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ORIGINAL ARTICLE Small molecules originating from microbes (SMOM) and their role in microbes – host relationship N. V. Beloborodova and G. A. Osipov From the Research group of Academician Yu.Isakov, Russian Academy of Medical Sciences, Sadovo-Kudrinskaya St., 15, 103001 Moscow, Russia Correspondence to: N. V. Beloborodova, Research group of Academician Yu.Isakov, Russian Academy of Medical Sciences, Sadovo-Kudrinskaya St., 15, 103001 Moscow, Russia. Tel: +095 254 1039; Fax: +095 255 9300; E-mail: [email protected] Microbial Ecology in Health and Disease 2000; 12: 12–21 A hypothesis proposed, according to which non-specific purulent-inflammatory processes are mainly caused by disturbances at low-molecular level —i.e. by small molecules originating from microbes (SMOM), while SMOM homeostasis decompensation takes the leading role in complex polyfactorial pathogenesis of sepsis. The hypothesis is logically justified by understanding the reasonableness for creation within the ontogenesis the system of signal molecules, which would serve as mediators for informational exchange between microorganisms and host’s cells. To check this hypothesis we’ve studied blood of 25 healthy donors and 161 patients with various conditions (peritonitis, endocarditis, infections of urinary tract etc.) using Gas chromatography-Mass spectrometry. In all cases we detected SMOMs-fatty acids (hydroxy-acids, branched, unsaturated and cyclopropanoic acids), aldehydes, alcohol’s and phenyl-carbonic substances, that are never normally synthesized by mammals, but are structural components or metabolites of microorganisms. The quantitative and qualitative characteristics of SMOMs varied significantly in patients from those of healthy people. Substances of low molecular weight — that are the structural components of normal endogenous microflora and dominate in blood of healthy people — have disappeared or were present in very low quantities. Meanwhile concentration of bacterial molecules that are rarely found in healthy persons, has increased by 10–100 times. Other SMOMs, never present in healthy people, were also found. The data obtained stand pro the hypothesis on constant presence of SMOMs in blood of healthy and ill individuals and allow to formulate the Concept on Homeostasis of small molecules originating from microbes; serious break up of this homeostasis (decompensation of SMOM homeostasis) serves as inductor of general inflammatory response, septic shock and multisystem organ failure. Key words: microbial markers, phenylcarbonic compounds, gas chromatography–mass spectrometry, fatty acids, homeostasis, human blood, non-antibiotics, sepsis. INTRODUCTION Sterile human organism comes into the world teemed with various microorganisms (air, water, soil etc.). Life-threatening specific infections (diphtheria, plague, cholera, botulism, tetanus, and others) are caused by pathogenic bacteria, possessing large variety of pathogenic factors. Pathogenic bacteria life cycles are characterized by production of highly toxic substances, that are intended to kill the host, whose death leads finally to death of bacteria themselves, i.e. from biological sense this rout of evolution of micro-universe comes out as a dead lock. Coming true nowadays and based on this approach is the forecast, dating to 40 years ago: with time life threatening infections would be of secondary importance in comparison with those caused by host’s own opportunistic flora (autochthonous infection) (1). In the end of the 1990s sepsis has become the leading cause of lethality among ICU patients from high-risk groups with different pathologies, such as surgical or oncologic-hematological diseases, trauma, burns, premature birth etc. It’s well known nowadays that it is not © Taylor & Francis 2000. ISSN 0891-060X pathogenic bacteria, that cause sepsis, but the conflict, arising from inadequate immune reactions to organism’s own opportunistic microflora, colonizing opened biocenoses. There’s still serious lack of knowledge on basic relationship between the superior-host’s and inferior-microbe’s