Bacterial Lysates Research Articles

Возможности современной противорецидивной терапии инфекций мочевых путей у детей: исследование CRUTIL

Актуальность. Рецидивирующие инфекции мочевого тракта представляют серьезную проблему в детской нефрологии, влияя на качество жизни и увеличивая нагрузку на систему здравоохранения. Учет индивидуальных факторов и разработка подходов к противорецидивной терапии, особенно у детей без видимых объективных причин для рецидива инфекции, является актуальной задачей современной нефрологии. Материалы и методы. Проспективное открытое многоцентровое рандомизированное в параллельных группах исследование CRUTIL (Children’s Recurrent Urinary Tract Infections on bacteria Lysate) проведено у 83 детей в возрасте от 3 до 15 лет (8,0 ± 2,2 года) с рецидивирующими инфекциями мочевого тракта. Дети были рандомизированы на 3 группы: первая — 22 ребенка, получавших дополнительную терапию лизатом Urivac, вторая — 28 детей, получавших дополнительную терапию лизатом Uro-Vaxom, и третья (контрольная) — 33 ребенка, получавших стандартную терапию. Продолжительность иммуностимулирующей терапии составила 6 месяцев, пациенты этих групп также получали противорецидивное лечение однократной дозой уроантисептика на ночь в течение 18 месяцев, продолжительность наблюдения составила 24 месяца. Результаты. В первой группе детей, получавших 6-валентную вакцину лизатов бактерий Urivac, безрецидивное течение к окончанию исследования наблюдалось у 87 % (19 детей). В группе, получавшей монолизаты бактерий Uro-Vaxom, безрецидивное течение наблюдалось у 72 % (20 детей) (отношение шансов (ОШ) 2,5; P > 0,05; минимальное значение ожидаемого явления — 4,84). Среди тех, кто не получал уроантисептики на ночь и лизаты бактерий, безрецидивное течение документировали у 13 детей (40 %) (р ≤ 0,05; OШ = 0,26 с группой 6-валентной вакцины). 15% повышение эффективности при использовании 6-валентного лизата бактерий было получено за счет элиминации Pseudomonas aeruginosa и Enterococcus faecalis. В дальнейшем с 24-го по 30-й месяц наблюдения в группе Uro-Vaxom еще 3 детей имели рецидив (P ≤ 0,05 с группой, получавшей Urivac). Выводы. Лизаты бактерий существенно повышают эффективность терапии при рецидивирующих инфекциях мочевого тракта у детей. Лучшие результаты в формировании безрецидивного течения рецидивирующих инфекций мочевого тракта демонстрирует назначение профилактической дозы уроантисептика однократно на ночь и применение 6-валентной вакцины Urivac.

2D Strategy for the Construction of an Enzyme-Activated NIR Fluorophore Suitable for the Visual Sensing and Profiling of Homologous Nitroreductases from Various Bacterial Species.

Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives. The activity of NTRs in bacteria facilitates the metabolic activation and antibacterial activity of 5-nitroimidazoles. Therefore, NTR activity correlates with the drug susceptibility and resistance of pathogenic bacteria. As such, it is important to develop a rapid and visual assay for the real-time sensing of bacterial NTRs for the evaluation and development of antibiotics. Herein, an activatable near-infrared fluorescent probe (HC–NO2) derived from a hemicyanine fluorophore was designed and developed based on two evaluation factors, including the calculated partition coefficient (Clog P) and fluorescence wavelength. Using HC–NO2 as the special substrate of NTRs, NTR activity can be assayed efficiently, and then, bacteria can be imaged based on the detection of NTRs. More importantly, a sensitive in-gel assay using HC–NO2 has been developed to selectively identify NTRs and sensitively determine NTR activity. Using the in-gel assay, NTRs from various bacterial species have been profiled visually from the “fluorescence fingerprints”, which facilitates the rapid identification of NTRs from bacterial lysates. Thus, various homologous NTRs were identified from three metronidazole-susceptible bacterial species as well as seven unsusceptible species, which were confirmed by the whole-genome sequence. As such, the evaluation of NTRs from different bacterial species should help improve the rational usage of 5-nitroimidazole drugs as antibiotics.

