MONOCYTE SUBSETS IN HEALTHY ADULTS AND SEPSIS PATIENTS

Monocytes play a key role in the development of immune response in bacterial infection, because of their phagocytic, antigen-presenting and secretory functions. There are three subpopulations of monocytes: “classical” CD14+CD16-, “intermediate” CD14+CD16+, and “nonclassical” CD14+dimCD16+. These monocyte subtypes have different phenotypes and functions. The ratio of appropriate subpopulations varies with development of the antibacterial response. The aim of the present research was to study phenotypes of the monocyte subpopulations in the patients with sepsis, and changes in the monocyte subpopulation ratio, depending on the presence of bacteria in circulating blood of the patients, as well as to estimate contribution of the monocyte subpopulations to the cytokine production. We observed 16 patients with sepsis (10 men and 6 women; mean age, 58±14 years, SOFA 9.4±2.1; a total of 44 blood samples) examined in dynamics. The control group included healthy adults (n = 23, 12 men and 11 women; mean age, 51±13 years). Laboratory studies included bacteriological cultures, determination of absolute and relative numbers of subpopulations of classical, intermediate and non-classical monocytes and their expression of HLA-DR and CD64, determination of IL-6, TNFα, IL-1β, IL-10 concentration in blood serum. Absolute number of monocytes was increased in the sepsis patients, the ratio of classical monocytes was also increased, like as relative and absolute numbers of  intermediate cells. Meanwhile, the subpopulation of non-classical monocytes did not change significantly. The monocyte subpopulation ratio depended on the presence of bacteria in blood, i.e., a higher proportion of intermediate cells was observed in the samples positive for bacteria in blood cultures. The ratio of subpopulations was restored after elimination of bacteria from the circulation. The expression density of LPS receptor (CD14), IgG receptors (CD16 and  CD64) was found to be increased, especially in the subpopulations of intermediate and nonclassical monocytes. In all subpopulations of monocytes, expression of HLA-DR is reduced, most notably in classical monocytes, least in intermediate cells. There was a significant increase in serum levels of IL-6, IL-1β, TNFα and IL-10 cytokines. Direct correlation between the absolute number of classical monocytes and IL-6 concentration was revealed, as well as intensity of multiple organ dysfunction. Increase in absolute amount of classical monocytes and IL-6 concentration might serve as an indirect criterion for evaluation of endothelial activation, an active producer of IL-6 and myeloid cell growth factors. A direct correlation between the percentage of CD14+CD16+ cells and IL-10 concentration in blood serum indicates to an important role of intermediate monocytes in IL-10 production. IL-10 suppresses the antigen-presenting function of intermediate cells, namely, expression of HLA-DR molecules, as suggested by inverse correlation between the IL-10 concentration and HLA-DR expression density on CD14+CD16+ cells. We have also determined an inverse correlation between the degree of multi-organ dysfunction  and relative amount of HLA-DR+ monocytes. The larger was a classical monocyte subpopulation, the more noticeable was a decrease of this index. The studies in ratios of monocyte subpopulations help to understand the mechanisms of antibacterial protection in sepsis. Monitoring of classical monocyte numbers and serum concentrations of IL-6 is necessary for a comprehensive assessment of inflammatory response in sepsis. Determination of HLADR expression on monocytes allows us to evaluate the intensity of immune suppression in critically ill patients

Sensitivity Optimization and Experimental Study of the Long-Range Metal Detector Based on Chaotic Duffing Oscillator

Sensors based on chaotic oscillators have a simple design, combined with high sensitivity and energy efficiency. Among many developed schemes of such sensors, the promising one is based on the Duffing oscillator, which possesses a remarkable property of demonstrating chaotic oscillations only in the presence of a weak sine wave at the input. The main goal of this research was to evaluate the maximal sensitivity of a practically implemented metal detector based on the Duffing oscillator and compare its sensitivity with conventional sensors. To achieve high efficiency of the Duffing-based design, we proposed an algorithm which performs a bifurcation analysis of any chaotic system, classifies the oscillation modes and determines the system sensitivity to a change in different parameters. We apply the developed algorithm to improve the sensitivity of the electronic circuit implementing the Duffing oscillator, serving as a key part of a three-coil metal detector. We show that the developed design allows detecting the presence of metal objects near the coils more reliably than the conventional signal analysis techniques, and the developed detector is capable of sensing a large metal plate at distances up to 2.8 of the coil diameter, which can be considered a state-of-the-art result. © 2022 by the authors

A Model for the Development of the Rhizobial and Arbuscular Mycorrhizal Symbioses in Legumes and Its Use to Understand the Roles of Ethylene in the Establishment of these two Symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis–outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants

THE NEW METALL-BETA-LACTAMASE’S INHIBITOR EFFICACY IN A MODEL SYSTEM IN VITRO

The Enterobacteriaceae antibiotics resistance depends on a combination of several mechanisms, such as the beta-lactamases overproduction, the microbial cell reduction outer membrane permeability (usually associated with loss of protein porin), the presence of efflux systems. Particularly noteworthy are the metallo-beta-lactamases (MBL) whose presence causes resistance of gram-negative microorganisms to all beta-lactam antibiotics (in some cases except aztreonam). Currently there are no MBL inhibitors permitted for use in the clinic. The effective inhibitors search for carbapenem-resistant bacteria’ MBL authorized for use in the clinic and reinforcing effects of carbapenems, served as the basis for the present study. The work was carried out in three stages: 1) creating a model system using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, to evaluate the increasing of minimal inhibitory concentrations (MIC) of carbapenems against previously sensitive Gram-negative microorganisms strains in vitro; 2) evaluation of MBL promising inhibitors in the presence of the same standard enzyme reagent; 3) evaluation of the ability of the identified inhibitors increase the carbapenems effects against clinical isolates of Gram-negative microorganisms producing MBL, in terms of the their MIC and fractional inhibitory concentration index (FIC index). The checkerboard array was modified to evaluate the combined use of carbapenems and potential MBL inhibitor — a drug from the group of bisphosphonates — etidronic acid. Using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, we created a model system that allows to assess the prospects of new inhibitors MBL gram-negative microorganisms. A dose-dependent effect of increasing the meropenem level MIC from reagent MBL quantity in a model system against previously antibiotic sensitive reference strains of microorganisms was revealed. MBL enzyme inactivation was noted in the presence of even small doses of bisphosphonate, in the tests the appearance of logarithmic phase of P. aeru ginosa ATCC 27853 growth was shown delayed up to 12 hours compared to the control. In this case the maximum dose of etidronic acid 50 000–100 000 μg/ml completely inhibited the MBL, there was no a log phase microbe’s growth due to the effect of meropenem on the reference level of sensitivity (2 μg/ml). The synergistic effect (FIC index < 0.5) of combined meropenem with etidronic acid use was identified against clinical isolates Gram-negative microorganisms resistant to carbapenems and producing MBL, wherein the enhancing action of the antibiotic was more 8–512 times compared with the initial MIC levels

TIME-KILL ASSAY: AN EFFICACY OF SYNERGY BETWEEN CARBAPENEMS AND CLODRONIC ACID

Abstract. Currently, a search for augmenting antibiotics activity is still crucial due to elevated frequency of detecting carbapenem-resistant Gran-positive bacterial isolates. To resolve this, it might be reasonable to combine carbapenems metal-â-lactamase (MâL) inhibitors. Unfortunately, no MâL inhibitors approved for treatment of carbapenem-resistant infections are currently available. Pathogenic bacteria may survive antibiotic attack, exert tolerance and persistence accompanied with the ongoing infectious process. In connection with this, determining dependence between antimicrobialrelated bactericidal effect and exposure time on microbes at 4, 8, 12 and 24 hours after the onset, a so called time-kill assay, is necessary. A synergy between both agents was noted upon reduced microbial population by ≥ 3 log10. A checkerboard array followed by seeding the microplate well contents onto a dense nutrient medium at various time points were used to assess a synergistic efficacy of carbapenems applied together with clodronic acid against MâL-producing VIMgenotype P. aeruginosa 532/14 clinical isolate obtained from patients with infectious complications (minimal inhibitory concentrations [MIC] for imipenem or meropenem were 512 μg/ml), microbial burden 106 CFU/ml. Optical density was measured at two wavelengths (490 and 630 nm) in ELx800 reader, within 4–24 hour exposure time to determine time of logarithmic growth phase emerging in test culture. It is noteworthy that magnitude of optical density is a difference between two bichromatic measurements resulting in remarkably reduced inaccuracy due to scratches or fingerprints left on the plate. It was found that clodronic acid exhibited a synergic bactericidal effect with carbapenems against a clinically resistant MâL-producing VIM-genotype P. aeruginosa 532/14 strain. Upon that, imipenem-related antimicrobial activity was evident as early as 8 hours after the onset decreasing cell growth down to 1.4 log10 compared to control, whereas 12 hours later it resulted in total inhibition of test strain by decreasing growth of the test strain by 6 log10. Meropenem in combination with clodronic acid showed a more pronounced activity: complete absence of P. aeruginosa 532/14 growth by 8 hours of incubation, growth suppression by 3.2 log10, which reached 6 log10 12–24 hours after the onset. Time-kill assay allows to identify efficient combinations of carbapenems and MâL inhibitors, which is of great importance for increasing therapeutic efficacy of patients with severe purulent-septic complications