Proteomics in immunity

Tyrosinkinase 2 (Tyk2) gehört zur Familie der Janus Kinasen (Jak). Tyk2 wurde ursprünglich als essentielle Komponente in der Typ-I Interferon (IFN) Signaltransduktions-Kaskade identifiziert und ist in die Signaltransduktion vieler weiterer Zytokine und einiger Wachstumsfaktoren involviert. Tyk2-defiziente Mäuse zeigen erhöhte Sensitivität bei Infektion mit vielen unterschiedlichen Pathogenen (z.B. Maus Cytomegalovirus, Listeria monocytogenes). Im Gegensatz dazu sind sie resistent gegen Lipopolysaccharid (LPS)- und Ischämie/Reperfusion-induzierten septischen Schock. Makrophagen spielen eine zentrale Rolle bei der Erkennung von und der Antwort auf LPS und somit in der Pathogenese des septischen Schocks. Makrophagen zeigen in Abwesenheit von Tyk2 eine reduzierte Aktivierung der IFN-Signaltransduktion nach Behandlung mit LPS und folglich eine erniedrigte Expression zumindest mancher IFN-induzierbarer Gene. Ziel dieser Arbeit war es, die molekulare Rolle von Tyk2 in Makrophagen detaillierter zu untersuchen, wobei der Fokus auf die Ermittlung von neuen, Tyk2-abhängigen Antworten der Wirtszellen gelegt wurde. Ein Proteomics-Ansatz zur vergleichenden Analyse von Proteinmustern in Zelllysaten von Wildtyp- versus Tyk2-defizienter primärer Makrophagen wurde ausgewählt. Als zentrale Technik hierfür wurde zweidimensionale „Fluorescence Difference Gel Electrophoresis“ (2D-DIGE) verwendet. Experimentelle Bedingungen für die spezifischen Probentypen wurden optimiert und die Reproduzierbarkeit sowie die minimal detektierbaren Unterschiede statistisch ermittelt. Diese optimierte Methode wurde dann eingesetzt, um Kandidaten für die massenspektrometrische Identifizierung zu ermitteln. Wir konnten zeigen, dass die Proteinmuster in Gesamtzell- und Kern-Lysaten zwischen Wildtyp- und Tyk2-defizienten Knochenmark-Makrophagen, sowohl vor als auch nach der Behandlung mit LPS, signifikant unterschiedlich sind. In 27 differentiell exprimierten Spots wurden mittels Massenspektrometrie 23 verschiedene Proteine identifiziert. Diese Proteine gehören zu diversen funktionellen Klassen (z.B. Immunantwort, oxidative Stressantwort, Apoptose, Metabolismus und Transkription/Translation) und ihre Expression ist durch Tyk2 entweder positiv oder negativ reguliert. Weitere detaillierte Untersuchungen an ausgewählten Proteinen ergaben, dass Tyk2 die Proteinexpression auf mRNA- und/oder Proteinebene beeinflusst. Mittels 1D und 2D Westernblot Analysen fanden wir, dass Tyk2 die LPS-abhängigen Veränderungen im Proteinmuster von Peroxiredoxin 1 (PRDX1), nicht aber dessen Gesamtproteinmenge, beeinflusst. Die subzelluläre Verteilung von Elongationsfaktor 2 (EF2) scheint abhängig von Tyk2 zu sein, da Genotyp-spezifische Unterschiede nur in Kern- und nicht in Gesamtlysaten detektierbar sind. Wir konnten weiters zeigen, dass Tyk2 die Expression von Plasminogen Activator Inhibitor 2 (PAI2) und pro-Interleukin-1beta (pro-IL-1beta) Protein, nicht aber deren mRNAs, negativ reguliert. Im Gegensatz dazu ist die Anwesenheit von Tyk2 für eine effiziente mRNA Expression von N-myc Interactor (NMI) erforderlich, dessen transkriptionelle Regulation durch IFNs bekannt ist. Die Tatsache dass IL-1beta eine wichtige Rolle in der Immunität spielt und seine Expression bisher nicht in direkten Zusammenhang mit Tyk2 gebracht wurde, hat uns veranlasst den molekularen Mechanismus der erhöhten pro-IL-1beta Proteinexpression näher zu analysieren. Wir konnten zeigen, dass die erhöhte intrazelluläre Menge von pro-IL-1beta nicht durch defekte Prozessierung und/oder Sekretion verursacht wird und dass in Abwesenheit von Tyk2 auch die extrazelluläre Menge von IL-1beta erhöht ist. Die Analyse des Proteinabbaus nach der Applikation des translationellen Inhibitors Cycloheximid oder einer radioaktiven Protein-Markierung ergab, dass die Stabilität von pro-IL-1beta in beiden Genotypen ähnlich ist. Interessanterweise beobachteten wir eine erhöhte Assoziation der IL-1beta mRNA mit Polysomen in Abwesenheit von Tyk2. Dies lässt Differenzen in der translationellen Effizienz und eine neue Rolle von Tyk2 in der translationellen Kontrolle annehmen. Zusammenfassend zeigen wir, dass mittels 2D-DIGE mit unserem experimentellen Aufbau eine hohe Reproduzierbarkeit erzielt wird. Durchschnittlich können signifikante Unterschiede in der Proteinexpression schon ab 30% Differenz in Zelllysaten primärer Makrophagen festgestellt werden. In Bezug auf Tyk2 weisen unsere