Prospective Study in Children with Complicated Urinary Tract Infection Treated with Autologous Bacterial Lysates.

Antimicrobial bacteria resistance is an important problem in children with recurrent urinary tract infections (rUTI), thus it is crucial to search for alternative therapies. Autologous bacterial lysates (ABL) may be a potential treatment for rUTI. Twenty-seven children with rUTI were evaluated for one year, urine and stool cultures were performed, 10 colonies of each culture were selected and those identified as Escherichia coli were characterized by serology. For patients who presented ≥105 UFC/mL, an ABL was manufactured and administered orally (1 mL/day) for a month. Twelve children were monitored for ≥1-year, 218 urine and 11 stool samples were analyzed. E. coli (80.5%) was the main bacteria isolated from urine and feces (72%). E. coli of classical urinary serotypes (UPEC), O25:H4, O75:HNM, and O9:HNM were identified in patients with persistent urinary infection (pUTI). In 54% of patients treated with ABL, the absence of bacteria was observed in urine samples after 3 months of treatment, 42% of these remained without UTI between 10–12 months. It was observed that the use of ABL controlled the infection for almost 1 year in more than 60% of the children. We consider it necessary to develop a polyvalent immunogen for the treatment and control of rUTI.

Bacterial YedK represses plasmid DNA replication and transformation through its DNA single-strand binding activity

The SOS response-associated peptidase (SRAP) is an ancient protein superfamily in all domains of life. The mammalian SRAP was recently reported to covalently bind to the abasic sites (AP) in single stranded (ss) DNA to shield the chromosome integrity. YedK, the Escherichia coli SRAP, is not functionally characterized. Here we report the fortuitous pull-down of YedK from bacterial cell lysates by short (<20 bp) double stranded (ds) DNAs, further enrichment of YedK was observed when single stranded (ss) DNA was added. YedK can bind multiple DNA substrates, particularly with a high affinity to DNA duplex with single strand segment. As a SRAP protein, the involvement of YedK in SOS response was extensively examined, however yedK mutant of Escherichia coli showed no difference from the wild type strain upon the treatments with UV and various DNA damaging reagents, indicating its non-essentiality or redundancy in E. coli. Surprisingly, yedK mutants derived from Escherichia coli and Samonella enterica both showed an increased plasmid DNA transformation efficiency compared to the wild types. In accordance with this, induction of YedK effectively decreased the copy number of plasmid DNA. Site-directed mutagenesis of YedK demonstrated that residues involved in single strand DNA binding and cysteine residue at position 2 from N-terminus can discharge the repression of the plasmid transformation efficiency.

The role of immunomodulatory preparations in asthma treatment

Asthma remains one of the most prevalent respiratory tract disorders. The disease affects both adults and children and remains the most common cause of respiratory morbidity. Considering its significant impact on patients’ quality of life and the treatment burdened with side effects, a new therapy approaches affecting the clinical course of asthma are needed. Here we describe the current results that have been obtained on using immunomodulatory preparations in asthma. The analysis of previously published studies was conducted by using the PubMed and Scopus databases. Probiotics, bacterial lysates (BLs) and pidotimod are immunomodulatory compounds affecting both adaptive and innate immunity. The therapy based on probiotics might modulate the intestinal microbiota and regulate the inflammatory response. Bacterial lysates promote immune response by reversing Th1/Th2 unstable balance, which leads to reduction of allergen-induced airway hyperresponsiveness during asthma exacerbations. Pidotimod stimulates PRRs and increases the release of antimicrobial peptides which also leads to the improvement in the rate of respiratory tract infections. Some studies showed the beneficial effect of described preparations in asthma course. Regrettably, findings do not correspond with each other and the data referring to immunomodulatory compounds is still limited, thus there is an urgent need to conduct more, large sample studies.The conclusion we can only draw is that immunomodulatory compounds may offer an alternative approach for symptom reduction and prophylaxis against both infections and exacerbations of asthma.

