Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all ...available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from
with a single sgRNA targeting the
gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from
with a single guide RNA against
almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Introduction. SARS-CoV-2 infection causes immune disorders that create conditions for the reactivation of human herpesviruses (HHVs). However, the estimates of the HHVs effect on the course and ...outcome of COVID-19 are ambiguous. Аim – to study the possible relationship between the HHV reactivation and the adverse outcome of COVID-19. Materials and methods. Postmortem samples from the brain, liver, spleen, lymph nodes and lungs were obtained from 59 patients treated at the Moscow Infectious Diseases Hospital No.1 in 2021–2023. The group 1 comprised 39 patients with fatal COVID-19; group 2 (comparison group) included 20 patients not infected with SARS-CoV-2 who died from various somatic diseases. HHV DNA and SARS-CoV-2 RNA were determined by PCR. Results. HHV DNA was found in autopsy samples from all patients. In group 1, EBV was most often detected in lymph nodes (94%), HHV-6 in liver (68%), CMV in lymph nodes (18%), HSV in brain (16%), VZV in lung and spleen (3% each). The detection rates of HHVs in both groups was similar. Important differences were found in viral load. In patients with COVID-19, the number of samples containing more than 1,000 copies of HHV DNA per 100,000 cells was 52.4%, in the comparison group – 16.6% (p 0.002). An association has been established between the reactivation of HSV and HHV-6 and the severity of lung damage. Reactivation of EBV correlated with increased levels of liver enzymes. Conclusion. Reactivation of HHVs in patients with fatal COVID-19 was associated with severe lung and liver damages, which indicates a link between HHV reactivation and COVID-19 deaths.
Introduction. Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines.
The aim of the ...work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum.
Materials and methods. BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response by antigen-specific lymphocyte proliferation and by production of IFN- in vitro.
Results. Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN- in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins.
Conclusion. Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.
Abstract Objective Genetic instability manifested as loss or gain of whole chromosomes (aneuploidy) is a newly described feature of the human brain. Aneuploidy in the brain was hypothesized to be ...involved in schizophrenia pathogenesis. To gain further insights into the relationship between aneuploidy in the brain and schizophrenia pathogenesis, a molecular-cytogenetic study of chromosome 1 aneuploidy was performed. Methods Interphase multiprobe fluorescence in situ hybridization (FISH) with quantitative FISH (QFISH) and interphase chromosome-specific multicolor banding (ICS-MCB) were used to define aneuploidy rate in 12 unaffected and 12 schizophrenia brains. Results In the unaffected brain ( n = 12; 22,794 cells analyzed), average frequencies of stochastic chromosome 1 loss and gain were 0.3% (95%CI 0.2–0.4%) and 0.3% (95%CI 0.2–0.4%), respectively. The threshold level for stochastic chromosome gain and loss (the mean + 3SD) in the normal brain was 0.7%. Average rate of aneuploidy in the schizophrenia brain ( n = 12; 28,482 cells analyzed) was 0.9% (95%CI 0.3–1.5%) for chromosome 1 loss and 0.9% (95%CI 0.2–1.7%) for chromosome 1 gain. Significantly increased level of mosaic aneuploidy involving chromosome 1 was revealed in two schizophrenia brains (3.6% and 4.7% of cells with chromosome 1 loss and gain, respectively). Stochastic aneuploidy rate for chromosome 1 in the schizophrenia brain without two outliers ( n = 10) reached 0.6% (95%CI 0.3–0.9%) for loss and 0.5% (0.2–0.9%) for gain and was higher than in controls ( P = 0.005 and P = 0.001, respectively). Conclusions Our findings support the hypothesis suggesting that subtle genomic imbalances manifesting as low-level mosaic aneuploidy may contribute to schizophrenia pathogenesis.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Introduction. A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a ...weak immune response and require the use of adjuvants.
The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid.
Materials and methods. An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated.
Results. Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60.
Conclusions. Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.
After suffering pulmonary embolism (PE), doctors are confronted with various consequences of the disease, from asymptomatic residual pulmonary thrombosis to the formation of chronic thromboembolic ...pulmonary hypertension (CTEPH). There is also a subgroup of patients who have undergone pulmonary embolism, who experience shortness of breath during physical exertion, absent before pulmonary embolism, or shortened dyspnea preceding PE, combined with residual thrombosis of pulmonary artery (PA) and normal average pressure in PA at rest during catheterization of the right heart (CRH). This condition is defined as chronic thromboembolic pulmonary disease or post thromboembolic syndrome. Pathogenetic aspects of this condition are not fully investigated. It is important to predict the development of postembolic syndrome and to develop algorithms for the diagnosis, treatment and rehabilitation of patients with symptoms and residual pulmonary thrombosis. In case of the development of pulmonary vasculopathy in some patients who have undergone pulmonary embolism, a severe life-threatening condition forms - CTEPH, characterized by an increase in pressure in the pulmonary artery, right heart failure due to the presence of organized blood clots that have entered the pulmonary vascular bed during PE. The volume of thrombotic masses does not always correlate with clinical symptoms, which indicates the importance of microvascular remodeling. If CTEPH is suspected, a diagnostic algorithm is required, including ventilation-perfusion scintigraphy, CT angiopulmonography and catheterization of the right heart. Treating a patient with CTEPH is a difficult task fora doctor. The timely referral of the patient to the center where they are involved in treatment, including surgery and CTEPH is extremely important. Timely performed thrombendarterectomy in some cases allows to completely cure the patient. In the case of inoperable CTEPH or residual pulmonary hypertension after thrombendarterectomy, balloon angioplasty of the PA is used as well as drug treatment with specific drugs that reduce the pressure in the PA (riociguat), endothelin receptor antagonists (bosentan, macitentan), prostanoids (inhalant illoprost) phosphodiesterase-5 inhibitor and combined therapy. In this article we considered some consequences directly related to PE: asymptomatic residual pulmonary thrombosis, chronic thromboembolic pulmonary disease, chronic thromboembolic pulmonary hypertension.
The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics ...and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.
PDF