Ergebnisse auf einen regulativen Einfluss auf mehrere Ebenen der Proteinexpression und auf neue Zusammenhänge zwischen Tyk2 und diversen zellulären Proteinen hin.Tyrosine kinase 2 (Tyk2) is a member of the Janus kinase (Jak) family and was originally identified by its essential role in type I interferon (IFN) signalling. Tyk2 is involved in signal transduction via various cytokines and some growth factors. Tyk2-deficient mice show increased sensitivity to infection with a number of different pathogens (e.g. Murine Cytomegalovirus, Listeria monocytogenes). In contrast, they show high resistance to lipopolysaccharide- (LPS) and ischemia/reperfusion-induced shock. Macrophages play a crucial role in the recognition of and response to LPS, and consequently, in the pathogenesis of endotoxin shock. In the absence of Tyk2, macrophages show a decreased activation of the IFN signalling cascade upon LPS treatment and, accordingly, reduced induction of at least some IFN target genes. Aim of this study was to investigate the molecular role of Tyk2 in macrophages in more detail with the main focus on the identification of novel Tyk2-dependent host cell responses. A proteomic approach was chosen for the comparative analysis of protein patterns in lysates from wild-type (WT) versus Tyk2-deficient primary macrophages. The key method was two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) using minimal labelling with cyanine dyes. Experimental conditions for the specific sample type were optimised and reproducibility and the minimal detectable differences (effect size) were determined by statistical analyses. The method was applied to select candidates, which were then subjected to identification by mass spectrometry. We could show that protein patterns in whole cell lysates and nuclear extracts are significantly different between WT and Tyk2-deficient bone marrow-derived macrophages (BMM) both in the LPS-treated and the untreated state. Twenty-three different proteins derived from 27 differentially expressed spots were identified by mass spectrometry. The identified proteins belong to distinct functional categories (e.g. immune response, oxidative stress response, apoptosis, metabolism, and transcription/translation) and their expression is either positively or negatively regulated by Tyk2. More detailed analysis of selected proteins revealed that Tyk2 influences protein expression at the mRNA and/or at the protein level. We show that Tyk2 influences LPS-dependent changes in the peroxiredoxin 1 (PRDX1) spot pattern but not the total PRDX1 expression level, as displayed by 1D and 2D western blot analysis. Subcellular distribution of elongation factor 2 (EF2) appeared dependent on Tyk2, since genotype specific differences were only detected in nuclear but not in whole cell extracts. We could furthermore show that Tyk2 negatively regulates plasminogen activator inhibitor 2 (PAI2) and pro-interleukin-1beta (pro-IL-1beta) protein but not mRNA expression. In contrast, N-myc interactor (NMI), a protein known to be transcriptionally regulated by IFN, was dependent on the presence of Tyk2 for efficient mRNA and protein expression. Since IL-1beta plays an important role in immunity and Tyk2 has not yet been linked to IL-1beta expression, we further analysed the mechanistic basis of enhanced pro-ILbeta protein expression in Tyk2-deficient macrophages. We could show that enhanced levels of intracellular pro-IL-1beta are not due to defective processing and/or secretion and that also extracellular IL-1beta is enhanced in the absence of Tyk2. Similar protein stability in both genotypes could be demonstrated by using the translational inhibitor cycloheximide and monitoring pro-IL-1beta degradation and pulse/chase labelling experiments. Interestingly, we found increased association of IL-1beta mRNA with polysomes in the absence of Tyk2, arguing for differences in translational efficiency and suggesting a novel role of Tyk2 in translational control. In summary, we show that high reproducibility can be achieved with 2D-DIGE technology with the developed experimental set-up. Detection of differences in protein expression in cell lysates derived from primary macrophage cultures of as low as 30% (on average) could be demonstrated. With respect to Tyk2, our results imply regulatory roles of Tyk2 at multiple levels of protein expression and establish novel connections between Tyk2 and several cellular proteins