Recombinant SARS-CoV-2 Nucleocapsid Protein: Expression, Purification, and Its Biochemical Characterization and Utility in Serological Assay Development to Assess Immunological Responses to SARS-CoV-2 Infection.

The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay’s specificity. We expressed and purified from bacterial cells two recombinant forms of SARS-CoV-2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Bacterial lysate add‐on therapy to reduce exacerbations in severe asthma: A double‐blind placebo‐controlled trial

BackgroundAsthma exacerbations are frequently induced by respiratory tract infections (RTIs). Bacterial lysates have been described to possess immune‐modulatory effects and reduce RTIs as well as asthma symptoms in children. However, whether bacterial lysates have similar effects in adult asthma patients is unknown.AimsTo reduce asthma exacerbations by add‐on bacterial lysate therapy in adults with severe asthma and to characterize the clinical and immune‐modulatory effects of this treatment.MethodsAsthma patients (GINA 4) with ≥2 annual exacerbations in the previous year were included. The intervention regimen consisted of OM‐85/placebo for 10 consecutive days per month for 6 months during two winter seasons. Primary end‐point was the number of severe asthma exacerbations within 18 months. The study was approved by the national and local ethical review board and registered in the Dutch Trial Registry (NL5752). All participants provided written informed consent.ResultsSeventy‐five participants were included (38 OM‐85; 37 placebo). Exacerbation frequencies were not different between the groups after 18 months (incidence rate ratio 1.07, 95%CI [0.68–1.69], p = 0.77). With the use of OM‐85, FEV1% increased by 3.81% (p = 0.04) compared with placebo. Nasopharyngeal swabs taken during RTIs detected a virus less frequently in patients using OM‐85 compared to placebo (30.5% vs. 48.0%, p = 0.02).In subjects with type 2 inflammation adherent to the protocol (22 OM‐85; 20 placebo), a non‐statistically significant decrease in exacerbations in the OM‐85 group was observed (IRR = 0.71, 95%CI [0.39–1.26], p = 0.25). Immune‐modulatory effects included an increase in several plasma cytokines in the OM‐85 group, especially IL‐10 and interferons. Peripheral blood T‐ and B cell subtyping, including regulatory T cells, did not show differences between the groups.ConclusionAlthough OM‐85 may have immune‐modulatory effects, it did not reduce asthma exacerbations in this heterogeneous severe adult asthma group. Post hoc analysis showed a potential clinical benefit in patients with type 2 inflammation.

Study of influence of bacterial lysate on cytokine indicators in experimental periodontis in rats

The aim of the research: to optimize the treatment of patients with a disturbed cytokine profile by a bacterial lysate in an experimental model of periodontitis against the background of changes in the local and systemic immune response.&#x0D; Materials and methods: prospective study has been conducted on experimental periodontitis in 42 rats for 90 days. The animals were treated by solution of «Respibron» and the reference drug «Imudon». The concentration of cytokines in the blood serum was determined by the enzyme immunoassay, circulating immune complexes – precipitation method is measured on a spectrophotometer, C-reactive protein – using a latex diagnosticum. All interventions and euthanasia of animals were carried out in compliance with the European principles.&#x0D; Results: the obtained data from the study indicate a high degree a state of decompensation of experimental periodontitis. An immuno-inflammatory reaction preceding periodontitis is accompanied by the disturbances of the cytokine balance, local and systemic immune response. Applying of bacterial lysates led to the compensation of bacterial dysbiosis, restoration of the balance of the activity of pro- and anti-inflammatory cytokines, and a decrease the hyperactivity of immunological markers.&#x0D; Conclusion: the studies carried out have shown that the use of bacterial lysate is a pathogenetically substantiated component of therapeutic tactics. The normalizing effect of the lysate on the parameters of the immune response and cytokine profile has been reliably shown. Considering the immuno-inflammatory reaction at the experimental periodontitis, accompanied by the cytokine imbalance, study the pharmacological correction by bacterial lysate should continue from the point of view of establishing the mechanisms of development, protection, and restoration of tissues.&#x0D; &#x0D; It has been proved that by the end of the 90-day period of observation of the course of experimental periodontitis, the power of natural compensatory mechanisms acquired a state of decompensation. It has manifested by increased pro-inflammatory cytokine IL-1β in 1.7 times, and TNF-α in 3 times, decreased anti-inflammatory cytokine IL-10 in 1.6 times; increased the concentration of CIC in the blood in 2 times, C-reactive protein in 4.4 times at the local level in the gum tissue homogenate of the control pathology group of animals.&#x0D; The powerful corrective effect of bacterial lysates «Respibron» and «Imudon» on the indicators of the cytokine series on the model of chronic experimental periodontitis has been reliably established. The normalizing effect is due to the compensation of bacterial dysbiosis, restoration of the balance of the activity of pro- and anti-inflammatory cytokines, and a decrease in the hyperactivity of immunological markers.&#x0D; The therapeutic effect of «Respibron» (bacterial lysate of 13 strains of microorganisms) can be assessed as expressive and more powerful in comparison with «Imudon».&#x0D; &#x0D; The obtained results make it possible to continue the study of the course of experimental periodontitis from the point of view of establishing the mechanisms of development, protection, and restoration of tissues under conditions of pharmacological correction with bacterial lysate «Respibron».

Bacterial lysates (OM-85 BV): a cost-effective proposal in order to contrast antibiotic resistance.

SummaryBackgroundChronic Obstructive Pulmonary Disease (COPD) is one of the most frequent pathologies in which antibiotics are used because 50% of the exacerbations are attributable to a bacterial infection. The aims of our study were: i) to perform a meta-analysis on the efficacy of the bacterial lysate OM-85 BV in preventing acute exacerbations in patients with COPD; ii) to evaluate whether this preventive treatment can lead to significant savings for the National Health Service (NHS).MethodsA systematic research was conducted in the electronic database MEDLINE (PubMed) in June 2017-July 2020, collecting evidences without time restrictions. Only randomized controlled trials (RCTs) were included. The keywords used were “OM 85 BV AND chronic bronchitis” and “OM 85 BV AND COPD”. A cost-effectiveness analysis (CEA) was performed considering the costs for a treatment with OM-8BV, the costs for the treatment of an acute exacerbation and the number of prevented exacerbations.Results59 publications were found, but the meta-analysis was conducted on 13 studies that met the inclusion criteria. OM-85 BV is responsible of a statistically significant reduction in the mean number of COPD exacerbations (p < 0.01; WMD = -0.86; CI 95%: -1.38, -0.34) and in the days of antibiotic therapy (p < 0.01; WMD = -9.49; CI 95%: -11.93, -7.05). The cost-effectiveness ratio with a negative value is in favor to treatment.ConclusionsOM-85 BV is effective in reducing exacerbations, and could lead to significant savings for the NHS. Moreover, reducing the number of exacerbations it could avoid an over-use of antibiotics and the consequent antibiotic resistance.

Effective Use of Linear DNA in Cell-Free Expression Systems.

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

Bacterial lysate treatment in allergic disease: A systematic review and meta-analysis.

ObjectiveThe aim of this review was to assess the efficacy of bacterial lysate treatment in patients with allergic disease.MethodRandomized controlled trials (RCTs) of bacterial lysate therapy for patients with allergic diseases (asthma, atopic dermatitis, and allergic rhinitis) were searched using PubMed, EMBASE, Cochrane, China National Knowledge Infrastructure, Chinese Biomedical literature, and Wanfang databases up to March 2020. Based on the guidelines of the Cochrane collaboration, risk of bias was assessed.ResultsThis meta‐analysis based on 19 studies comparing bacterial lysate‐treated patients with a control group showed a 24% (RR: 1.24, 95% CI [1.19, 1.30]) increase in improvement of allergy symptom control. In addition, the improvement of asthma symptom control was 22% (RR: 1.22, 95% CI [1.14, 1.26]) higher in the bacterial lysate treatment group. Moreover, the levels of immunoglobulin (IgA and IgG), T lymphocyte subtype (CD3+, CD4+, CD4+/CD8+, Th1), and cytokines (IFN‐γ, IL‐2, and IL‐12) were increased in the treated group compared with controls. There was no significant difference in adverse event rate between the two groups.ConclusionTreatment with bacterial lysate improves symptom control in patients with allergic diseases on the basis of routine therapy. No adverse risk was found in this meta‐analysis.

STUDY OF THE INFLUENCE OF BACTERIAL LYSATE ON INDICATORS OF LYSOSOMAL PHOSPHATASE ACTIVITY IN EXPERIMENTAL PERIODONTITIS IN RATS

Introduction. One of the stages of the pathophysiological study of the parameters of mineral metabolism of alkaline (ALP) and acidic (AP) phosphatases – enzymes of bone tissue of osteoblast and osteoblast membranes in experimental periodontitis against the background of the use of bacterial lysate Respibron® was carried out. Materials and methods. Experimental periodontitis in rats was in the modification of O.I. Sukmansky. There were rats with experimental periodontitis and rats treated by Respibron (10 mg/kg 1 time per day) or Imudon for 30 days. Determination of alkaline (ALP) and acidic (AP) phosphatases activity (pH 4.8 and 10.5) was performed according to the methodological recommendations of AP Levitsky et al. Discussion of results. It was found that by the end of the 90-day period of the experiment, a statistically stable course of chronic damage to the periodontium was observed in terms of ALP and AP, which exceeded the intact ones by 1.93 and 1.4 times, respectively, which reflects the hypoergic state of chronic periodontal inflammation. A powerful corrective effect of the drugs Respibron® and Imudon® on markers of osteoblast and osteoblast activity in experimental periodontitis has been proven. Established a stimulating effect on osteoregeneration Respibron® in the hypoergic course of the inflammatory process. The therapeutic effect of Respibron® is more expressive and powerful compared to Imudon®. Conclusions.The results obtained make it possible to continue the study of the course of experimental periodontitis in order to establish the mechanisms of development, protection, and restoration of tissues under the conditions of the pharmacological correction Respibron®.

Use of the Bacterial Lysate OM-85 in the Paediatric Population in Italy: A Retrospective Cohort Study.

Background: In Italy, the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®) is registered for the prophylaxis of recurrent respiratory tract infections (RTIs) in adults and children above one year of age, but there are limited data on its use in the paediatric population. We aim to estimate the impact of OM-85 treatment on RTIs and antibiotic prescriptions in children. Methods: This study included children aged 1 to 14 years enrolled in Pedianet, a paediatric general practice research database, from January 2007 to June 2017, having at least one prescription of OM-85. Children with less than 12 months of follow-up before (PRE period) and after (POST period) the OM-85 prescription were excluded. The frequency of antibiotic prescriptions and the frequency of RTI episodes in the PRE and POST periods were compared through the post-hoc test. Subgroup analysis was performed in children with recurrent RTIs. Results: 1091 children received 1382 OM-85 prescriptions for a total follow-up of 619,525.5 person-years. Overall, antibiotic prescriptions decreased from a mean of 2.8 (SD (standard deviation) 2.7) prescriptions in the PRE period to a mean of 2.2 (SD 2.6) prescriptions in the POST period (p < 0.0001). RTIs decreased from a mean of 3.4 (SD 2.9) episodes in the PRE period to a mean of 2.5 (SD 2.6) episodes in the POST period (p < 0.0001). No change in antibiotic class was noted, and co-amoxiclav remained the preferred therapy in 28% of cases, followed by amoxicillin. These results were confirmed among children with recurrent RTIs. Conclusions: OM-85 is effective in preventing both antibiotic prescriptions and RTIs in children.

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1.

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B1 (AFB1) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB1 was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB1 were strongly affected by the metal ions in which Cu2+ stimulated the degradation and Zn2+ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB1, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.

Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions, the RNA is protected by viral capsid proteins, membrane proteins, and nucleocapsid proteins. To utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment in vitro. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids.

Spinal fluid IgG antibodies from patients with demyelinating diseases bind multiple sclerosis-associated bacteria

A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases (DD). The anti-microbial ELISA assays follow on prior human brain tissue RNA sequencing studies that established multiple sclerosis (MS) microbial candidates. Lysates included in the ELISA panel were derived from Akkermansia muciniphila, Atopobium vaginae, Bacteroides fragilis, Lactobacillus paracasei, Odoribacter splanchnicus, Pseudomonas aeruginosa, Cutibacterium (Propionibacterium) acnes, Fusobacterium necrophorum, Porphyromonas gingivalis, and Streptococcus mutans. CSF responses from patients with demyelinating diseases (DD, N = 14) were compared to those with other neurological diseases (OND, N = 8) and controls (N = 13). Commercial positive and negative control CSF specimens were run with each assay. ELISA index values were derived for each specimen against each of the 10 bacterial lysates. CSF reactivity was significantly higher in the DD group compared to the controls against Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Four of the 11 tested DD group subjects had elevated antibody indexes against at least one of the 10 bacterial species, suggesting intrathecal antibody production. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.Key messagesA panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases, including multiple sclerosis and acute disseminated encephalomyelitis.CSF reactivity was significantly higher in the demyelination group compared to the controls against the bacteria Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium.Several of the demyelination subjects had elevated antibody indexes against at least one of the 10 antigens, suggesting at least limited intrathecal production of anti-bacterial antibodies.This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.

Immunomodulation options and frequency of respiratory infections in children

Frequent respiratory tract infections in the paediatric population are associated mainly with immune system immaturity. A child aged up to 5 years can have up to 8 respiratory infections in a year. Immunomodulation through the elimination of negative environmental factors is of limited efficacy in the paediatric population. For this reason, in certain cases there is a need for pharmacological stimulation of immune response mechanisms in order to limit the number of infections. Modulators include medication, vitamins and trace elements, probiotics and plant formulations. Unfortunately, there are insufficient scientific data which confirm the efficacy of many such agents. Bacterial lysates are an exception: their effects have been confirmed in well-designed studies. Such formulations consist of killed bacterial cells or fragments of their organelles. Bacterial lysates contain antigens of the most common human respiratory pathogens. The efficacy of a bacterial lysate is the resultant of two components: patient-specific (individual immune reactivity) and formulation-specific (composition, dosing regime). To date, no reliable studies comparing the efficacy of different formulations have been conducted. One of the most thoroughly studied products is the one containing OM-85 lyophilisate (BV, Broncho-Vaxom®).

STUDY OF THE INFLUENCE OF BACTERIAL LYSATE ON INDICATORS OF LYSOSOMAL PHOSPHATASE ACTIVITY IN EXPERIMENTAL PERIODONTITIS IN RATS

Introduction. One of the stages of the pathophysiological study of the parameters of mineral metabolism of alkaline (ALP) and acidic (AP) phosphatases – enzymes of bone tissue of osteoblast and osteoblast membranes in experimental periodontitis against the background of the use of bacterial lysate Respibron® was carried out. Materials and methods. Experimental periodontitis in rats was in the modification of O.I. Sukmansky. There were rats with experimental periodontitis and rats treated by Respibron (10 mg/kg 1 time per day) or Imudon for 30 days. Determination of alkaline (ALP) and acidic (AP) phosphatases activity (pH 4.8 and 10.5) was performed according to the methodological recommendations of AP Levitsky et al. Discussion of results. It was found that by the end of the 90-day period of the experiment, a statistically stable course of chronic damage to the periodontium was observed in terms of ALP and AP, which exceeded the intact ones by 1.93 and 1.4 times, respectively, which reflects the hypoergic state of chronic periodontal inflammation. A powerful corrective effect of the drugs Respibron® and Imudon® on markers of osteoblast and osteoblast activity in experimental periodontitis has been proven. Established a stimulating effect on osteoregeneration Respibron® in the hypoergic course of the inflammatory process. The therapeutic effect of Respibron® is more expressive and powerful compared to Imudon®. Conclusions.The results obtained make it possible to continue the study of the course of experimental periodontitis in order to establish the mechanisms of development, protection, and restoration of tissues under the conditions of the pharmacological correction Respibron®.

Contribution of Heptose Metabolites and the cag Pathogenicity Island to the Activation of Monocytes/Macrophages by Helicobacter pylori.

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose–dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori–colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.