General dynamics of the URT microbiome and microbial signs of recovery in COVID-19 patients.

COVID-19 is caused by an airborne virus, SARS-CoV-2. The upper respiratory tract (URT) is, therefore, the first system to endure the attack. Inhabited by an assemblage of microbial communities, a healthy URT wards off the invasion. However, once invaded, it becomes destabilised, which could be crucial to the establishment and progression of the infection. We examined 696 URT samples collected from 285 COVID-19 patients at three time-points throughout their hospital stay and 100 URT samples from 100 healthy controls. We used 16S ribosomal RNA sequencing to evaluate the abundance of various bacterial taxa, α-diversity, and ß-diversity of the URT microbiome. Ordinary least squares regression was used to establish associations between the variables, with age, sex, and antibiotics as covariates. The URT microbiome in the COVID-19 patients was distinctively different from that of healthy controls. In COVID-19 patients, the abundance of 16 genera was significantly reduced. A total of 47 genera were specific to patients, whereas only 2 were unique to controls. The URT samples collected at admission differed more from the control than from the samples collected at later stages of treatment. The following four genera originally depleted in the patients grew significantly by the end of treatment: Fusobacterium, Haemophilus, Neisseria, and Stenotrophomonas. Our findings strongly suggest that SARS-CoV-2 caused significant changes in the URT microbiome, including the emergence of numerous atypical taxa. These findings may indicate increased instability of the URT microbiome in COVID-19 patients. In the course of the treatment, the microbial composition of the URT of COVID-19 patients tended toward that of controls. These microbial changes may be interpreted as markers of recovery.

Modified Estrogen-Progesterone Induction Method of Mammary Gland Hyperplasia in Rats.

In female Wistar rats, mammary gland hyperplasia (MGH) was modeled according to a modified protocol involving estrogen-progesterone induction and taking into account the duration of the estrous cycle of this animal species. MGH was induced over four 7-day cycles; each cycle included subcutaneous administration of 17ß-estradiol (0.5 mg/kg) for 4 days, injection of progesterone (5 mg/kg) on day 5, then 2 days without injections. In females with MGH, a significant increase in the height and diameter of the nipples of the mammary glands was recorded, two types of changes were observed in the gland tissue: tubuloalveolar and lobuloalveolar hyperplasia. The study confirmed the development of MGH in rats by a modified method.

Induction of cross-reactive, mucosal anti-SARS-CoV-2 antibody responses in rheumatoid arthritis patients after 3rd dose of COVID-19 vaccination.

Systemic vaccination against SARS-CoV-2 elicited high titers of specific antibodies in the blood and in the oral cavity. Preexisting autoimmune diseases, such as rheumatoid arthritis, and biological treatments, like B cell depletion, are known to exhibit higher risk of severe COVID-19 manifestation and increased frequency of breakthrough infections after vaccination. We hypothesized that such increased risk is associated with an aberrant induction of secreted antibodies in the oral cavity. Here we evaluated the levels of secreted antibodies in the oral cavity against the SARS-CoV-2 Spike protein during the course of vaccination in RA patients with or without B cell depletion. We found that total salivary IgG levels were correlated with number of B cells in the blood. Anti-Spike IgG responses 7 days after second vaccination were induced in the oral cavity of all healthy individuals, while only 6 out 23 RA patients exhibited anti-Spike IgG in their saliva regardless of B cell depleting therapy. Importantly, both salivary and serologic anti-Spike IgG and IgA responses towards WT and omicron Spike variants were efficiently induced by third vaccination in RA patients with or without B cell depletion to the levels that were similar to healthy individuals. Altogether, these data advocate for the necessity of three dose vaccination for RA patients to mount anti-Spike antibody responses at the mucosal surfaces and annotate the reduction of secreted salivary IgG by B cell depletion.

[Iron deficiency syndromes and their correction with nutritional support in elderly patients with colo-rectal cancer.]

