[The association of aromatic microbial metabolites, inflammatory and autoimmune biomarkers with clinical dynamics in severe diseases of the central nervous system]. / Svyaz' aromaticheskikh mikrobnykh metabolitov, vospalitel'nykh i autoimmunnykh biomarkerov s klinicheskoi dinamikoi pri tyazhelykh zabolevaniyakh tsentral'noi nervnoi sistemy.

OBJECTIVE: To identify the association between the changes in the profile of microbial metabolites, inflammatory and autoimmune markers and the dynamics of neurological status in chronic critically ill patients with diseases of the central nervous system (CNS). MATERIAL AND METHODS: Sixty serum samples from 37 patients, aged 19-77 years, with severe CNS diseases were studied. The changes in clinical condition were assessed with NIHSS, the Rankin scale, the Glasgow Coma Scale, the FOUR Coma Scale and the Rivermead Mobility Index. The levels of aromatic microbial metabolites (AMM) and several inflammatory and autoimmune biomarkers, including the contents of procalcitonin (PCT) and S100, the activity of leukocyte elastase (LE) and a1-proteinase inhibitor a1-PI, the levels of autoantibodies to S100b and MBP were measured. Serum from 60 age- and sex-matched healthy people with no signs of neurological and somatic pathology was used as a control. RESULTS: All patients were divided into groups depending on the neurological dynamics: A - positive (n=16), B - without dynamics (n=15), C - negative (n=6). The study revealed a profile of AMM, as well as inflammatory and autoimmune biomarkers associated with the severity of neurological disorders. A significant increase in acute phase proteins, S-100 level and a decrease in the functional activity of neutrophils (via LE activity) were observed in the serum of patients. The different dynamics of neurological status was associated with the multidirectional changes in the microbial metabolites profile and biomarkers. The correlations between the clinical and biological parameters indicate that AMM might modulate immune reactions in patients with different dynamics of neurological status. CONCLUSION: The results suggest the involvement of AMM and the level of immune activation via biomarkers in the pathogenesis of neurological dysfunction. Dynamic changes in the profile of microbial metabolites and the level of activation of the immune system may be a promising tool for prediction of neurological recovery.

Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

[PARTICIPATION OF AROMATIC MICROBIAL METABOLITES IN THE DEVELOPMENT OF SEVERE INFECTION AND SEPSIS.]

Background Actuality of the problem of infection in critically ill patients remains high. Addition a local infectious processes have a tendency to rapid generalization and sepsis. Neutrophilsfunction failure plays a key role in the patho- genesis of infectious complications and sepsis. The hypothesis about the role of microbial metabolites in the formation of immunosuppression, in particular in the inhibition of phagocytic activity of neutrophils as the first line of defense against bacteria is acquiring more and more supporters. Among the microbial metabolites aromatic compounds, such as phenylcarbinol acid (PCA) are of particular interest. Their concentration in the blood reaches a maximum in case of generalized infection, and correlates with mortality in patients in critical conditions, and PCA biological activity confirmed by earlier researches in vitro. The purpose of the study was to reveal the relationship of the PCA with the severity of bacterial infections and to assess the effect of PCA on neutrophils phagocytic activity. MATERIALS AND METHODS: At the first stage the levels of PCA (GCh-FID method) in the serum ofpatients (n=57) with a documented bacterial infection of varying severity, confirmed APACHE II and SOFA scales were analyzed comparing with healthy donors (n=72). During the second stage the effect of clinically-relevant concentrations of PCA on neutrophils phagocytic activity ofperipheral blood was analyzed in vitro. RESULTS: The levels ofphenylacetic (PAA), phenyllactic (PLA), hydroxyphenylacetic (p-HPAA) and hydroxyphenyllactic (p-HPLA) acids in patients were significantly higher than in healthy donors, reaching the highest values in severe infection. High direct correlation of PCA levels with the indicators ofscales APACHE II and SOFA was detected. A significant decrease in the number ofphagocytic neutrophils under the influence PAA, p-HPAA and PLA was identified in vitro experiment on average 11%. The most significant influence p-HPAA, PLA and p-HPLA have made on the intensity of the neutrophils absorption capacity reducing this parameter by an average of 26%. Also noted that phenylpropionic acid (PPC) which is in blood of healthy donors, disappears in critical ill patients. CONCLUSION: Phenolcarbonic acids level in the blood serum shows the severity of bacterial inflammatory process. Ex- periment in vitro shows that the PCA in clinically-relevant concentrations is able to suppress the neutrophilsphagocytic activity.