The association between periodontal conditions, inflammation, nutritional status and calcium-phosphate metabolism disorders in hemodialysis patients

Objectives: To analyze the association between periodontal conditions and inflammation, nutritional status and calcium-phosphate metabolism disorders in hemodialysis (HD) patients. Material and Methods: We analyzed 128 HD patients divided into two groups: dentate (n = 103) and edentulous (n=25). The following items were assessed: baseline characteristics, age at the start and duration of HD, biochemical data: C-reactive protein (CRP), serum albumin, calcium, phosphorus, alkaline phosphatase, parathormone. A single dentist performed a complete dental/periodontal examination, including parameters of oral hygiene and gingival bleeding. Results: One person had healthy periodontium, 62.14% of the patients had gingivitis, and 36.9% had moderate or severe periodontitis. The age at HD onset had a positive impact on periodontal status and negatively correlated with the number of teeth. A positive correlation between age and CRP level and negative correlations between age and serum albumin and phosphorus were found. Pocket depth (PD) was negatively correlated with serum albumin. The number of teeth was negatively correlated with serum CRP. Conclusions: High prevalence and severity of periodontal disease are observed in hemodialysis patients. There is a high probability that periodontal disease may be present at the early stages of chronic kidney disease (CKD) before the hemodialysis onset

Pro- and anti-inflammatory cytokines and growth factors in patients undergoing in vitro fertilization procedure treated with prednisone

Embryo implantation is a key moment in pregnancy. Abnormal production of pro- and anti-inflammatory cytokines, their receptors and other immune factors may result in embryo implantation failure and pregnancy loss. The aim of this study was to determine the profile of selected pro- and anti-inflammatory factors in the blood plasma of patients undergoing in vitro fertilization (IVF) and control women who achieved pregnancy after natural conception. The examined patients were administered steroid prednisone. We present results concern the plasma levels of IFN-ɣ, BDNF, LIF, VEGF-A, sTNFR1 and IL-10. We found that IVF patients receiving steroids differed significantly from patients who were not administered such treatment in terms of IFN-γ and IL-10 levels. Moreover, IVF patients differed in secretion of all tested factors with the fertile controls. Our results indicated that women who secrete at least 1409 pg/ml of sTNFR1 have a chance to become pregnant naturally and give birth to a child, while patients after IVF must achieve a concentration of 962.3 pg/ml sTNFR1 in blood plasma for successful pregnancy. In addition, IVF patients secreting VEGF-A above 43.28 pg/ml have a greater risk of miscarriage or a failed transfer in comparison to women secreting below this value. In conclusion, fertile women present a different profile of pro- and anti-inflammatory cytokines, and growth factors compared to patients with recurrent implantation failure (RIF)

Clinical examples of 3D dose distribution reconstruction, based on the actual MLC leaves movement, for dynamic treatment techniques

AbstractAimTo present practical examples of our new algorithm for reconstruction of 3D dose distribution, based on the actual MLC leaf movement.BackgroundDynaLog and RTplan files were used by DDcon software to prepare a new RTplan file for dose distribution reconstruction.Materials and methodsFour different clinically relevant scenarios were used to assess the feasibility of the proposed new approach: (1) Reconstruction of whole treatment sessions for prostate cancer; (2) Reconstruction of IMRT verification treatment plan; (3) Dose reconstruction in breast cancer; (4) Reconstruction of interrupted arc and complementary plan for an interrupted VMAT treatment session of prostate cancer. The applied reconstruction method was validated by comparing reconstructed and measured fluence maps. For all statistical analysis, the U Mann–Whitney test was used.ResultsIn the first two and the fourth cases, there were no statistically significant differences between the planned and reconstructed dose distribution (p=0.910, p=0.975, p=0.893, respectively). In the third case the differences were statistically significant (p=0.015). Treatment plan had to be reconstructed.ConclusionDeveloped dose distribution reconstruction algorithm presents a very useful QA tool. It provides means for 3D dose distribution verification in patient volume and allows to evaluate the influence of actual MLC leaf motion on the dose distribution