51 patients with operated colorectal cancer T1N0M0, T3N1M0 and T4N0M1 at the age of 67±2,3 years receiving adjuvant chemotherapy and nutritional support (NS) were examined. Nutritional status was assessed using alimentazione-volume diagnosis to the points on L.N.Kostyuchenko, nutritional risk - with NRI, body composition - with bioimpedance method, the iron metabolism - with basic markers (serum ferritin, transferrin saturation with iron, erythrocyte indices: erythrocyte saturation of iron, the average concentration of hemoglobin in the erythrocyte, mean corpuscular volume, hemoglobin, etc.), traditional settings, the staging of tumors - according to TNM. Iron deficiency before the development of anemia can be corrected with NS. Upon the occurrence of anemia requires additional pharmacological treatment iron supplementation, preferable with iron III hydroxide olygoisomaltazat 1000 + NS) for the prevention of toxic-metabolic complications.

Cytokines as Mediators of Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Cytokines play a pivotal role in maintaining homeostasis of the immune system and in regulation of the immune response. Cytokine dysregulation is often associated with development of various pathological conditions, including autoimmunity. Recent studies have provided insights into the cytokine signaling pathways that are involved not only in pathogenesis of autoimmune neuroinflammatory disorders, such as multiple sclerosis, but also in neurodegenerative states, for example, Alzheimer's disease. Understanding the exact molecular mechanisms of disease pathogenesis and evaluation of relevant experimental animal models are necessary for development of effective therapeutic approaches.

[Auditory evoked potentials in children with auditory neuropathy spectrum disorder]. / Slukhovye vyzvannye potentsialy u detei s zabolevaniem spektra auditornykh neiropatii.

The objective of the present study was to estimate peculiarities of the auditory brainstem evoked potentials (ABR), auditory steady-state responses (ASSR) and cortical auditory evoked potentials (CAEP) in the children presenting with bilateral auditory neuropathy spectrum disorder (ANSD). The study included 100 patients with bilateral ANSD diagnosed based on the positive response of otoacoustic emissions (OAEs) and/or cochlear microphonic (CM) detection, while no synchronous neural activity was detected in the ABR test. Cochlear microphonic was the main clue for the ANSD diagnosing, because OAE was absent in both ears of 49 children. ABR testing revealed no response bilaterally in 72 cases (out of 100). In contrast to ABR, the ASSR thresholds were detectable at all the four main frequencies in both ears in 73 % of the cases (47 out of the 64 tested ones). Both ABR and ASSR in most cases were incomparable with the behavioral audiometric thresholds. 28 children underwent CAEP testing. In 7 cases out of 8 with mild hearing loss detectable CAEP were recorded. CAEP registration in l7 children making use of the hearing aids and in 3 children after cochlear implantation revealed, in the majority of the cases, the concordance between CAEP detectability with behavioral thresholds and rehabilitation outcomes with fairly good speech intelligibility. It is concluded that the ABR registration with CM evaluation is the most informative test for ANSD diagnosis. However, ABR as well as ASSR is useless for the estimation of the behavioral thresholds. The results of this study suggest that the presence or absence of CAEPs can provide some indication of the audibility of a speech sound in the children with ANSD; however this method requires further investigation.

Inhibitory effect of streptococci on the growth of M. catarrhalis strains and the diversity of putative bacteriocin-like gene loci in the genomes of S. pneumoniae and its relatives.

S. pneumoniae is a facultative human pathogen causing a wide range of infections including the life-threatening pneumoniae or meningitis. It colonizes nasopharynx as well as its closest phylogenetic relatives S. pseudopneumoniae and S. mitis. Both the latter, despite the considerable morphological and phenotypic similarity with the pneumococcus, are considerably less pathogenic for humans and cause infections mainly in the immunocompromized hosts. In this work, we compared the inhibitory effect of S. pneumoniae and its relatives on the growth of Moraxella catarrhalis strains using the culture-based antagonistic test. We observed that the inhibitory effect of S. mitis strains is kept when a hydrogen peroxide produced by cells is inactivated by catalase, and even when the live cells are killed in chloroform vapors, in contrast to the pneumococcus whose inhibiting ability disappeared when the cells die. It was suggested that this effect may be due to the production of bacterial antimicrobial peptides by S. mitis, so we examined the genomes of our strains for the presence of bacteriocin-like peptides encoding genes. We observed that a set of bacteriocin-like genes in the genome of S. mitis is greatly poorer in comparison with S. pneumoniae one; moreover, in one S. mitis strain we found no bacteriocin-like genes. It could mean that there are probably some additional opportunities of S. mitis to inhibit the growth of competing neighbors which are still have to be discovered.