Normal level of sepsis-associated phenylcarboxylic acids in human serum.

Previous studies showed that large amounts of phenylcarboxylic acids (PhCAs) are accumulated in a septic patient's blood due to increased endogenous and microbial phenylalanine and tyrosine biotransformation. Frequently, biochemical aromatic amino acid transformation into PhCAs is considered functionally insignificant for people without monogenetic hereditary diseases. The blood of healthy people contains the same PhCAs that are typical for septic patients as shown in this paper. The overall serum PhCAs level was 6 µM on average as measured by gas chromatography with flame ionization detection. This level is a stable biochemical parameter indicating the normal metabolism of aromatic amino acids. The concentrations of PhCAs in the metabolic profile of healthy people are distributed as follows: phenylacetic ≈ p-hydroxyphenyllactic > p-hydroxyphenylacetic > phenyllactic ≈ phenylpropionic > benzoic. We conclude that maintaining of stable PhCAs level in the serum is provided as the result of integration of human endogenous metabolic pathways and microbiota.

[Influence of Microbial Metabolites of Phenolic Nature on the Activity of Mitochondrial Enzymes].

The aim of this work was to study the effect of microbial metabolites of phenolic nature on the activity of enzymes of the tricarboxylic acid cycle in isolated mitochondria, and determine metabolites of the tricarboxylic acid cycle as potential biomarkers of mitochondrial dysfunction in the blood of patients with sepsis. It is shown that microbial metabolites of phenolic nature have an inhibitory effect on the activity of dehydrogenases, determined by the reduction of dichlorophenolindophenol and nitroblue tetrazolium in liver mitochondria and liver homogenates. This effect is more pronounced in oxidation of the NAD-dependent substrates than succinate oxidation, and at lower concentrations of microbial metabolites than inhibition of respiration. By gas chromatography-mass spectrometry it was found that the content of the tricarboxylic acid cycle metabolites in the blood of patients with sepsis decreased compared to healthy donors. The data obtained show that the microbial phenolic acids can contribute significantly to the dysfunction of mitochondria and suppression of general metabolism, characteristic of these pathologies.

[Sepsis: a new look at the problem].

The recent proceedings of congresses and forums on sepsis were used to write this review. The available definitions of sepsis and ideas on its etiology and pathogenesis are critically analyzed. There is information on new concepts of sepsis and data on a search for new targets, diagnostic and therapeutic approaches, and biomarkers. It is hypothesized that there is a mechanism of action of bacteria on mitochondrial dysfunction and human hormonal regulation with low-molecular weight exometabolites, namely aromatic microbial metabolites.

[Biological properties of some sepsis-associated low molecular aromatic microbial metabolites].

Physico-chemical and biological properties of sepsis-associated low molecular aromatic compounds are summarized and analysed in the review. Phenylcarbonic acids (PCAs), such as para-hydroxyphenyllactic acid (p-HPLA), phenyllactic acid (PLA), para-hydroxyphenylacetic (p-HPAA), phenylacetic acid (PAA), benzoic acid (BA), and phenylpropionic acid (PPA) are shown to have biorequlatory activity and be able to affect both bacteria and eukaryotic cells. In the review there are presented data on the diagnostic and pathogenetic value of PCAs, their bacteriostatic and bacteriocidal properties and biosynthesis by clinically significant bacterial species, as well as description of the mechanisms of microbial resistance to PCAs, the pathways of PCAs metabolism by prokaryotes, PCAs membrane transport and excretion pathways in humans, the data on the use of PCAs in clinical practice. The authors are of the opinion that PCAs of microbial origin share the metabolic and signal pathways in integration of the microbiome and man. On the basis of the literature data and personal studies the authors validated the hypothesis of possible development of new therapeutic strategies, grounded on regulation of the local and systemic balance of aromatic microbial metabolites in the human body.