organisms. Better knowledge of this relationship will provide physicians with new tools of regulating host’s relationship with it’s own microflora in different pathologic conditions. Let’s refer to a number of well known and the most recent data on the issue. The most important is considered the discovery of cytokine signal system, which is responsible for informational exchange between host’s cells and allows to explain a number of phenomena. For example, cytokines TNF, IL-1, IL-6 are the inductors of acute inflammatory phase of bodily response an integral part of comprehensive human internal protective system against all foreign substances and pathogenic microorganisms. Gene-engineering technique allowed discovering a number of other cytokines (IL-2, IL-10) with anti-inflammatory Microbial Ecology in Health and Disease SMOM and microbe – host relationships 13 Table I Cytokine-inducing bacterial modulines-from published materials (2–4) Target LPS and other bacterial modulines Monocytes/macrophages Epithelial cells Fibroblasts Osteoclasts Lymphocytes functions, that are responsible for blocking inflammatory reactions to residential microflora. Bacterial modulins — is a definition, offered to outline a new type of virulent factors of those bacterial structures, which possess cytokine-inducing potential (2). Lipopolysaccharides (LPS) are the focus of attention during the last 30–40 years. LPS as well as other proteins and phospholipids are built into macromolecular endotoxin complex of gram-negative bacteria (3, 4). It’s clear today, that a number of other bacterial complexes, besides LPS, are also capable of inducing cytokine secretion. During the last 5–10 years the components, stimulating synthesis of cytokine—such as carbohydrates, proteins and lipids — are the subject of thorough investigation (Table I). Medical literature of last years is the evidence of active search for other bacterial modulines. For gram-negative bacteria these are the external membrane proteins: porines, lipid A-bound proteins (LAP) or endotoxin-bound proteins, fimbrial (pili) proteins (5). Among other membrane-bound bacterial modulines are non-identified superficial proteins, protein A and heat-shock proteins of some microorganisms (6). Because lipoproteins of cytoplasmatic membrane act as bacterial modulines in both gram-negative and gram-positive bacteria, some researchers got involved in studying interleukin-stimulating effects of synthetic analogues and structural derivatives of lipoproteins (7). There’s a growing number of publications resulting from studies of bacterial membranous glycoprotein, which stimulates not only macrophagal secretion of TNF, but its synthesis as well (8). Monocytal and epithelial cell synthesis and secretion of cytokines is influenced by extracellular proteins—such as proteases, exotoxins and others, at the same time some lipids and polysaccharides also have cytokine-stimulating properties. For example, protein-free membranous poliol – lipid of Mycoplasma fermentans stimulates macrophagal secretion of IL-6 and TNF-a (9). As to polysaccharides— there’s not only in vitro, but also experimental evidence of increasing TNF-a concentration—as it was demonstrated for Streptococcus B group-specific polysaccharide in newborn rats (10). Results Cell reaction modifications Secretion of eicosanoids and cytokines Chemotaxis Metabolic activity Proliferation Inhibition of proliferation Differentiation Apoptosis and others Teichoic and lipoteichoic acids — principal components of cellular wall in gram-positive bacteria (Streptococcus pyogenes, Staphylococcus aureus, S. epidermidis) — together with peptidoglycan initiate development of cytokine induced septic shock, caused by gram-positive bacteria. Host’s enzymes quickly destroy peptidoglycan, but its fragments have cytokine-inducing properties (11). It’s important to point out, that according to experimental findings at least one of the bacterial modulines (membranous protein 39-kDa of Proteus mirablis) is capable of inhibiting LPS-induced synthesis of interleukin IL-1 and macrophagal production of substances with free oxygen radicals (12). Data