[Analysis of the Specificity of IgA Antibodies Produced in the Mouse Small Intestine].

Intestinal microbiota controls multiple aspects of body homeostasis. The microbiota composition changes easily in response to internal or external factors, which may result in dysbiosis and associated inflammatory reactions. Thus, maintaining the microbiota composition by the host immune system is crucial, and one of the main mechanisms for microbiota control is production of immunoglobulin A (IgA) at mucosal surfaces. The molecular mechanisms regulating the interactions between the immune system and microbiota remain obscure. A panel of hybridoma cell lines was constructed to produce monoclonal IgA antibodies specific to various commensal bacteria present in intestinal microbiota. The panel can be used to further understand the mechanisms whereby the adaptive immune system controls the microbiota composition.

[Cytokine neutralization at specific cellular source : A new therapeutic paradigm? German Version]. / Zytokinneutralisierung an spezifischen zellulären Quellen : Ein neues Therapiemodell?

BACKGROUND: Currently, treatment of autoimmune diseases is based on manipulation of general control mechanisms, including those mediated by immunoregulatory cytokines. This approach is non-curative and may cause unwanted side effects due to numerous beneficial and non-redundant functions of a particular cytokine. METHODS: Techniques of reverse genetics, such as conditional gene targeting, were employed to uncover the contributions of two proinflammatory and immunomodulatory cytokines, tumour necrosis factor (TNF) and interleukin 6 (IL-6), in various disease states. RESULTS: Several non-redundant functions of TNF from distinct cellular sources were identified. TNF from myeloid cells is pathogenic in several autoimmune diseases, whereas TNF produced by T cells showed non-redundant protective functions in experimental arthritis and in a Mycobacterium tuberculosis infection model. To test the idea of selective pharmacological inhibition of "bad" TNF produced by myeloid cells while sparing "good" TNF produced by T lymphocytes, a myeloid-specific TNF inhibitor (MYSTI) was designed - a recombinant mini-antibody with dual specificity that can bind to the surface molecule F4/80 on myeloid cells and to TNF. In vitro experiments confirmed retention of TNF on the surface of TNF-producing cells and in vivo experiments indicated that MYSTI can protect mice from lethal TNF-mediated hepatotoxicity. MYSTI is also effective in experimental arthritis. CONCLUSION: The proposed therapeutic strategy may be more effective than systemic anti-cytokine therapy in several human autoimmune diseases, as it would preserve the potentially beneficial effects of the same cytokine produced by other cell types. Such bispecific biological agents may become interesting tools for experimental studies and, eventually, drug development.

Cytokine neutralization at specific cellular source : A new therapeutic paradigm?

BACKGROUND: Currently, treatment of autoimmune diseases is based on manipulation of general control mechanisms, including those mediated by immunoregulatory cytokines. This approach is non-curative and may cause unwanted side effects due to numerous beneficial and non-redundant functions of a particular cytokine. METHODS: Techniques of reverse genetics, such as conditional gene targeting, were employed to uncover the contributions of two proinflammatory and immunomodulatory cytokines, tumour necrosis factor (TNF) and interleukin 6 (IL-6), in various disease states. RESULTS: Several non-redundant functions of TNF from distinct cellular sources were identified. TNF from myeloid cells is pathogenic in several autoimmune diseases, whereas TNF produced by T cells showed non-redundant protective functions in experimental arthritis and in a Mycobacterium tuberculosis infection model. To test the idea of selective pharmacological inhibition of "bad" TNF produced by myeloid cells while sparing "good" TNF produced by T lymphocytes, a myeloid-specific TNF inhibitor (MYSTI) was designed-a recombinant mini-antibody with dual specificity that can bind to the surface molecule F4/80 on myeloid cells and to TNF. In vitro experiments confirmed retention of TNF on the surface of TNF-producing cells and in vivo experiments indicated that MYSTI can protect mice from lethal TNF-mediated hepatotoxicity. MYSTI is also effective in experimental arthritis. CONCLUSION: The proposed therapeutic strategy may be more effective than systemic anti-cytokine therapy in several human autoimmune diseases, as it would preserve the potentially beneficial effects of the same cytokine produced by other cell types. Such bispecific biological agents may become interesting tools for experimental studies and, eventually, drug development.