[The role of thiol antioxidants in restoring mitochondrial functions, modified by microbial metabolites].

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acethylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. The partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acethylcysteine, the full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K3). It was found that the protective effect of thiol antioxidants became prooxidant one, if the medium contained free iron and compounds with a quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.

[Exometabolites of some anaerobic microorganisms of human microflora].

Some exometabolites produced by basic representatives of human anaerobic microflora were investigated, detected by gas chromatography-mass spectrometry (GC-MS). In vitro besides lactic acid Bifidobacterium and Lactobacillus generate substantial amounts of phenyllactic and p-hydroxyphenyllactic acids. Clostridium produced 2-hydroxybutyric acid and to a lesser extent lactic and phenyllactic acids. In contrast to C. perfringens, C. sporogenes generates substantial amount of phenylpropionic and p-hydroxyphenylpropionic acids and less p-hydroxyphenyllactic acid. C. perfringens produced minor amounts of 2-hydroxyglutaric acid. Bacteroids are potent producers of succinic and fumaric acids; they also contribute to production of significant portion of lactic acid. E. lentum generate lactic, phenyllactic and succinic acids and form a characteristic only for ones (from studied microorganisms) 2-hydroxyhexanic and 2-hydroxy-3-methylbutyric acids.

Microbial origin of phenylcarboxylic acids in the human body.

In previous studies we demonstrated increased amounts of phenylcarboxylic acids (PCA) in serum of patients with sepsis. This observation prompted the present study of the ability of the human microbiome bacteria to produce PCA in vitro. PCA were detected in culture media by gas chromatography-mass spectrometry. Increased amounts of phenyllactic and p-hydroxyphenyllactic acids were produced by Klebsiella pneumonia, Escherichia coli, and Staphylococcus aureus. Certain strict anaerobes (bifidobacteria, lactobacteria, eubacteria) have also been found to actively produce these PCA, but these bacteria are not etiologically linked to sepsis. Thus our results demonstrate the ability of sepsis-related bacteria to produce PCA and provide experimental support for the theory that the accumulation of PCA in the blood of patients with sepsis results from microbial degradation of phenylalanine and tyrosine.

[Etiology of postoperative bacteremias in an intensive care unit: an association with the level of procalcitonin].

The etiology of bacteremia determines the choice of adequate therapy for severe infections. The clinical manifestations of gram-negative and gram-positive bacterial infections are similar while biological markers may serve as a guide for the early diagnosis of the nature of a pathogen. The paper analyzes data on the rate and etiology of bacteremia in 1360 patients at the A. N. Bakulev Research Center of Cardiovascular Surgery over a period of 5 years (2002-2006). The purpose of the study was to assess an association between the level of procalcitonin (PCT) and the etiology of bacteremia. A hundred and fifty consecutive cases of documented bacteremia were analyzed in detailed in 113 cardiac surgical patients in the postoperative period, by comparing them with the level of on concurrent blood sampling for culture and PCT. In the gram-negative bacteremia group, plasma PCT levels were statistically significantly higher than in the gram-positive bacteremia group (5.40 and 0.86 ng/mg, respectively; p = 0.000000). On constructing a characteristic curve, the authors determined the PCT cut-off value of 5.2 ng/ml for the differential diagnosis of gram-negative and gram-positive bacteremia with the maximum (86%) specificity and more than 50% specificity. Thus, when patients with suspected infections have plasma PCT levels, the leading value of gram-negative microorganisms may be supposed just before obtaining blood culture results.

Toxic effects of microbial phenolic acids on the functions of mitochondria.