of a number of experiments show potential capability of microbial derivatives either to fuel inflammatory process or to inhibit it — in particular through regulation of macrophagal activity. This is an important detail within widely accepted role of macrophages as key factor in development of polyorganic failure in sepsis (13). Cumulative results of SMOM studies are not giving yet full picture of human – autochthonous bacteria relationship. This relationship with bacteria, colonising human’s intestines, skin, mucous membranes of respiratory and urinary tracts, etc., is symbiotic. Bacterial concentration is maximal on marginal surfaces which restrict inner media from the outer world: skin concentration reaches 105 – 6 CFU/g, intestinal mucous concentration exceeds 1011 – 12 CFU/g. The contacting surface of sterile inner media in human organism is huge: it’s length is 4 – 6 m, while smaller intestines total surface reaches 180 – 200 m2. It is already proved that invasion of saprophyte bacteria or their fragments from mucous surfaces into internal media happens more often, than it was thought before. Numerous data on penetrability of mucous membranes for microorganisms and large molecules, on continued migration of incorporated into macrophages microbes into the bloodstream, on direct bacterial translocation into inner media under certain conditions (like stress, shock, haemodynamic disorders, endotoxemia, etc.), on quite widely spread short-term episodes of transient bacteremia, that is observed, for example, after tooth extraction and even after brushing teeth — has been accumulated. 14 N. V. Beloborodo6a and G. A. Osipo6 Phagocytosis and natural autolysis of microbial cells are the principal sources of microbial fragments— lipids, amino acids, peptides, carbohydrates, and small molecules (monomers of lipids, proteins and carbohydrates), which are the products of microbial cells disintegration. Theoretically justified is the bloodstream bacterial molecules’ role as regulators of pro- and anti-inflammatory activity of cells and providers of homeostasis, rather than as cytokine-inducing factors of virulence. We present the hypothesis on constant SMOM circulation in the bloodstream of both healthy and ill people. It is coming out of ontogenetically justified necessity in special signal molecules to exchange information between host and microorganisms. In other words, we are offering the direction where the ABC of the ancient dialogue language between macro- and micro-universe should be looked for. Gas chromatography–mass spectrometry (GC-MS) method was used (14–17). AIMS OF THE STUDY To detect simple by chemical structure non-protein molecules originating from microbes with molecular weight up to 500 [small molecules originating from microbes—SMOM] in human blood under different health conditions. MATERIAL AND METHODS Two groups were studied. The first one was comprised of 25 healthy volunteer-donors that were examined and underwent standard laboratory tests. Blood was collected from 25 donors. The second group was comprised of patients with different pathologies: n =20 with peritonitis; n=32 with endocarditis; n= 109 with local inflammatory and post-operative infectious processes. Their blood was taken on the pick of clinical and laboratory manifestations of infectious process during diagnostic or curative venipunctures. The blood was conserved in heparinised test-tube and frozen under −5°C. Gas chromatography–mass spectrometry (GC-MS) was used to identify bacterial chemical substances in patient’s blood: fatty acids, alcohol and phenylcarbones. Whole blood (50 ml) was dried and subjected to acid methanolysis during 1 h, using 0.4 ml of 1M HCl in methanol under the temperature of 80°C. At this stage fatty acids are extracted from complex glycerol and cholesterol ethers in the form of methyl ethers. Fraction of fatty acids’ methyl ethers with other lipid components was extracted twice from reactive mixture with 200 ml of hexane, dried and derivated with 20 ml of N,O-bis (trimethylsilyl)-trifluoracethamide (BSTFA) for 15 min at 80°C to form trimethylsilyl derivatives of hydroxy acids, alcohol’s and sterols. 1–2 ml of reactive mixture was injected into GC-MS system (QP2000 Shimadzu, Japan). Chromatographic separation of the sample was performed in a capillary column with methyl-silicone phase Ultra-1 (Hewlett-Packard); length 25 m, inner diameter 0.2 mm, film thickness 0.25mm. Temperature program starting at 120°C with further increase up to 300 – 320°C, 5°C per min. Principal lipid components of the sample were identified on the basis of their spectra obtained by quadrupole mass spectrometer (electron ionization 70 eV), operating at full scan mode. The same pattern was used for identification of carbonic and phenylcarbonic acids and spirits. For detection of specific markers of bacteria high sensitivity mode of selected ion monitoring (mass-fragmentography) was used. Five groups of ions were switched in course of gas chromatographic separation. To detect small quantities of C10 – C20 fatty acids prominent m/z 87 ion was used; chosen group intervals allowed to avoid peaks of dominating in microbial lipid acid’s profile of C16 and C18 acids. For verification of b-hydroxy-acids two types of ions were used: characteristic for homologue row ion m/z 175 and acid-specific ion of M-15 type, for verification of a-acids-specific ion of M-59 type. Specific ions were used also to identify fatty alcohol’s and sterols. Additional identification criteria were the following: chromatographic relative retention time and selected ions’ peak ratios. Carbonic and phenylcarbonic acids and alcohol’s were extracted from acidified whole blood (pH 2) with diethyl ether. Ether extract was taken to dryness at the temperature up to 40°C, the dry residue was treated with 20 ml of BSTFA for 15 min at 80°C to form trimethylsilyl derivatives of acids and alcohol’s. Two microliters of mixture was injected. Full scan spectrum, similar chromatographic column (as in fatty acid analysis) was used. Temperature program: 7 min at 80°C with further ramp up to 250°C. Eluted substances were identified by its mass spectra using standard library search program (NBS library). Areas of selected ion trace peaks were integrated automatically or manually and fixed in the protocol. Afterwards all data were processed by based in EXCEL tables evaluation program. Pure component’s calibration data were used by adding of known quantity of tridecanoic acid or 12-OH-octadecanoic acid. Blood concentration of heptadecanic, i.e. margarinic acid were used in routine analysis. Its content is approximately constant in humans and equal to 20 9 5mg/ml (unpublished, our own measurements). This approach allowed to avoid repeated calibration. RESULTS In blood of healthy donors were identified foreign for human body low-molecular chemical substances: hydroxy-, branched, non-saturated and cyclopropanoic fatty acids (Table II). Hard to ignore the relative qualitative and quantitative stability of definite SMOMs: for example, h18, i14 and a19 were found in 88 – 94% blood samples of healthy donors, SMOM and microbe – host relationships 15 Table II Detection frequency and concentration range (ng/ml) of fatty acids–structural components of bacterial cells, and metabolic sterols identified in blood of healthy donors (n = 18) Name Abbreviation* Detection frequency Range (ng/ml) No. of persons % of total Hydroxy-acids Hydroxy-decanoic Hydroxy-lauric 2-Hydroxy-lauric Hydroxy-myristic 2-Hydroxy-myristic Hydroxy-iso-pentadecanoic Hydroxy-palmitic Hydroxy-iso-heptadecanoic Hydroxy-stearic 10-Hydroxy-stearic Hydroxy-iso-eicozanoic h10** h12 2h12 h14 2h14 hi15 h16 hi17 h18 10h18 hi20 10 11 11 18 13 3 18 13 16 18 2 55 61 61 100 72 17 100 72 88 100 11 0–50 0–40 0–30 0–45 0–100 0–10 10–590 0–70 7–150 1–77 0–10 Branched fatty acids anteiso-Tridecanoic iso-Myristic iso-Pentadecanoic anteiso-Pentadecanoic iso-Palmitic iso-Heptadecanoic anteiso-Heptadecanoic anteiso-Nonadecanoic Tuberculostearic a13 i14 i15 a15 i16 i17 A17 a19 10Me18 11 17 18 18 18 18 18 16 18 61 94 100 100 100 100 100 88 100 0–20 0–110 60–380 50–470 90–720 150–2000 630–3600 0–15 3–25 Unsaturated fatty acids 9,10-Tetradecenoic iso-Heptadecenoic Heptadecenoic cis-Vaccenic 14:1 i17:1 17:1 18:1D11 8 6 18 14 44 33 100 78 0–120 0–10 60–310 0–620 Cyclopropanoic acids cyclo-Heptadecanoic cyclo-Nonadecanoic 17cyc 19cyc 9 18 50 100 0–40 2–60 14 3 4 78 17 23 0–150 0–30 0–100 Alcohols Coprostanol Cholestendiol Metabolite 428 * Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of double bonds; h-hydroxy-acid; a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. For example, 2hi15 means 2 -hydroxy-iso-pentadecanoic acid. ** 3-hydroxy-acids—if position of hydroxyl is not indicated. while such molecules as h14, h16, 10h18, i15,a15, i16, i17, a17, 10Me18, 17:1, 19cyc were found in all 100% cases. In the second group—i.e. in patients with manifested infectious processes—were revealed accentuated qualitative and quantitative deviations in the spectrum of low molecular SMOMs (Table III). The main statistics are presented at Figs. 1 and 2 where distribution of microbial markers values in blood of donors and patients is shown. As far as concentrations of markers differ substantially, their values were normalized to interval for optimization of comparison: minimal value was subtracted from each measurement, and residue was divided into interval. Data on 25 donors and 161 patients frequency distributions are shown as ‘box and whiskers’. The box is the part of distribution that lies between the upper and lower quartiles (25 and 75% of distribution), and whiskers correspond to 9 1.5 quartile distance. Data between + 1.5 and + 3 quartile distance are designated by circles while asterisks indicate figures exceeding 3 quartile. Distribution diagrams of patients show configurations with extra right sided skewness and left-sided tailing when compared with symmetrical diagrams of donors. Extreme values exceeding +1.5 quartile distance are rarely found in donors, but common in patients. Such values probably indicate inflammation, caused by corresponding microbes. 16 N. V. Beloborodo6a and G. A. Osipo6 The markers form two distinct groups, termed as ‘zero’ and ‘nonzero’. Markers showing generally zero level are included in the ‘zero’ group. For instance hi20 (Chlamydia), hi17 (B. fragilis), hi15 (Pre6otella melaninogenica), E242 (Corynebacterium urealyticum), h10 (Pseudomonas) and other markers are assigned to pathogens or to rarely detected commensals. ‘Nonzero’ are the markers which show distribution with nonzero mode and are found both in patients and in donors. Statistical comparison between the controls and study patients could be applied only to ‘nonzero’ group. Altogether thirteen markers were found corresponding to the criteria of ‘Nonzero’ group: 10h18, a19, i14, i15, 19cyc, h14, a15, 17:1, i16, 18:1D11, h16, a17, h18. The Mann – Whitney test has shown valuable differences at p B0.03 for five markers: a19, i14, i15, h14, a15. But this test should preferably be applied to normal, Gaussian frequency distribution. Because of real right-side tailing of distribution, we apply also Kolmogorov-Smirnov test as more convenient one. According to this test two more markers 19cyc and 18:1D11 have shown valuable difference at pB 0.05 between the patient and control groups. As a result of these tests six following markers, i.e. 10h18, 17:1, i16, h16, a17 and h18 are remaining out of statistical confirmation, that are assigned mainly Table III Concentration (ng/ml) of fatty acids— structural components of bacterial cells — and metabolic sterols identified in blood of patients (n = 59) Name Abbreviation* Detection frequency No. of persons % of total Hydroxy-acids Hydroxy-decanoic Hydroxy-lauric 2-Hydroxy-lauric Hydroxy-myristic 2-Hydroxy-myristic Hydroxy-iso-pentadecanoic Hydroxy-palmitic Hydroxy-iso-heptadecanoic Hydroxy-stearic 10-Hydroxy-stearic Hydroxy-iso-eicozanoic h10** h12 2h12 h14 2h14 hi15 h16 hi17 h18 10h18 hi20 15 37 17 48 11 30 59 28 59 59 8 25 63 29 81 19 51 100 47 100 100 13 0–96 0–120 0–90 0–114 0–90 0–120 6–300 0–90 6–1800 6–1380 0–120 Branched fatty acids anteiso-Tridecanoic iso-Myristic iso-Pentadecanoic anteiso-Pentadecanoic iso-Palmitic iso-Heptadecanoic anteiso-Heptadecanoic anteiso-Nonadecanoic Tuberculostearic a13 i14 i15 a15 i16 i17 a17 a19 10Me18 54 59 59 59 59 59 59 59 54 91 100 100 100 100 100 100 100 91 0–126 50–540 60–1380 120–1620 12–3000 40–5200 102–6900 6–2040 0–108 Unsaturated fatty acids 9,10-Tetradecenoic iso-Heptadecenoic Heptadecenoic cis-Vaccenic 14:1 i17:1 17:1 18:1D11 51 33 59 34 86 56 100 58 0–2160 0–60 60–2460 0–1440 Cyclopropanoic acids cyclo-Heptadecanoic cyclo-Nonadecanoic 17cyc 19cyc 29 57 49 97 0–60 6–120 26 20 10 44 33 16 0–540 0–6160 0–360 Alcohols Coprostanol Cholestendiol Metabolite 428 Range * Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of double bonds; h-hydroxy-acid; a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. For example, 2hi15 means 2 -hydroxy-iso-pentadecanoic acid. ** 3-hydroxy-acids—if position of hydroxyl is not indicated. SMOM and microbe – host relationships 17 Fig. 1. Distribution of microbial markers values (blood, donors n = 25). For better comparison of markers which concentration are differ substantially in scale their values were normalized to interval: minimal value was subtracted from each measurement, and residue was divided to interval. to C. perfringens, C. albicans, actinomycetes, B. urealyticum, corineforms and H. pylori, respectively. But we have no doubt about microbial origin of this substances. Three of them are hydroxy acids and come from gramnegative microbes’ LPS. Heptadecenoic acid 17:1 shows much enough circle and asterisk points (Fig. 2, patient), as well as a17, 10h18, h16 and i16. More data are needed for statistically reliable results for this markers. Blood levels of H. pylori h18 markers show no difference between patients with stomach ulcer and donors, for this marker is known to accumulate rather in urine, than in blood (our unpublished data). Thorough search for volatile bacterial phenyl derivatives in healthy people’s blood gave the following results (Table IV). Four acids from this list—phenylacetic, phenylpropi- onic and their hydroxy-derivatives– are known as anaerobic bacteria metabolites (18). The table shows that blood of healthy people contains benzoic acid, benzaldehyde and two phenol alcohols — phenylmethanol and phenoxyethanol. These substances are rarely found in unhealthy, while phenylacetic and phenylpropionic acids, as well as their hydroxy-derivatives and phenylpentadecan, are characteristic for the disease. Concentration of phenylcarbonic substances in blood of healthy people seems to be individual, for it fluctuates from barely detectable quantities (10 ng/ml) to 10 mg/ml. We are far from considering the details of phenol substances quantitative analysis as faultless as it is with fatty acids, that is why explanations for these fluctuations are not offered. 18 N. V. Beloborodo6a and G. A. Osipo6 DISCUSSION In summary Table V certain SMOMs, found in human blood, are related with maximal probability to definite groups of microorganisms. These data are based on the results of our investigation on chemical composition of bacterial cell fatty acids (16, 17), as well as on digest of scientific literature on the issue, in particular on chemodifferentiation of bacteria (15). Presence of SMOMs in healthy people could be explained by natural mechanisms of phagocyte digestion up to eventual products of bacteria, normally colonising mucous membranes of respiratory and gastrointestinal tracts. It is well known, that symbiotic microflora plays an important role in health maintenance, which is explained by a number of factors, such as colonization resistance mechanisms, synthesis of vitamins, etc. Bacterial cells are also destroyed by such mechanisms as autolysis, or enzymatic hydrolysis by blood protein complement, etc. Cells are cleaved up to monomers of biologic polymers. It has been estimated, that every minute bloodstream/interstitial space exchange of plasma reaches up to 70% of total plasma volume, including SMOMs. That is why small fragments of microbial biopolymers, secreted into interstitial space, practically instantly get into bloodstream. Taken into account, that phagocytes stay mostly extravascularly— i.e. interstitially, easily migrating through vascular wall, it is natural to presume, that microbial markers as well as phagocytes and transporting proteins get continuously and quickly Fig. 2. Distribution of microbial markers values (blood, patients n =161). See Fig. 1 comments. SMOM and microbe – host relationships 19 Table IV Phenyl-carbonic substances in blood of healthy donors and patients Name Phenylmethanol Benzoic acid Benzaldehyde Phenoxyethanol Phenylacetic acid Phenylpropionic acid Hydroxy-phenylacetic acid Hydroxy-phenylpropionic acid Phenyl-pentadecane Number of positives Healthy donors (n = 18) Patients (n =10) No. of persons % No. of persons % 18 16 9 14 0 0 0 0 0 100 88 50 78 0 0 0 0 0 2 5 0 0 6 4 3 3 3 20 50 0 0 60 40 30 30 30 into bloodstream independent of inflammatory site. I.e. range of microbial markers reflects the spectrum of microbial species within the human body, independent of their habitat or inflammatory process location. Decreased concentration of some SMOMs under certain inflammatory conditions, and especially in case of treated (by broad spectrum antibiotics) sepsis, might reflect ‘micro- ecological’ breaks in affected human organism. This did not affect the patient distribution curves against donors in its left ‘healthy’ branch, but results in frequency of simultaneous decreasing several markers in patients. Although, this phenomenon could be interpreted differently, i.e. microbial–immunologic homeostasis of a healthy human organism is provided through balanced concentration of various SMOMs; some of them are responsible for activation of macrophages, others—for their inhibition. It is likely, that SMOMs, that are found in maximum concentrations in blood of healthy people, inhibit macrophage reaction to all foreign agents. Results of the research allow to presume inhibiting properties in branched fatty acids and some phenyl-carbonic substances. Hypothesis on existence of microbe-induced inflammatory process inhibitors looks natural, for such a mechanism is really needed to preserve immunologic homeostasis under constant inflow of bacteria into human organism, as for example, bacterial translocation from mucous membranes. Clinically manifested inflammation develops only when pro-inflammatory SMOMs significantly prevail over anti-inflammatory SMOMs. Results of this research enabled us to formulate the Concept of homeostasis of small molecules originating from microbes (SMOM) in human organism, which is an important regulating mechanism of the relationship between host’s organism and endogenous microflora. It should be pointed out, that in a healthy person markers of normally predominating intestinal bacteria — such as Bacteroides, Eubacterium and Bifidobacterium— are absent or barely found (traces), which favours the theory on immunology tolerance towards indigenous anaerobes and, consequently, low probability of their phagocytosis. Quite obvious, that presence of markers in the blood does not indicate bacteriemia: small molecules can penetrate into bloodstream from all sites of natural replication — i.e. from microflora loci in any part of the body. Apparently high concentration of SMOMs is explained by their accumulation in monocytes, macrophages and neutrophils. It is well known, that neutrophils are natural accumulators of different microbial markers, for each cell is capable to ‘digest’ hundreds of bacteria during its short life, which lasts for 6 – 8 h in bloodstream and up to two days in interstitial space or on mucous membranes. As it is seen in Table V, SMOMs prevailing in blood of healthy people are the markers of such bacteria, as Bacteroides, Bacillus, Candida, Enterococcus, Clostridium, and diphteroids i.e. basic representatives of endogenous human microflora. But these SMOMs might be as well crossmarkers, for are found in other microbes. Markers of potentially pathogenic microbes, such as Peptostreptococcus anaerobius, Clostridium perfringens, Staphylococcus, Candida, Pseudomonas, Eubacterium, Klebsiella and others were dominating in blood of patients with infectious manifestations. In this research no markers of absolutely pathogenic bacteria (such as tuberculostearinic acid —of Mycobacterium tuberculosis; hydroxydodecanoic acid —of Neisseria, Moraxella; 2,3-dihydroxy-iso-myristinic acid— of Legionella, and also markers Brucella, Francisella, Treponema) were found in either group of healthy people, or patients with non-specific purulent-septic conditions. Quantitative analysis of SMOMs revealed significant deviations in case of disease. For a number of SMOMs deviations were fixed in both directions — towards elevation and decrease. That is especially characteristic for branched and unsaturated acids, components of bacilli and lactobacilli cells, corine- and propionic bacteria, streptococci and candida. 20 N. V. Beloborodo6a and G. A. Osipo6 Basic postulates of the Concept of SMOM homeostasis could be formulated as follows: 1. Molecules of microbial origin are always present in blood of healthy and ill people. Some molecules activate host’s cells (first of all monocytes, macrophages), thus inducing put out of eicosanoids (prostaglandines, leucotriens, tromboxans), interleikins and other cytokines, while other inhibit cell’s activity; admitted, that some molecules could stay indifferent. 2. In healthy organism normal symbiotic microflora is considered to be the source of SMOMs. 3. Healthy organism preserves balance between SMOMs, having pro- and anti-inflammatory activity. Stability of this balance is reached thanks to functional interchangeability of functionally identical SMOMs. Healthy state is provided by domination of inflammation inhibitors over inflammation inductors. Excess of inhibitors allows to avoid inflammatory reactions in case of accidental bacterial intrusion — as it is seen in translocation. 4. Bacterial molecules — markers of pathogenic bacteria — are missing in blood of healthy people and also in Table V Database of chemical substances, related to those microorganisms, in which they are found the most often 1 Code* Chemical name Characteristic microorganism 1 2 3 4 5 6 7 8 9 10 11 11 12 13 14 15 16 17 10:0 a13 i14 14:1D9 i15 à15 i16:0 i17:1 i17:0 a17:0 17:1 17cyc 17:0 18:1D11 i18 19cyc a19 10Me18 Decanoic anteiso-Tridecanoic iso-Myristic 9,10-Tetradecanoic iso-Pentadecanoic anteiso-Pentadecanoic iso-Palmitic iso-Pentadecenoic iso-Heptadecanoic anteiso-Heptadecanoic Heptadecenoic cyclo-Heptadecanoic Heptadecanoic cis-Vaccenic iso-Octadecanoic cyclo-Nonadecanoic anteiso-Nonadecanoic 10-Methyl-octadecanoic (tuberculostearic) hydroxyl-acids Streptococcus Bacillus cereus Peptostreptococcus anaerobius, Streptomyces, Bacillus Streptococcus pneumonia, Clostrydium Propionibacterium, Bacteroides, Staphylococcus Staphylococcus, Bacillus, corynebacteria Bacillus, Streptomyces, Bacteroides, Bre6ibacterium Fla6obacterium Bacillus, Pre6otella, Propionibacterium Corynebacterium (diphteroids) Candida albicans Enterobacteriaceae Human cells, invariant Serratia, Enterobacteriaceae, Pseudomonas, Bacillus subtilis, Peptostreptococcus, Bifidobacterium, Enterococcus, Lactobacillus Staphylococcus Mycobacterium, Corynebacterium xerosis group, C. urealyticum Hydroxy-decanoic Hydroxy-dodecanoic Hydroxy-lauric Hydroxy-tridecanoic Hydroxy-myristic Hydroxy-acids 18 h10 19 h12:1 20 h12 21 h13 22 h14 23 24 hi15 h16 Hydroxy-iso-pentadecanoic Hydroxy-palmitic 25 26 27 28 29 30 31 32 hi17 h18 10h18 hi20 h22** 2h12 2h14 2hi15 Hydrohy-iso-heptadecanoic Hydroxy-stearic 10-Hydroxy-stearic Hydroxy-iso-eicosanoic 3-Hydroxy-docosanoic 2-Hydroxy-lauric 2-Hydroxy-myristic 2-Hydroxy-iso-pentadecanoic Pseudomonas Pseudomonas aeruginosa Pseudomonas, Neisseria, Vibrio, Acinetobacter E. coli, Bacteroides hypermegas, Selenomonas Enterobacteriaceae, Fusobacterium, Neisseria, Haemophilus, Moraxella, Campylobacter Pre6otela Bacteroides urealyticum, Brucella, Wolinella, Fusobacterium, Bordetella, Campylobacter Bacteroides fragilis Helicobacter pylori, Francisella, Brucella Clostridium perfringens Chlamydia trachomatis Chlamydia trachomatis Pseudomonas aeruginosa, Acinetobacter Klebsiella Fla6obacterium Coprostanol (cholestanol) Ergosterol Eubacterium Candida, Aspergillus Sterols 33 34 * Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of double bonds; h-hydroxy-acid; a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. 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