Microbiota Induces Expression of Tumor Necrosis Factor in Postnatal Mouse Skin.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates many important processes in the body. TNF production in a physiological state supports the structure of lymphoid organs and determines the development of lymphoid cells in hematopoiesis. However, chronic TNF overexpression leads to the development of various autoimmune disorders. Sites of TNF production in the naïve state remain unclear due to the lack of in vivo models. In the present study, we used TNF-2A-Kat reporter mice to monitor the expression of TNF in different tissues. Comparative analysis of tissue fluorescence in TNF-2A-Kat reporter mice and wild type mice revealed constitutive expression of TNF in the skin of naïve adult mice. In the skin of TNF-2A-Kat reporter mouse embryos, no statistically significant differences in the expression of TNF compared to wild type animals were observed. Furthermore, we established that local depletion of microflora with topical antibiotics leads to a reduction in the fluorescence signal. Thus, we assume that the skin microflora is responsible for the expression of TNF in the skin of mice.

[Antibiotic Resistance of MRSA in the Russian Federation].

The results of the multicentre trial on estimation of MRSA antibiotic susceptibility to 17 antibiotics are presented. 474 nonrepeting isolates of MRSA (mecA+), collected in 2011-2014 in 10 cities of the Russian Federation were used in the trial. The antibiotic susceptibility was determined by the method of serial microdilutions in broth with estimation of the MICs in accordance with the international standards CLSI 2014 and EUCAST 2014. The highest levels of the MRSA resistance were stated against ciprofloxacin--92%(MIC50 32 mcg/ml), gentamicin--85% (MIC50 128 mcg/ml), erythromycin--54% (MIC50 32-mcg/ml) and clindainycin - 45% (MIC50 0.03 mcg/ml), as well as against rifampicin--38% (MIC50 0.06 mcg/ml). The frequency of MRSA isolated at the vancomycin dose of 2 mcg/ml equaled 26%. No correlation of the decrease in susceptibility to vancomycin and rifampicin was observed. In 5% of MRSA isolated from infected surgical wounds in patients with bone infection or sepsis, there was observed a decrease in the susceptibility to ceftarolin (MIC 2-4 mcg/ml). Co-trimoxasole, fusidic acid (MIC50 0.06 mcg/ml) and mupirocin (MIC50 0.5 mcg/ml) showed high antibacterial activity, 93-98% of the isolates being susceptible to the drugs. No resistance to linezolid and tigecycline was detected. By the associate resistance spectrum, most of the MRSA isolates were characterized by resistance to drugs of 3-7 groups (56%). The phenotypes with simultaneous resistance to drugs of 8-10 groups amounted to 6%. As a whole, 70 variants of associate resistance combinations were detected.

[Antibiotic Resistance of Coagulase-Negative Staphylococci Isolated at Hospitals of St. Petersburg and Moscow].

Antibiotic susceptibility of 119 coagulase-negative staphylococci isolated at hospitals of St. Petersburg and Moscow was investigated and estimated at the local laboratories as oxacillin resistant. The following species were identified: Staphylococcus epidermidis, S. haemolyticus, S. hominis, S.capitis, S. simulans, S. pettenkoferi, S. lentus, S. carnosus and S. warneri. The oxacillin resistance was confirmed in 79.8% of the isolates. The frequency of the associated resistance to non-beta-lactams was much higher in the oxacillin resistant isolates vs. the oxacillin susceptible ones. When the CLSI and EUCAST susceptibility criteria were used, 1-3% difference in the resistance levels was recorded. Among the oxacillin resistant isolates the frequency of resistance to gentamicin, ciprofloxacin, erythromycin, moxifloxacin, tetracycline and clindamycin equaled 90, 88, 88, 63, 43 and 26% respectively. Two linezolid resistant isolates of S. epidermidis with lower susceptibility to tedizolid were isolated. Eight isolates of S. epidermidis showed lower resistance to mupirocin. The MIC of ceftarolin for oxacillin resistant coagulase-negative staphylococci varied from 0.5 to 2.0 mcg/ml, while for the oxacillin susceptible ones it was lower than 0.25 mcg/ml. No resistance to tigecyclin and vancomycin was observed.

An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina.

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1ß, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1ß was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1ß. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1ß.