Pregnancy of a 47-year old woman treated by in vitro fertilization using oocyte from a donor (IVF-OD)

Abstract The paper presents a case of pregnancy, childbirth and postpartum of a 47-year-old woman with secondary infertility and obstetric history. In vitro fertilization with oocyte derived from a 21-year donor (IVF-OD) was performed due to premature ovarian failure and age of the patient. The treatment resulted in unifetal pregnancy, complicated by, among others: hypertension, cardiac problems, varicose veins in the lower limbs, elevated concentration of bile acids and anxiety neurosis. A healthy newborn was delivered at 37 hbd by cesarean section. Due to the development of techniques of assisted reproduction, pregnancies of perimenopausal women will pose a growing problem in modern obstetrics.

Microwave-assisted synthesis and characterization of novel chitosan-based biomaterials for pelvic organ prolapse treatment

Pelvic organ disorders affect up to one in four women in the United States. The prevalence of pelvic organ prolapse (POP) is increasing with each year, particularly in the setting of prolonged life expectancy and an aging population. Current treatment approaches, including polypropylene monofilaments are associated with numerous painful and worrisome side-effects. Therefore, scientists are looking for new solutions. A promising alternative to the current treatment is tissue engineering, which can be utilized to re-create support to the vagina and pelvic organs. Tissue engineering requires the use of three-dimensional scaffolds, derived from biocompatible materials. Chitosan is a natural polymer, obtained from shellfish exoskeletons. It is known for its biodegradability, lack of cytotoxicity and non-pyrogenicity. Due to the presence of free hydroxyl and amino groups, it may undergo various modifications. In this paper, we describe a new type of chitosan-based biomaterials, which can be used as a new alternative scaffold that may provide support to prolapse organs. The chitosan scaffold was obtained under microwave radiation using multifunctional amino and organic acids. We discuss the scaffold's characteristics, with an emphasis on its chemical structure and morphology. Fourier transform infrared spectroscopy (FT-IR) analysis confirmed cross-linking processes with preservation of free amino groups. Moreover, mechanical durability, the stability and swelling ability of the scaffolds in a simulated body fluid were investigated. All of the prepared scaffolds demonstrated very good antioxidant activity and biodegradability. Importantly, the biocompatibility of chitosan scaffolds was examined on human vaginal VK2/E6E7 cell line. No evidence of toxicity was documented, and the cells maintained their presence on the studied materials. These results allude to the lack of toxicity of the scaffolds, and indicate that chitosan-based scaffold should be further investigated in in vivo studies as they may be a promising alternative treatment to pelvic organ prolapse.Web of Science70349147

Facial and oral manifestations following COVID-19 vaccination: a survey-based study and a first perspective

(1) Background: Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) emerged in Wuhan, China, in late 2019. The development of effective and safe vaccines against SARS-CoV-2 has been extremely fast. The list of orofacial adverse effects of BNT162b2 and mRNA-1273 vaccines based on the clinical trials are reported to be rare. The aim of this study was to investigate the facial and oral manifestations of COVID-19 vaccination using a survey-based study. (2) Methods: The questionnaire was developed using Google Forms and sent anonymously to a total of 700 subjects (medical personnel) in Poland, Italy, and other EU countries. (3) Results: 223 people answered the questionnaire, mainly vaccinated with BNT162b2. Only 3.1% and 5.4% experienced oral and facial symptoms, respectively. General diseases presence and age have significant influence on the probabil- ity of oral symptoms occurrence after the second dose. Facial symptoms are correlated with general disease; autoimmune pathologies and age, at first and second dose, respectively. Gender, smoking and regular medication intake have significant influence on the probability of taking an absence day. Gender, age, and smoking have a significant influence on the duration of symptoms after second dose. (4) Conclusions: Based on the results of this preliminary survey, there is no observed significant correlation between vaccine administration for COVID-19 and facial and oral manifestations

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases