STUDY OF EXPRESSION OF HBD-1 AND HBD-2 GENES IN EPITHELIAL CELLS OF MUCOUS UPPER AIRWAY IN NEWBORNS WITH PNEUMONIA DEPENDING ON THE CAUSATIVE AGENT

Β-defensins play an important role in protecting the fetus from infection, so the expression of these antimicrobial peptides in the respiratory tract in newborns is really important. In this regard, we set a task of studying the expression of the HBD-1 and HBD-2 genes in the epithelial cells of the mucous of the upper airway in newborns with pneumonia and in healthy newborns, depending on the causative agent. Also, the polymorphic marker G(-20) A in the DEFB1 gene was associated with infectious pathology of newborns (in particular pneumonia). Methods: The microflora and the factors of congenital immunity on the mucous membranes of the upper airway have been studied in two groups: newborns with ventilator-associated and congenital pneumonia. The biological material was scrapings of epithelial cells of the mucous membrane of the upper airway of newborns and puerperas and blood. Results: It was found that the expression of the HBD-2 gene increases 2.3-fold in children who have an infectious agent, but there are no clinical manifestations of pneumonia. A significant decrease in HBD-2 (3.2 times) in patients with pneumonia caused by K. pneumonia was shown. The frequencies of alleles of the DEFB1 gene in the fetal infection group and in the comparison group: allele G - 0.66, 0.79, allele A - 0.34, 0.21, respectively. The frequencies of the genotypes of the test marker in mothers in the ventilator-associated, congenital pneumonia and the comparison group were as follows: GG - 0.78, 0.58, 0.58; AA is 0, 0.25, 0; AG - 0.22, 0.17, 0.42, respectively. In newborns allele G dominated among alleles (frequency was higher than 0.73 in all groups) and genotype GG (frequency exceeded 0.52). Conclusion: In the course of the study, it was confirmed that β-defensins protect the mucous from infectious agents. The results indicate that the genetic marker G (-20) A of the DEFB1 gene is associated with the risk of developing the child's UTI

СHANGES OF TLR GENE EXPRESSION IN CERVICAL EPITHELIUM FROM THE PATIENTS WITH UROGENITAL INFLAMMATORY DISEASES

Toll-like receptors (TLR) are involved into innate immune recognition of microorganisms, including pathogenic bacteria. The aim of this work is to assess relative levels of TLR2 and TLR9 expression in cervical epithelial cells of women with inflammatory diseases of pelvic organs. The total group of patients consisted of 47 persons, including 20 women with urogenital infections, and 27 women comprised a control group. Using real-time PCR, we identified the opportunistic pathogens and gene expression levels of TLR2, TLR9 in the cervix epithelial cells. It was shown that expression of TLR9 in the group with urogenital infections was 13.7-fold higher than in control group. We have revealed that a significant increase of TLR9 expression in the mucosal epithelium from cervical canal correlated with detection of infectious pathogens in the samples

Microbiological assessment of the effectiveness of standard therapy in atopic dermatitis

Background. Atopic dermatitis is an inflammatory skin disease characterized by recurrent lesions and intense pruritus. Nowadays there is a stepwise approach to the treatment of atopic dermatitis, which is defined by disease intensity and complications such as secondary skin infections. However, the current management of atopic dermatitis may not always lead to the expected outcome due to not only immune dysregulation of both adaptive and innate immunity but also imbalance of the skin microbiome. Aims. The aim of the study was to evaluate changes in the composition of the skin microbiome in both lesional and non-lesional skin in patients with atopic dermatitis during standard treatment. Materials and methods. Twenty patients with atopic dermatitis and twenty six healthy controls over 18 years old were included into the study. All microbiome samples were obtained from lesional and non-lesional skin sites of atopic dermatitis patients before and after therapy. Whereas samples from healthy controls were taken once from a flexor surface of the elbow. Species identification of clinical isolates were identified using MALDI Biotyper Sirius (Bruker Daltonics). Results. At baseline, the prevalence of S. aureus colonization among patients with atopic dermatitis was 34.20% in lesional skin and 32.50% in non-lesional skin. After treatment, there was a significant decrease in the prevalence of S. aureus carriage in both lesional and non-lesional skin areas (р 0.05). However, no significant difference was observed in the proportion of all other staphylococci (р 0.1). Interestingly, S. aureus was not found in healthy controls. Conclusions. The results of the study demonstrated the effectiveness of standard therapy for managing patients with atopic dermatitis as it had a positive impact on the skin microbial community and showed a decrease in S. aureus proportion after the treatment

Investigation of local expression of NLRP3 inflammasome complex genes in modeling retinal degeneration <i>in vivo</i>

Neurodegenerative ophthalmopathology is one of the main causes of irreversible blindness and disability in the world. In the pathogenesis of diseases of this group, more and more attention has recently been paid to the role of local inflammation caused by the activation of innate immunity and the mechanisms of its genetic regulation. In recent years, works have appeared in the field of experimental ophthalmology that have demonstrated the possibility of NLRP1, NLRP3 inflammasome complexes assembling when exposed to hyperglycemia, oxygen deprivation of retinal cells, as well as modeling compressive stress similar to that in glaucoma [15]. However, the mechanism of inflammasome involvement in the development of neurodegenerative eye diseases remains unclear. The aim of the study was to investigate the local expression of genes encoding proteins of the NLRP3 inflammasome complex (NLRP3, CASP-1) in an experimental model of retinal degeneration in rabbits. The studies were performed on samples of tissue complex (TC) of the retina/retinal pigment epithelium (RPE) (retina/RPE TC), isolated from the eyes of 14 New Zealand albino rabbits, in which degenerative retinal lesion was modeled by a single subretinal injection of 0.01 mL of 0.9% sodium chloride solution, and 7 healthy rabbits without eye damage. The formation of retinal degeneration was judged on the basis of changes in morphofunctional parameters obtained during specialized ophthalmological research methods (optical coherence tomography, fundus autofluorescence, electroretinography) at follow-up periods of 1, 3 and 6 months. The level of expression of NLRP3 and CASP-1 genes in the retina/RPE TC was evaluated by reverse transcription polymerase chain reaction (RT-PCR). According to the results of the study, a statistically significant increase in NLRP3 gene expression (p &lt; 0.001) was noted in the retina/RPE TC of experimental animals, which may indicate the involvement of NLRP-3 inflammasome components in the development of neurodegenerative retinal lesions. At the same time, the expression of the gene encoding CASP-1 was detected only in the retina/RPE TC of experimental eyes and is probably due to local inflammatory mechanisms in the retinal tissue.The high level of NLRP3, CASP-1 mRNA, detected in all retina/RPE TC samples of experimental eyes at late stages of the experiment (3 and 6 months), allows us to assume the formation of mechanisms (for example, activated glial phenotype) that support inflammation in retinal tissue. This should be taken into account in actively developing transplantation methods for the treatment of retinal degeneration

Influenza virus infection and postviral bacterial pneumonia pathogenesis induced by different subtypes of influenza virus in mice

Secondary bacterial infections after influenza virus infection further increase morbidity and mortality due to influenza. Despite of seasonal influenza vaccination, antiviral drugs and antibiotics are widely used in viral/bacterial pneumonia therapy. Therefore, further comprehensive study of the infection pathogenesis is relevant. Murine models for influenza virus infection were reproduced with different virus subtypes A/California/04/2009MA (pandemic H1N1 2009), A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/69 (H3N2), Anadyr/177/2009 (H1N1) and for post-influenza bacterial pneumonia caused by the Gram-positive Staphylococcus aureus. After the infection occurs, its pathogenic features were detected by daily monitoring the mortality (survival) and morbidity rate (body weight loss) and, in addition, viral pathogenesis also was evaluated by assessing virus replication (viral titer) and humoral immune responses (production of pro- and anti-inflammatory cytokines) in respiratory tract of infected mice including during antiviral (oseltamivir) and antibacterial (cefuroxime) therapy. Mortality and virus titer in the infected mice did not differ significantly between the groups of different influenza A virus subtypes. However, production of cytokines (IL-10, IFNg, TNFa) and weight gain proved to be different. Mortality of the mice reached 100% after secondary bacterial infection, whereas IFNg and TNFa levels in mice lung increased reached maximal values in the treated groups. Viral subtype A/California/04/2009MA of influenza A was most pathogenic in mouse model of secondary bacterial pneumonia. Antiviral and antibacterial treatment caused a decrease in mortality, reduced viral titers in lungs, and retain body weight gain of mice. According to these points, the treatment groups did not significantly differ from each other. At the same time, it should be noted that the cytokine production significantly decreased in the treated groups, and IL-10 and IFNg levels in lungs were different, that may be due to therapeutic mechanisms of these drugs. Thus, antiviral therapy for influenza infection and combination therapy for viralbacterial pneumonia can be an effective tool to reduce mortality of influenza

Hyperexpression of TLR2 and TLR4 in patients with ischemic stroke in acute period of the disease

Pathogenesis of ischemic stroke  is actively  involved  in the  system  of innate immunity. Under conditions of cerebral  ischemia, a number of biologically  active  substances are  released  that  interact with innate immunity receptors, in particular TLR2  and  TLR4, which  exacerbate inflammation in brain  tissue. Identification of predictor markers  at the level of the innate immunity system may foresee the clinical course of ischemic stroke and ensure timely treatment. Our objective was to study expression of TLR2 and TLR4 receptors in peripheral blood leukocytes  in patients with ischemic stroke in the dynamics of the disease. 27 people  were included in the study. The main  group consisted of patients with ischemic stroke of varying severity (n = 19). Patients of the main  group were divided into two subgroups:  with an NIHSS index value of &lt; 10 (n = 10) and &gt; 10 (n = 9). The control group included healthy  donors  with no history  of acute  and chronic inflammatory diseases (n = 8). Peripheral blood  leukocytes  were used as the  test material. To determine expression  of the TLR2  and TLR4  genes, RT-PCR in real time was used. Surface  expression  of TLRs was determined by flow cytometry. A study of the TLR2 and TLR4 gene expression showed that on the 1st, 3rd  and 7th  day post-stroke, the TLR4 gene expression  in patients was significantly  increased, when compared to the control group (p &lt; 0.01), whereas TLR2 gene expression on the 3rd  day of the disease was not statistically different from the control group. A study of surface expression  of receptors showed that the average TLR2 fluorescence intensity on the patients’ peripheral blood monocytes was significantly  increased on the 1st  and 3rd  day of disease when compared to the control group.  The  surface  expression  of TLR4  on monocytes has a statistically significant  increase  only on day 7. Assessment  of surface expression  of TLRs in subgroups  with different  severity values by NIHSS showed that  patients with a NIHSS index &gt; 10 had a significantly  higher  level of surface of TLR2  expression  over the observation period, while the largest difference in TLR4  expression  in the subgroups  was observed  on the 1st day of the disease (p &lt; 0.05). Patients with ischemic stroke showed an increase  in TLR2 and TLR4 expression at the gene and protein level, compared to healthy  donors. These indices can be considered possible predictors for clinical  prognosis  of ischemic stroke

CHANGES OF INNATE IMMUNITY INDEXES IN SEVERE ASTHMA IN CHILDREN

At the present time, the role of innate immunity in pathogenesis of bronchial asthma (BA) is actively studied, in particular, significance of TLRs and cytokines. The study included 42 patients with severe bronchial asthma (from 3 to 12 years old), and 67 healthy children at the same age. Expression of TLR2, TLR4, and TLR9 genes was evaluated by PCR-RT from the scrapings of nasal mucosa; cytokines (IL-33, TSLP, IL-4, TGF-β1 and IL-28B) were assayed in nasal swabs by ELISA technique. The main results were as follows: an increased gene expression of TLR2, TLR4, TLR9 genes was revealed in the nasal mucosa scraps from the patients with bronchial asthma as compared to healthy children. We have also measured the contents of important cytokines secreted by the respiratory epithelium in the course of TLRs activation. The study of IL-33, TSLP, IL-4 in nasal samples revealed significantly increased concentrations of these cytokines in the patients with severe BA against the control group. A study of TGF-βlevels in nasal cavity swabs revealed a significant decrease of this regulatory cytokine in the group of pediatric patients with asthma. Worth of note, evaluation of antiviral IL-28B cytokine in the group of patients with severe BA showed a significant downward trend, in comparison to the control indexes. Hence, one may conclude on some disturbances of local innate immunity system in the patients with severe BA which manifest as hyperexpression of TLRs genes, increased production of proinflammatory and epithelial cytokines, decreased production of antiviral IL-28B cytokine, and TGF-β1

The impact of polymorphic variants of interferon receptor genes on COVID-19 severity and antibiotic resistance

Single nucleotide substitutions in gene sequence associated with conformational changes in protein receptor or in expression of interferon receptors may explain variations in human susceptibility to infection and severity of COVID-19 along with other well-known risk factors. The study aimed to investigate associations between polymorphic variants of interferon receptor genes, COVID-19 severity and prevalence of antibiotic resistance genes in the gut microbiota. Materials and methods. The study was conducted using a random sample of Arkhangelsk population aged 42 to 76 years (n = 305). The research involved gathering COVID-19 data from the Federal Register, conducting blood tests for SARS-CoV-2 antibodies and polymorphic interferon receptor gene variants, and identifying antibiotic resistance genes in stool samples. Results. During the first 12–15 months of the COVID-19 pandemic, 17.4% of the study participants had symptomatic COVID-19, while 32.8% were asymptomatic. By the Autumn of 2022, symptomatic COVID-19 cases rose up to 36.4%, while asymptomatic cases increased to 61.3%. We reveal an association between the CC genotype of the IFNAR1 gene rs2257167 variant, the presence of the T allele of IFNAR2 gene rs2229207 variant, the CCTT haplotype and symptomatic COVID-19. The GCTC haplotype was associated with pneumonia and COVID-19 severity. In November 2022, macrolide resistance genes were observed in 98.4% of cases, whereas those to beta-lactams and glycopeptides — in 26.9% and 13.8% cases, respectively. Resistance to three classes of antibiotics was observed in 4.9% and was more frequently detected in individuals with the ССТТ haplotype. Genes encoding beta-lactamases were more often found in individuals with the GCTC haplotype, those who had COVID-19 with pneumonia and those who received hospital treatment. Glycopeptide resistance genes were associated with the CC genotype of the rs2257167 variant of IFNAR1 gene. Conclusion. We identified genetic determinants of susceptibility, symptomatic infection and COVID-19 severity. The associations between polymorphic variants of interferon receptor genes and COVID-19 severity can be used to identify people with a genetic predisposition to severe infection and to determine priority groups for vaccination, including the prevention of antibiotic resistance in complicated course of viral infections

Действие противовирусных миРНК на выработку цитокинов in vitro

Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.Цели. Оценить динамику уровня экспрессии генов IL-1β и IL-28β (IFN-λ3) в результате комплексного нокдауна некоторых клеточных генов, чьи продукты экспрессии играют важную роль в репродукции вируса гриппа.Методы. Вируссодержащую жидкость и клеточный лизат отбирали в течение 3-х дней с момента трансфекции и заражения и оценивали интенсивность вирусной репродукции методами титрования по цитопатическому действию. Концентрацию вирусной рибонуклеиновой кислоты (вРНК) и изменение экспрессии IL-1β и IL-28β (IFN-λ3) определяли методом обратной транскрипции и полимеразной цепной реакции в режиме реального времени (ОТ-ПЦР-РВ). Для вычисления статистически значимых различий между группами использовали непараметрический критерий Манна-Уитни.Результаты. Использование каждого комплекса малых интерферирующих РНК (миРНК) приводило к снижению вирусной репродукции на 1-е сутки при множественности заражения 0.001. Применение комплексов A (FLT4.2 + Nup98.1) и D (FLT4.2 + Nup98.1 + Nup205) приводило к снижению вирусного титра на 2.8 lgТЦД50/мл и на 2.1 lgТЦД50/мл относительно применения неспецифической миРНК L2 и вирусного контроля (р ≤ 0.05). результате трансфекции комплексов B (Nup98.1 + Nup205) и C (FLT4.2 + Nup205) вирусный титр также снижался на 1.5 lgТЦД50/мл и 1.8 lgТЦД50/мл соответственно относительно неспецифической миРНК L2 и вирусного контроля (р ≤ 0.05). При проведении ОТ-ПЦР-РВ также было отмечено достоверное уменьшение концентрации вРНК. При использовании комплексов B, C и D концентрация вРНК снижалась на 1-е сутки в 14.5, 4.1 и 15.0 раз соответственно. На 2-е сутки в клетках с комплексами B и D наблюдалось уменьшение концентрации вРНК в 17.1 и 18.3 раз (р ≤ 0.05). Наряду со снижением вирусного титра и вРНК наблюдалось повышение экспрессии генов IL-1β и IL-28β на 1-е сутки при использовании всех комплексов миРНК относительно неспецифического и вирусного контроля (р ≤ 0.05). На 2-е сутки также наблюдалось повышение экспрессии в клетках с комплексами A и D, а на третьи – в клетках с комплексом D (р ≤0.05).Выводы. Исследование показало, что применение комплексов миРНК приводит к выраженному противовирусному эффекту при одновременном подавлении активности клеточных генов (FLT4, Nup98 и Nup205). Параллельно с этим было выявлено, что при трансфекции комплексов, блокирующих образование продуктов экспрессии, необходимых для вирусной репродукции, повышается уровень экспрессии генов IL-1β и IL-28β. Данные результаты свидетельствуют о том, что используемые миРНК обладают не только противовирусной, но также и иммуномодулирующей активностью, что способствует более эффективному иммунному ответу организма