[Long-term adaptation of the patients to electrical stimulation in the course of rehabilitation following cochlear implantation].

The objective of the present work was to study long-term adaptation of the patients to electrical stimulation in the course of rehabilitation following cochlear implantation. The following parameters were determined: variations of threshold auditory sensation of electrical stimulation (ESS), alteration of the maximum electrical stimulation comfort level (MCL), changes in the dynamic range (DR) of the stimulus and changes in the threshold auditory nerve action potentials (ANAP). The observations on ESS dynamics and DR of the stimulus were collected during 2 years after the first connection of the processor in a group of 27 patients. The data obtained from the 3rd, 12th, and 20th stimulation channels were included in the study. The observations on ANAP dynamics were collected during 1 year after surgery in a group of 112 patients. It was shown that ESS variations largely occurred within the first two weeks after connection and did not reach statistical significance. In contrast, MCL and DR changes showed a stably increasing tendency throughout the entire period of observations, with the enhancement of MCL of DR amounting to 25-28% and roughly 50% of the initial values respectively. Threshold auditory sensation of electrical stimulation significantly decreased by the time of the first connection of the implant compared with the surgical data. Further changes in threshold ESS were relatively small and did not reach statistical significance.

Microbiota, intestinal immunity, and mouse bustle.

The composition of the intestinal microbiota is regulated by the immune system. This paper discusses the role of cytokines and innate immunity lymphoid cells in the intestinal immune regulation by means of IgA.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10-C12) than those in lipid A from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.

Modern anti-cytokine therapy of autoimmune diseases.

The emergence of genetically engineered biological agents opened new prospects in the treatment of autoimmune and inflammatory diseases. Cytokines responsible for regulation of a wide range of processes during development of the normal immune response are among the most successful therapeutic targets. Studies carried out in recent decades and accompanied by rapid development of biotechnology have promoted establishing in detail the role and place of cytokines in autoimmune and inflammatory pathologies. Nevertheless, mechanisms that underlie anti-cytokine therapy are still not fully understood. This review examines the role of such cytokines as TNF, IL-1, and IL-6 in the development of inflammatory processes and the action mechanisms of their inhibitors.

Experimental models of arthritis in which pathogenesis is dependent on TNF expression.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint damage as well as systemic manifestations. The exact cause of RA is not known. Both genetic and environmental factors are believed to contribute to the development of this disease. Increased expression of tumor necrosis factor (TNF) has been implicated in the pathogenesis of RA. Currently, the use of anti-TNF drugs is one of the most effective strategies for the treatment of RA, although therapeutic response is not observed in all patients. Furthermore, due to non-redundant protective functions of TNF, systemic anti-TNF therapy is often associated with unwanted side effects such as increased frequency of infectious diseases. Development of experimental models of arthritis in mice is necessary for studies on the mechanisms of pathogenesis of this disease and can be useful for comparative evaluation of various anti-TNF drugs. Here we provide an overview of the field and present our own data with two experimental models of autoimmune arthritis - collagen-induced arthritis and antibody-induced arthritis in C57Bl/6 and BALB/c mice, as well as in tnf-humanized mice generated on C57Bl/6 background. We show that TNF-deficient mice are resistant to the development of collagen-induced arthritis, and the use of anti-TNF therapy significantly reduces the disease symptoms. We also generated and evaluated a fluorescent detector of TNF overexpression in vivo. Overall, we have developed an experimental platform for studying the mechanisms of action of existing and newly developed anti-TNF drugs for the treatment of rheumatoid arthritis.

[Search for new modes of antibiotic prophylaxis of septic complications after percutaneous nephrolithotripsy].

The risk of infectious and inflammatory complications after PNL is based on presence of microorganisms in the form of biofilms inside the stone. Destruction of stones during surgery or lithotripsy may be a trigger for the growth of microorganisms that are integrated into the biofilms, and the migration of bacteria and their toxins in the blood flow under pressure of irrigation fluid can cause septic complications. The danger of infectious and inflammatory complications after percutaneous interventions for kidney stones requires a search for specific antibiotics for antimicrobial prophylaxis and efficient modes of their administration. The results of a comparative study of pharmacokinetic parameters of ciprofloxacin, and the effectiveness of two modes of administration (bolus dosing and prolonged intravenous administration) at a dose of 1000 mg are presented.