Low-molecular-weight phenolic acids (PhAs) phenylacetate, phenyllactate, phenylpropionate, p-hydroxyphenyllactate, and p-hydroxyphenylacetate are essentially the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora. In sepsis, the concentrations of some of these acids in the blood increase tens of times. Assuming that these compounds can cause the mitochondrial dysfunction in sepsis, we examined their effects on respiration, the induction of pore opening, and the production of reactive oxygen species (ROS) in mitochondria. It was found that phenylpropionate and phenylacetate produce a more toxic effect on mitochondria than the other phenolic acids. At concentrations 0.01-0.1 mM they decreased the rate of oxidation of NAD-dependent substrates and activated the Ca2+- and menadione-induced opening of the cyclosporin A-sensitive pore and the production of ROS. The disturbances caused by these PhAs are similar to those observed in mitochondria in sepsis, and hence the rise in their level may be one of the causes of mitochondrial dysfunctions. Phenyllactate, p-hydroxyphenyllactate, and p-hydroxyphenylacetate inhibited the production of ROS and pore opening, acting as antioxidants. Thus, the ability of PhAs to affect the mitochondrial functions, as well as an increase in their concentrations in sepsis (the total concentration of these PhAs in the blood is close to 0.1 mM), suggests that PhAs can be directly involved in the development of mitochondrial failure.

[Sepsis-induced immune paralysis: pathogenesis, diagnosis, and possible modes of correction].

Recent clinical and experimental studies indicate that the pathogenesis of a septic process in critically ill patients may be divided into two stages: (1) a systemic inflammatory reaction and (2) immune paralysis. Verification of biological markers of both stages is the key point for choosing adequate drug therapy. Immune paralysis virtually shows no clinical symptoms, at the same time this condition can be diagnosed at the earliest stages, by applying a number of laboratory techniques. For the time being, the determination of the level of IL-10 and TNF-alpha and their ratio in the serum are most suitable for this purpose in routine practice. The estimation of the levels of mHLA-DR expression and apoptotic markers of different cell populations is of unquestionable practical value. The diagnosis of immune paralysis requires urgent pathogenetically substantiated therapy. Despite the fact that clear criteria for mediator-controlled therapy have not been defined so far, it is absolutely clear that it is precisely such an approach that is most justified and promising. Furthermore, immunological parameters in the use of new therapy options need meticulous monitoring for the evaluation of the efficiency and adequacy of drug action. All the issues in question--from diagnosis to criteria for the adequacy of therapy--require further development and they will be undoubtedly to have the most direct impact on the results of treatment in intensive care unit patients.

[Diagnostic and therapeutic aspects of pneumonias after cardiosurgical operations associated with artificial ventilation].

Artificial ventilation (AV)-associated pneumonias are the most common infectious complication in cardiosurgery. This prospective comparative study covered 50 patients with AV-associated pneumonias occurring after surgery under extracorporeal circulation (EC). All the patients received the routine perioperative antibiotic prevention regimen (cefuroxime or ceftriaxone). According to the initial therapy, the patients with evolving pneumonia, the patients were divided into 2 groups: 1) those were given cefuroxime (maxipim); 2) those receiving a combination of maxipim or clarithromycin (clacid). The analysis has indicated that if pneumonia develops after surgery under EC, then this most frequently occurs in the first 5 postoperative days, i.e. early AV-associated pneumonias are prevalent. In cases of concurrent pneumonia, the duration of EC, the length of stay in an intensive care unit, and the total period of hospi- talization considerably increase. For cardiosurgical patients, the laboratory guide for establishing the diagnosis of AV-associated pneumonia is the elevated blood cell levels of more than 15 x 10(9)/l, unlike those of more than 10 x 10(9)/l proposed for most patients. The etiology of AV-associated pneumonia is shown to vary with the timing of complication occurrence. There is evidence for the involvement of intracellular microorganisms (Chlamydia, Mycoplasma) in the development of early AV-associated pneumonias in at least every 10 patients. The advantages of a study of bronchoalveolar lavage samples over that of endotracheal aspirates for the etiological diagnosis of pneumonias were revealed. The advisability of prescribing a combination of a beta-lactam antibiotic (third- or fourth-generation cephalosporin) and a macrolide (clarythromycin) in early AV-associated pneumonias is warranted. The objective criterion for the adequacy of this combination is positive changes in the marker of severe bacterial infections (procalcitonin). An algorithm is offered for antibacterial therapy for AV-associated pneumonias developing after cardiosurgical operations, which considers the performed antibiotic prevention and the timing of pneumonia development.

