Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus

Funding Information: Expression and purification of HDV antigen was supported by Russian Foundation for Basic Research (grant 16-04-01490a). Evaluation of serum by Western blot and confocal microscopy was supported by Russian Science Foundation (grant 14-14-01021). Experiments in rabbits were supported by the Swedish Institute grants 09272_2013 and 19806_2016. Cross-border collaboration of the partners, exchange of the materials and standard operation procedures used in the study, and dissemination of the data were supported by the EU Twinning project VACTRAIN, contract nr 692293. Publisher Copyright: © 2016 by the authors; licensee MDPI, Basel, Switzerland.Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.publishersversionPeer reviewe

Генерализованный туберкулез при системной красной волчанке на фоне пульс-терапии метилпреднизолоном: трудности диагностики и лечения

Systemic lupus erythematosus (SLE) and a high-dosage corticosteroid therapy both are risk factors of tuberculosis development. The clinical case presented demonstrates the difficulties of diagnostics and simultaneous therapy of comorbid diseases, which are followed by a multisystemic tuberculosis on the one hand, and a highly active SLE resulting from tubercular regimen on the other. Our experience shows the importance of antituberculosis drug prescription in accordance with a personal chemotherapy acceptability. Высокая суммарная доза кортикостероидов при терапии системной красной волчанки (СКВ) и само заболевание являются факторами риска развития туберкулеза. Клинический случай освещает проблемы диагностики и одновременной терапии коморбидных заболеваний, сопровождающихся, с одной стороны, распространенным полиорганным туберкулезом, с другой, – чрезмерно выраженной активностью СКВ, усугубляющейся на фоне применения противотуберкулезных средств. Данный опыт подчеркивает важность назначения индивидуального режима химиотерапии туберкулеза с учетом переносимости противотуберкулезных препаратов и наличия побочных эффектов.

Assessment of physicians’ and senior medical students’ knowledge in treatment of patients with community acquired pneumonia: Current results of the KNOCAP project

Introduction. Community-acquired pneumonia (CAP) remains one of the most acute problems of bronchopulmonary pathology being the 4th in the mortality structure (after cardiovascular, cerebrovascular diseases and malignant neoplasms) and the 1st among all fatalities from infectious diseases. Thanks to the scientific progress achieved in the antibiotic therapy and vaccine prophylaxis, the death toll has decreased four times compared to its rate during the “pre-antibiotic era“. However, nowadays there is a steadily increasing trend in the pneumonia mortality rate in Russia. The only possible way to increase efficacy of CAP treatment is timely initiated rational antibiotic therapy, considering the possible etiologies, risk factors and the severity of the patient’s condition. Materials and methods. The article represents the results of anonymous prospective surveys within the framework of the KNOCAP multi-centered research project aimed at accessing the knowledge on the fundamental issues in diagnosis and treatment of community-acquired pneumonia. The survey involved 222 students in their fifth- and sixth years in medical institute from Belgorod, Dnepr (Dnipro), Voronezh, Kiev (Kyiv) and Saratov and 110 physicians from Krasnodar, Saratov, Belgorod and Dnepr.Results and discussion. According to the results of the survey, such levels of correct answers were given by doctors and students, respectively: the inadmissibility of antimicrobial therapy (AMT) delay in CAP - 82% and 59%; the main criterion for withholding AMT - 56% and 37%; “sequential therapy” - 61% and 59%. At the same time, only 24% of the students and 23% of the physicians surveyed correctly reported typical mistakes in the treatment of a non-severe CAP with 50% or more accuracy; and in case of initial treatment, the number of correct responses was less than 28% for students and 45% for doctors. Conclusion. The survey showed that both senior medical students majoring in Medical Care and general practitioners had a low level of knowledge in CAP treatment. Hence, curricula need to be adjusted both in medical universities and in health institutions for practitioners in order to inform them and, thus, improve the quality of their knowledge in this field

Assessment of physicians’ and senior medical students’ knowledge in treatment of patients with community-acquired pneumonia: Current results of the KNOCAP project

Community-acquired pneumonia (CAP) remains one of the most acute problems of bronchopulmonary pathology being the 4th in the mortality structure (after cardiovascular, cerebrovascular diseases and malignant neoplasms) and the 1st among all fatalities from infectious diseases. Thanks to the scientific progress achieved in the antibiotic therapy and vaccine prophylaxis, the death toll has decreased four times compared to its rate during the “pre-antibiotic era“. However, nowadays there is a steadily increasing trend in the pneumonia mortality rate in Russia. The only possible way to increase efficacy of CAP treatment is timely initiated rational antibiotic therapy, considering the possible etiologies, risk factors and the severity of the patient’s condition

Development, production and characterization of SARS-CoV-2 virus-like particles (Coronaviridae: <i>Orthocoronavirinae: Betacoronavirus: Sarbecovirus</i>)

Introduction. The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. Materials and methods. Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. Results. VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. Conclusion. The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Connective Tissue Growth Factor in Patients with Coronary Heart Disease: a Pilot Study

AIM. To determine the concentration of connective tissue growth factor and assess the relationship of this index with a number of pathological conditions in elderly patients with CHD. MATERIAL AND METHODS. The study enrolled 50 patients older than 75 years with a diagnosis of coronary heart disease (CAD); most of them (71%) were women. The patients ranged in age from 75 to 96 years (mean age -87.8 years). The concentration of connective tissue growth factor (CTGF) in blood was determined by enzyme immunoassay. RESULTS AND DISCUSSION. In the group of CHD patients, the average CTGF concentration was 357.2 pg/ml, whereas in healthy young adults it was 1076.7 pg/ml (p=0.07). Only 7.9% of patients with CHD had CTGF level exceeding 1000 pg/ml, whereas in young subjects CTGF level exceeded 1000 pg/ml in 25% of cases. In patients with clinically significant CHD, CTGF concentration was significantly higher than in patients without CHD (p=0.001). A negative correlation was registered between the CTGF levels and the systolic (r = -0.25; p = 0.1) and diastolic (r = -0.36; p = 0.02) blood pressure. In the subgroup of patients with pneumosclerosis, the median CTGF concentration reached 190.7 pg/l, without it – 34.7 pg/ml (p = 0.03). A significant inverse correlation was found between the CTGF levels and glucose (r = -0.34; p = 0.03), total cholesterol (r = -0.49; p = 0.002) and LDL cholesterol (r = -0.40; p = 0.01) concentrations. This pilot study found a significant effect of CTGF on echocardiographic parameters and the course of CHD, and higher levels of CTGF in patients with pneumosclerosis. However, a small sample of patients and extremely variable CTGF values do not currently allow unequivocal conclusions to be drawn at this time about the role of this factor in various comorbid conditions. CONCLUSION. Further research is needed to establish the clinical significance of CTGF

Generalized tuberculosis in association with systemic lupus erythematosus on a background of methylprednisolone pulse therapy: difficulties of diagnostics and treatment

Systemic lupus erythematosus (SLE) and a high-dosage corticosteroid therapy both are risk factors of tuberculosis development. The clinical case presented demonstrates the difficulties of diagnostics and simultaneous therapy of comorbid diseases, which are followed by a multisystemic tuberculosis on the one hand, and a highly active SLE resulting from tubercular regimen on the other. Our experience shows the importance of antituberculosis drug prescription in accordance with a personal chemotherapy acceptability