orchestrating the innate immune response?

Innate immunity is an evolutionarily ancient form of host defense that serves to limit infection. The invading microorganisms are detected by the innate immune system through germline-encoded PRRs. Different classes of PRRs, including TLRs and cytoplasmic receptors, recognize distinct microbial components known collectively as PAMPs. Ligation of PAMPs with receptors triggers intracellular signaling cascades, activating defense mechanisms. Despite the fact that Gram-negative bacteria and parasitic protozoa are phylogenetically distant organisms, they express glycoconjugates, namely bacterial LPS and protozoan GPI-anchored glycolipids, which share many structural and functional similarities. By activating/deactivating MAPK signaling and NF-κB, these ligands trigger general pro-/anti-inflammatory responses depending on the related patterns. They also use conservative strategies to subvert cell-autonomous defense systems of specialized immune cells. Signals triggered by Gram-negative bacteria and parasitic protozoa can interfere with host homeostasis and, depending on the type of microorganism, lead to hypersensitivity or silencing of the immune response. Activation of professional immune cells, through a ligand which triggers the opposite effect (antagonist versus agonist) appears to be a promising solution to restoring the immune balance.