[Systemic inflammation after operations on the open heart: significance of a microbial factor].

The given review of the literature discusses, from the present standpoints, the pathogenetic, clinical, and diagnostic aspects of a systemic inflammatory reaction developing during cardiac surgery under extracorporeal circulation. There is evidence that endogenous infection is of importance in developing systemic inflammation during cardiosurgical interventions. Current approaches to the early diagnosis of systemic bacterial inflammation, by applying the procalcitonin test and the chromatographic mass-chromatographic technique are proposed. The prevention of systemic inflammation and its sequels is considered.

[Chromatographic mass spectrometric determination of low-molecular-weight aromatic compounds of microbial origin in the serum from patients with sepsis].

The investigation quantitatively determined the content of low-molecular-weight aromatic compounds of microbial origin in the sera of 34 individuals by chromatographic mass spectrometry. An "Agilent Technogies 6890N" gas chromatograph with a 5973 mass selective detector was applied; chromatographic separation of components was effected on an Hp-5MS quartz capillary column. Aromatic small molecules originating from microbes (SMOM) were determined in the sera of 7 patients with sepsis. The diagnosis of sepsis was documented by the presence of the systemic inflammation syndrome and by that of bacteriemia and/or artificial ventilation-associated pneumonia along with the level of procalcitonin of higher than 10 ng/ml. The levels of aromatic SMOM were compared in 10 healthy donors, 8 preoperative cardiosurgical patients, and 9 patients with different abnormalities without sepsis treated in an intensive care unit (ICU). Serum phenylacetic and 3-phenylpropionic acids were found to be prevalent in the healthy donors and postoperative cardiosurgical patients. In ICU patients with different complications without sepsis, more than half the compounds under study were undetectable, the others were found in very low concentrations, which may be accounted for by antibiotic therapy. At the same time, almost the whole spectrum of the test compounds (other than 3-phenylpropionic acid) with the highest concentrations of 3-phenyllactic, p-hydroxyphenylacetic, 3-(p-hydroxyphenyl)lactic and 2-hydroxybutanic acids, was detectable in septic patients receiving a more intensive therapy. The differences were statistically significant (by the Mann-Whitney U-test; p < 0.05). By taking into account the potentially high biological activity of the test compounds, studies are to be continued in this area.

[Study of volatile fatty acids in the blood of patients with periodic disease].

Microecological "failures" are an important pathogenetic factor of different diseases and, in the authors' opinion, periodic disease (PD) is one of them. PD is a recessive disease characterized by fever attacks and neutrophil-mediated serous inflammation. A genetic factor has been established to be responsible for half the cases of PD, the influence of non-hereditary factors, particularly a role of the host automicroflora in the genesis of an inflammatory process, has been little studied. The authors' early studies indicate that there are changes in the qualitative and quantitative composition of microbial molecules in the blood of patients with PD. The anaerobic bacterial metabolites that are volatile fatty acids (VFAs) represent biologically active substances that affect the growth of the microflora, on the one hand, and the host's immunological responsiveness, on the other. Out of VFAs, only is acetic acid detectable in small quantities in the blood of healthy individuals. The other VFAs, namely propionic, valeric, butyric, and caproic acids and their isomers, are absent. Gas chromatography was used for qualiitative and quantitative determination of the metabolites of anaerobic microorganisms in the blood of patients with PD (n = 13) during an attack and remission and in that of healthy volunteers (Armenians) (n = 5) of a control group from one Yerevan region. The blood samples from all the patients with PD displayed a significantly higher concentration of caproic acid while the latter was absent in the blood of the controls. This finding suggests that there is a specific shift in the structure of the microbiocenosis in patients with PD. It is conceivable that caproic acid plays a certain role in the pathogenesis of the disease under study. Further studies will deal with the association of some microbial molecules with the manifestation of an attack of PD, which may provide the key to the goal-oriented regulation of detected homeostatic disorders and to the management of the frequency of its attacks.