Particularities of acute myocardial infarction in mycoplasma-infected patients
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Igor P. Arleevskiy1, Olga A. Chernova2,I
acute,myocardial,infarction;mycoplasmas;postinfarction,complication;atheromatous,plaques;risk,factor,Particularitiesofacutemyocardialinfarctioninmyco
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Particularities of acute myocardial infarction in mycoplasma-infected patients(pdf)
Correspondence to Dr. Maxim V.Trushin, Kazan Institute of Biochemistry and Biophysics, Lobachevskiy st. 2/31, PO BOX 30, 420111 Kazan, Russia
[Abstract] Objective To investigate hemocoagulation reactions and immune state of acute myocardial infarction in two groups of patients with mycoplasma infections and without ones. Methods The investigations were carried out in the medical ward of the Republican Clinical Hospital No.3 of Tatarstan Ministry for Public Health, Laboratory of Molecular Pathogenesis of the Kazan Institute of Biochemistry and Biophysics and Republican Center for Family, Maternity and Childhood Welfare of Tatarstan Ministry for Public Health, Russia. 76 patients with acute myocardial infarction (age of 25 to 75 years, average age of 54) were involved in the study. Diagnosis of acute myocardial infarction was confirmed according to the clinical, instrumental and laboratory data. Blood samples were taken for detection of mycoplasma infections. The presence of antibodies to microorganisms in blood serum of patients was detected by the enzyme-multiplied immunoassay. Contents of macroelements and microelements (iron, copper, zinc, strontium) in clinical material were detected by the atomic absorption spectroscopy. Results In general, an obvious trend to thrombophilia was observed in the mycoplasma-infected patients. A tendency to mild acute myocardial infarction pattern was also detected in patients with mycoplasma infections. The role of mycoplasmas in atherogenesis and acute myocardial infarction is discussed. Conclusions Taking into account the distribution and ability of mycoplasma infections to transferring by both horizontal and vertical (from mother-to-child) ways, an additional aspects of the cardiovascular disease prophylaxis seem to require a careful consideration.
[Key words] acute myocardial infarction;mycoplasmas;postinfarction complication;atheromatous plaques;risk factor
INTRODUCTION
Nowadays,atherosclerosis is considered as a response to the endothelial injury of the arteries associated with endothelial dysfunction[1~3]. Increasing vascular permeability for the components of the blood plasma, accumulating monocytes and platelets in the endothelium and subendothelial space follow the injury. Being chronic, these processes result in development of atherosclerotic plaques. Inflammatory process in the arterial paries favoring destabilization of the atheromatous plaques may determine development of acute myocardial infarction (AMI). In this connection, the cause of chronic inflammatory reactions appears to be significant [4～6]. However,the trigger agents for the inflammation still remain unclear[4]. Oxidazed low-density lipoproteins[6,7], tissue-factor[8] and some other factors are believed to be involved into rupture and thrombosis of the plaques[9, 10]. It is suggested that development of the cardiac events may be also related with persistent infections[5～10]. The causative agents of the persistent infection are suspected to be some viruses, bacteria and simplest eukaryotes, the microbes of one common feature——an ability to persist——but of quite different biology, however. In this line,the problem of influence of different persistent infections on occurrence of AMI seems to be of a great interest. Persistent infections biasing hemostasis and vascular system may promote atherogenesis and AMI according to the known immune and biochemical mechanisms of reactivity to infectious agents. Unique biology of a concrete causative agent of the infection as well as individual immunoreactivity seems to determine pattern of the disease.
Recently, it was suggested that mycoplasmas may be an essential infectious agent in the development of atherosclerosis, plaque destabilizing and AMI[11,12]. Mycoplasmas are unique cell-wall-free prokaryotes able to self-producing. In humans mycoplasmas may be involved into development of various diseases. Persistent mycoplasma infections are reported to be a wide spread. The limited biosynthesizing possibilities of the bacteria are not an obstacle for them to spread in various biosenosia when persisting in higher eukaryotes and circulating in nature due to unique adaptive abilities including ones allowing to affect signal systems and to avoid immune control of the host organism. The microorganisms are able to modulate host immunoreactivity and to suppress signal systems responsible for expressive inflammatory processes. In this line,pattern of AMI in patients with mycoplasma infections is of a special interest. Nevertheless the data are still absent[13].
The influence of mycoplasma infections on AMI was the object of our research. Previously, it was reported that shift of some parameters for hemostasis and immune systems and content of some macro-and microelements follows persistence of mycoplasmas in humans[14]. In this study comparative analysis for some hemocoagulation reactions, immune state of AMI and postinfarction complications has been done in two groups of patients with mycoplasma infections and without ones.
MATERIALS AND METHODS
The investigations were carried out in the medical ward of the Republican Clinical Hospital No.3 of Tatarstan Ministry for Public Health, Laboratory of Molecular Pathogenesis of the Kazan Institute of Biochemistry and Biophysics and Republican Center for Family, Maternity and Childhood Welfare of Tatarstan Ministry for Public Health.
76 patients with AMI (age of 25 to 75 years, average age of 54) residing in the medical ward of Republican Clinical Hospital No.3 were involved in the study. Persons were hospitalized for an acute myocardial infarction (with or without electrocardiographic evidence of ST-segment evlevation). Diagnosis of AMI was confirmed according to the clinical, instrumental and laboratory data including investigations of the most important myocardial markers-creatine kinase (CK) and its dimeric isoenzyme MB (CK-MB).
The experimental group was divided into two subunits: the mycoplasma-infected patients and non-infected ones (54 and 22 patients, respectively). Both subunits did not differ in the AMI localization, age, sex and applied therapy. In general, the experimental group consisted of 57 males (average age 52.3 years) and 19 females (average age 60.4 years). There were 40 control (healthy) patients (age of 30 to 50 years, average age 35 years). The differences between average ages, male sex, smoke status, body mass index and diabetes mellitus in the experimental and control groups were statistically insignificant. Patients were seen for follow visit at 30 days. Twelve-lead electrocardiographic investigations were made repeatedly in patients.
In patients with AMI and representatives of the control group, blood samples were taken for detection of mycoplasma infections (in %) and carrier state (in %) of antibodies to some microorganisms associated with persistent infections (including the TORCH group) cytomegalic virus, rubella, chlamydia, toxoplasma,features of hemocoagulation system, levels of IgG, IgА, IgМ and circulating immune complexes (CIC), concentrations of some macroelements and microelements (strontium, zinc, copper, iron) as well as frequencies of some AMI complications were monitored.
Clinical materials (blood and urine) were taken from the patients during the first three days of admission. The material from atheromatous plaques of coronary arteries of died patients was tested on a presence of mycoplasmas; levels of strontium and zinc were also detected. All procedures were performed according to the WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI Ethical Principles for Medical Research Involving Human Subjects[http://ohsr.od.nih.gov/guidelines/helsinki.html].
For detection of mycoplasmas, a polymerase chain reaction (PCR) with use of rDNA oligonucleotide sequences as universal primers for revealing the human-specific mycoplasmas[15] as well as transmissive micrography were applied[16]. Oligonucleotides were synthesized by the scientific-production company “Litech” (Moscow, Russia). Taq-polymerase (“Litech”, Moscow, Russia) was added to the reaction mix before beginning of the reaction in concentrations recommended by the producer. Reaction regimen was controlled with the use of “Tercyc” amplifier (“DNA-technology”, Moscow, Russia). Electroforetic separation of DNA fragments stained with bromide etidium was performed in 1%～2% agarose gel (“Dia-M”, Moscow, Russia) and analysed using gel-documentation system (“Litech”, Moscow, Russia).
The presence of antibodies to microorganisms in blood serum of patients was detected by the enzyme-multiplied immunoassay, a method of separation using the specificity of antibody-antigen binding and quantitation using enzyme reaction[17]. Commercial kits were provided by SPF “Litech” (Russia) and “Vector-Best” (Russia).
Contents of macroelements and microelements (iron, copper, zinc, strontium) in clinical material were detected by the atomic absorption spectroscopy[18] using atomic absorption spectrometer “СА10MP”. Investigation of the serum IgG, IgA, IgM content was performed using the method of radial immunodiffusion[19].
The level of the total complement was determined in the reaction of its fixation according to 50% hemolysis in standard units CH-50[20].
CIC were revealed by their sedimentation in polyoxyethylene glycol-6000[21]. Calculation of thrombocytes and thrombocyte aggretability were estimated by the “Biola” laser aggregometer[22].
Antithrombin Ⅲ(AT-Ⅲ) and activated partial thromboplastin time (aPTT) (a test that measures clotting time in plasma (the liquid portion of blood); it focuses on a specific pathway in the blood clotting process; normal values for aPTT is 25 to 35 seconds) were detected according to Abilgaard[23] and Caen[24], respectively.
Transmissive microscopy of atheroma samples was done according to Brown[16] with some modifications. Material was fixed with 2.5% glutaric aldehyde on phosphate mixture (pH 7.2), then, treated with 1% solution of osmium oxide during 4 h with addition of 2.5 mM saccharose. After dehydration in spirit of ascending concentration, 100% acetone and oxidopropylene, material was perfused by epon-812. Thin slices obtained using ultramicrotome LKB-Ⅲ (Sweden) were contrasted with the saturated water solution of uranyl acetate and then with the solution of zinc citrate.
Statistical analysis of the obtained data was performed using Statistica program. The data were presented as mean ± standard deviation. Data between three groups (Table 1) were analysed by ANOVA[25]; a P value of <0.05 was considered to indicate significance.
Table 1 Some Hemostatic,Immunologic Parameters and Content of Macroelements and Microelements in the Blood Serum of People under Study
Abbreviation: aPTT=activated partial thromboplastin time; SPA=spontaneous platelet aggregation; IPA=induced platelet aggregation; PR= prothrombin ratio; CIC=circulating immune complexes
RESULTS
As a result of system investigations, mycoplasma infections were revealed in 52% of patients. In the control group, 5% of patients were infected with mycoplasmas. All patients with AMI also had antibodies to some microorganisms associated persistent infections. In the control group,antibodies to cytomegalic virus and rubella were detected (12.5% and 2.5%, respectively).
In the mycoplasma-infected patients, in comparison with non-infected ones, antibodies to cytomegalic virus, toxoplasma and rubella were detected more often,while to chlamidia more rarely (38.5% and 27.3%; 26.9% and 13.6%; 15.4% and 9.3%, 3.8% and 4.6%, respectively;P<0.05). In the mycoplasma-infected group,pathologies of urogenital system were more frequent in comparison with the non-infected cohort (31.8% and 15%, respectively).
As a result of ultracytostructural investigation of coronary artery atheromas in the mycoplasma-infected patients, microbodies with sizes of 0.2～0.6 μm and their structures corresponding to vegetative form of mycoplasmas cells as well as ultramicroforms (nanocells) of mycoplasmas[26] were detected among villi at outer membrane of endotheliocytes and in cytoplasm of macrophages localized in the subendothelial stratum. In this case, statistically significant increased levels of strontium and zinc were detected in the atheromas of the mycoplasma-infected patients (12.28±2.53 μg/ml*; 44.6±2.02 μg/ml**;*P<0.05;**P<0.01, respectively) in comparison with the non-infected ones (2.49±0.29 μg/ml; 24.7±2.71 μg/ml, respectively). In blood serum of the mycoplasma-infected patients, the increased level of strontium was observed while the level of zinc was decreased (Table 1).
The increased levels of IgG, IgM and circulating immune complexes as well as the decreased content of total complement were found in serum of the mycoplasma-infected patients (Table 1).
In the mycoplasma-infected patients, thrombophilia was observed as evidenced by the decreased activated partial thromboplastin time, the increased spontaneous and induced platelet aggretability as well as by tendency to the increased level of fibrinogen (Table 1).
As a result of the investigation of frequency of postinfarction complications in the tested groups of patients a low rate of ventricular tachycardia, ventricular fibrillation, atrioventricular block, premature postinfarction stenocardia as well as a low hospital lethality were observed in the mycoplasma-infected patients(Table 2).
Table 2 Frequency of Postinfarction Complications in AMI Patients (in %)
DISCUSSION
Recently, a lot of reports on high level of distribution of persistent infections with mycoplasmas in humans have been published. The data found in our study that mycoplasmas were detected in 5% of the control group reflect the fact of a high level of distribution of the persistent infection in general but the difference with the control group-52% of mycoplasma infected patients with AMI allowed to implicate uncausal relations of the microbes with the disease. As to AB, the immunologic parameter unlike PCR is known to be not a perfect evidence of presence of infections in patients at the moment of testing. Thus, we may conclude only that prevalence of mycoplasma infections in patients with AMI was found.
Mycoplasmas are believed to be causative agents of many diseases in humans, animals and plants. They are the main contaminants of cell cultures and occur particularly in cells used in biotechnology for producing viral vaccines. It was suggested that mycoplasmas are associated with AIDS, malignancy, chronic fatigue syndrome, Gulf War Syndrome and a variety of other illness as either the primary agent or cofactor[27]. These microorganisms possess unique adaptive abilities allowing to overcome various defense systems of higher eukaryots and to persist in host tissues affecting homeostasis, immune state and hemocoagulation reactions[13].
It was shown in our studies that shift of some parameters for hemostasis and immune system, and content of some micro-and macroelements follow persistence of mycoplasmas in humans[14]. Chronic mycoplasma infections are usually accompanied by increasing vascular wall permeability, disturbance of microcirculation and thrombosis[28]. Indeed, an obvious trend to thrombophilia was observed in the mycoplasma-infected patients. These facts could not be explained by the anticoagulant therapy since it was equal in the groups of patients.
The more frequent cases of inflammatory processes of urogenital system in the mycoplasma-infected patients with AMI may be consequence of the microbe influence on the immune system, microbiocenosis and urogenital tract. It was marked that abnormalities in microbiocenosia[28], respiratory and urogenital tract[13,27] arouse due to mycoplasma persistence, which was followed by coinfections. In these connection the tendency of specific AB presents to some persistent infectious agents found in the examined groups of patients is of a separate interest.
The increased levels of IgG, IgM and circulating immune complexes as well as the decreased content of total complement found in the mycoplasma-infected patients may be an evidence of the immunoreactivity towards infection agents and of destroy its unspecific humoral mechanisms. The content of strontium in the blood serum and atherosclerotic plaques features in the mycoplasma-infected patients with AMI seems to be an evidence of the involvement of the element into pathogenic reactions induced by these microbes, but the fine mechanisms of the process remain to be clarified.
The limited biochemical abilities determine state of dependence of these microbes on higher organisms. Mycoplasmas may reside outside or inside of the host cell but always in the closest contact with the host cell membrane. Due to this fact mycoplasmas are obviously called the “membrane parasites”. It is the “membrane parasitism” that may determine the peculiarities of the pathogenesis in humans with persistence of mycoplasmas[26].
Mycoplasmas are cell-wall-free bacteria. For synthesis of own membrane, mycoplasmas need cholesterol. Extracting of the component from the host cell membranes promotes to destroy of the membrane structure, its antigenic profile and, as a consequence, arising immune response to the infected cell. In this line, the presence of mycoplasmas in the atherosclerosis plaques (also found in our study) may be a risk factor for destabilizing the plaques. Mycoplasmas may trigger destabilization of atheroma and development of AMI according to the known mechanisms of reactivity of nonspecific signal systems towards persistent agents[4,29] (Figure 1).
There are some findings allowed to propose active participation of mycoplasmas in vulnerable plaques, by entering the subendothelial space and creating conditions that favor fat accumulation, dysfunction of the immunological and endothelial response, inflammation and increasing apoptosis, which are fundamental ingredients for plaque rupture[11,12, 30,31]. The bacteria induce depression in the immune response[32, 33], mainly of T cells through possible mechanisms of apoptosis[34,35] with a reduction in the CD4 T cells, which may be a facilitating mechanism for other infections. Chronic oxidative stress is a characteristic of mycoplasma infections[36]. Epitopes of oxidazed LDL being antigenic promote to produce inflammatory reaction in artery wall suggesting a possible autoimmune reaction[37,38]. In this connection, antioxidant agents may be reasonable for preventing complications of atherosclerosis[39,40].
It was reported[41] that despite long-term treatment with a bactericidal antibiotic effective against C. pneumoniae, no reduction in the rate of cardiovascular events was observed in the microbe-infected patients with AMI. In this line, the authors insisted that efforts toward secondary prevention in patients with coronary syndromes should be aimed at proven therapies, including antiplatelet therapy, treatment with beta-blockers and angiotensin-converting enzyme inhibitors, and intensive lowering of lipid levels with the use of statins[41]. Meanwhile recently it was found that C. pneumoniae the most suspected bacterium into atherogenesis and AMI, presents in the plaques mainly with mycoplasmas[11,12]. In this line, coinfection with mycoplasma may be involved into non-efficacy of the antibiotic therapy for the patients with C. pneumoniae. Suppression of mycoplasma infection is a serious problem[13].
Mycoplasmas are able to adapt to unfavorable growth conditions-through transformation of the vegetative cell forms into ultramicroforms (UMF) which are resistant to stress factors and able to revert[26]. The dormant state of the bacterium is followed by shift of its virulence. Our data on transmissive microscopy showed that in the plaques part of the cell population of mycoplasmas were presented mainly as UMF. Unfavorable growth conditions (including antibiotic therapy) may result in prevalence of dormant state of bacteria (UMF) able to revert into active forms (vegetative cell forms) while disappearing stress factors.
Figure 1 Scheme of the possible mechanisms of development of acute myocardial infarction during persistence of mycoplasmas in humans
Abbreviations: EC-endotheliocyte; pM-persistent mycoplasmas; L-arg-L-arginine; NO-nitric oxide; PAF-platelet activating factor; АA-arachidonic acid; COG-cyclo-oxygenase; LOG-lipoxygenase; TO-thromboxan; LT-leukotriene; TC / аTC-thrombocytes / activated thrombocytes; PC / аPC-phagocytes / activated phagocytes; RO-reactive oxygen; ED-endothelial destruction; HCC / аHCC-hemocoagulation cascade/activation of hemocoagulation cascade; FL-fibrinolysis; AC-anticoagulation; AG-changed antigens of own cells; HSP-heat shock proteins; CD-collagen destruction; SMCP-proliferation of smooth muscle cells; AP-atheromatous plaque; TG-thrombogenesis; M-macrophages; Bc-B-cells; Tc-Т-cells (CD4+) whose proliferation (Th1 and Th2 producing proinflammatory and anti-inflammatory cytokines) may be induced or inhibited by mycoplasmas; I/APD-inflammation/atheromatous plaque destabilization; AMI-acute myocardial infarction. Solid line corresponds to activation (→) or inhibition (—<) events
Mycoplasmas are called the “elite” parasites due to their ability to modulate reactivity of specific and nonspecific signal pathways of the host organism. That may probably cause the suppression of pathological processes connected with the activation of superoxide dismutase (SOD) and NO-synthesizing systems producing forms of active oxygen[13,42]. The mycoplasma features provide the phenomenon revealed in our study-a tend to “mild AMI pattern” observed in the mycoplasma-infected patients, but other factors connected with peculiarities of interrelating the host-parasite signal systems due to unique biology of the microbe and individual immunoreactivity, can not be excluded. In this line, an “AMI pattern” arising on the background of other (non mycoplasma) persistent infections is of a special interest.
As it was marked above, the tendency of shifting hemocoagulation reactions and immune state as well as content of strontium in blood serum being similar in mycoplasma infected patients with or without AMI proved to be similar, but quite differ in comparison with the mycoplasma free patients. The data may reflect peculiarities of the mycoplasma influence on the respective systems, and the cardiovascular problems. Taking into account the distribution and ability of mycoplasma infections to transferring by both horizontal and vertical (from mother-to-child) ways[43], an additional aspects of the cardiovascular disease prophylaxis, presuming the detection of the persistent infections (as a possible risk factor for atherosclerosis, coronary disease and AMI) at the beginning seem to require a careful consideration.
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1 Kazan Medical Academy, Kazan, Russia
2 Kazan Institute of Biochemistry and Biophysics, Kazan, Russia
(Editor HOU)
Particularities of acute myocardial infarction in mycoplasma-infected patients(pdf)
Correspondence to Dr. Maxim V.Trushin, Kazan Institute of Biochemistry and Biophysics, Lobachevskiy st. 2/31, PO BOX 30, 420111 Kazan, Russia
[Abstract] Objective To investigate hemocoagulation reactions and immune state of acute myocardial infarction in two groups of patients with mycoplasma infections and without ones. Methods The investigations were carried out in the medical ward of the Republican Clinical Hospital No.3 of Tatarstan Ministry for Public Health, Laboratory of Molecular Pathogenesis of the Kazan Institute of Biochemistry and Biophysics and Republican Center for Family, Maternity and Childhood Welfare of Tatarstan Ministry for Public Health, Russia. 76 patients with acute myocardial infarction (age of 25 to 75 years, average age of 54) were involved in the study. Diagnosis of acute myocardial infarction was confirmed according to the clinical, instrumental and laboratory data. Blood samples were taken for detection of mycoplasma infections. The presence of antibodies to microorganisms in blood serum of patients was detected by the enzyme-multiplied immunoassay. Contents of macroelements and microelements (iron, copper, zinc, strontium) in clinical material were detected by the atomic absorption spectroscopy. Results In general, an obvious trend to thrombophilia was observed in the mycoplasma-infected patients. A tendency to mild acute myocardial infarction pattern was also detected in patients with mycoplasma infections. The role of mycoplasmas in atherogenesis and acute myocardial infarction is discussed. Conclusions Taking into account the distribution and ability of mycoplasma infections to transferring by both horizontal and vertical (from mother-to-child) ways, an additional aspects of the cardiovascular disease prophylaxis seem to require a careful consideration.
[Key words] acute myocardial infarction;mycoplasmas;postinfarction complication;atheromatous plaques;risk factor
INTRODUCTION
Nowadays,atherosclerosis is considered as a response to the endothelial injury of the arteries associated with endothelial dysfunction[1~3]. Increasing vascular permeability for the components of the blood plasma, accumulating monocytes and platelets in the endothelium and subendothelial space follow the injury. Being chronic, these processes result in development of atherosclerotic plaques. Inflammatory process in the arterial paries favoring destabilization of the atheromatous plaques may determine development of acute myocardial infarction (AMI). In this connection, the cause of chronic inflammatory reactions appears to be significant [4～6]. However,the trigger agents for the inflammation still remain unclear[4]. Oxidazed low-density lipoproteins[6,7], tissue-factor[8] and some other factors are believed to be involved into rupture and thrombosis of the plaques[9, 10]. It is suggested that development of the cardiac events may be also related with persistent infections[5～10]. The causative agents of the persistent infection are suspected to be some viruses, bacteria and simplest eukaryotes, the microbes of one common feature——an ability to persist——but of quite different biology, however. In this line,the problem of influence of different persistent infections on occurrence of AMI seems to be of a great interest. Persistent infections biasing hemostasis and vascular system may promote atherogenesis and AMI according to the known immune and biochemical mechanisms of reactivity to infectious agents. Unique biology of a concrete causative agent of the infection as well as individual immunoreactivity seems to determine pattern of the disease.
Recently, it was suggested that mycoplasmas may be an essential infectious agent in the development of atherosclerosis, plaque destabilizing and AMI[11,12]. Mycoplasmas are unique cell-wall-free prokaryotes able to self-producing. In humans mycoplasmas may be involved into development of various diseases. Persistent mycoplasma infections are reported to be a wide spread. The limited biosynthesizing possibilities of the bacteria are not an obstacle for them to spread in various biosenosia when persisting in higher eukaryotes and circulating in nature due to unique adaptive abilities including ones allowing to affect signal systems and to avoid immune control of the host organism. The microorganisms are able to modulate host immunoreactivity and to suppress signal systems responsible for expressive inflammatory processes. In this line,pattern of AMI in patients with mycoplasma infections is of a special interest. Nevertheless the data are still absent[13].
The influence of mycoplasma infections on AMI was the object of our research. Previously, it was reported that shift of some parameters for hemostasis and immune systems and content of some macro-and microelements follows persistence of mycoplasmas in humans[14]. In this study comparative analysis for some hemocoagulation reactions, immune state of AMI and postinfarction complications has been done in two groups of patients with mycoplasma infections and without ones.
MATERIALS AND METHODS
The investigations were carried out in the medical ward of the Republican Clinical Hospital No.3 of Tatarstan Ministry for Public Health, Laboratory of Molecular Pathogenesis of the Kazan Institute of Biochemistry and Biophysics and Republican Center for Family, Maternity and Childhood Welfare of Tatarstan Ministry for Public Health.
76 patients with AMI (age of 25 to 75 years, average age of 54) residing in the medical ward of Republican Clinical Hospital No.3 were involved in the study. Persons were hospitalized for an acute myocardial infarction (with or without electrocardiographic evidence of ST-segment evlevation). Diagnosis of AMI was confirmed according to the clinical, instrumental and laboratory data including investigations of the most important myocardial markers-creatine kinase (CK) and its dimeric isoenzyme MB (CK-MB).
The experimental group was divided into two subunits: the mycoplasma-infected patients and non-infected ones (54 and 22 patients, respectively). Both subunits did not differ in the AMI localization, age, sex and applied therapy. In general, the experimental group consisted of 57 males (average age 52.3 years) and 19 females (average age 60.4 years). There were 40 control (healthy) patients (age of 30 to 50 years, average age 35 years). The differences between average ages, male sex, smoke status, body mass index and diabetes mellitus in the experimental and control groups were statistically insignificant. Patients were seen for follow visit at 30 days. Twelve-lead electrocardiographic investigations were made repeatedly in patients.
In patients with AMI and representatives of the control group, blood samples were taken for detection of mycoplasma infections (in %) and carrier state (in %) of antibodies to some microorganisms associated with persistent infections (including the TORCH group) cytomegalic virus, rubella, chlamydia, toxoplasma,features of hemocoagulation system, levels of IgG, IgА, IgМ and circulating immune complexes (CIC), concentrations of some macroelements and microelements (strontium, zinc, copper, iron) as well as frequencies of some AMI complications were monitored.
Clinical materials (blood and urine) were taken from the patients during the first three days of admission. The material from atheromatous plaques of coronary arteries of died patients was tested on a presence of mycoplasmas; levels of strontium and zinc were also detected. All procedures were performed according to the WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI Ethical Principles for Medical Research Involving Human Subjects[http://ohsr.od.nih.gov/guidelines/helsinki.html].
For detection of mycoplasmas, a polymerase chain reaction (PCR) with use of rDNA oligonucleotide sequences as universal primers for revealing the human-specific mycoplasmas[15] as well as transmissive micrography were applied[16]. Oligonucleotides were synthesized by the scientific-production company “Litech” (Moscow, Russia). Taq-polymerase (“Litech”, Moscow, Russia) was added to the reaction mix before beginning of the reaction in concentrations recommended by the producer. Reaction regimen was controlled with the use of “Tercyc” amplifier (“DNA-technology”, Moscow, Russia). Electroforetic separation of DNA fragments stained with bromide etidium was performed in 1%～2% agarose gel (“Dia-M”, Moscow, Russia) and analysed using gel-documentation system (“Litech”, Moscow, Russia).
The presence of antibodies to microorganisms in blood serum of patients was detected by the enzyme-multiplied immunoassay, a method of separation using the specificity of antibody-antigen binding and quantitation using enzyme reaction[17]. Commercial kits were provided by SPF “Litech” (Russia) and “Vector-Best” (Russia).
Contents of macroelements and microelements (iron, copper, zinc, strontium) in clinical material were detected by the atomic absorption spectroscopy[18] using atomic absorption spectrometer “СА10MP”. Investigation of the serum IgG, IgA, IgM content was performed using the method of radial immunodiffusion[19].
The level of the total complement was determined in the reaction of its fixation according to 50% hemolysis in standard units CH-50[20].
CIC were revealed by their sedimentation in polyoxyethylene glycol-6000[21]. Calculation of thrombocytes and thrombocyte aggretability were estimated by the “Biola” laser aggregometer[22].
Antithrombin Ⅲ(AT-Ⅲ) and activated partial thromboplastin time (aPTT) (a test that measures clotting time in plasma (the liquid portion of blood); it focuses on a specific pathway in the blood clotting process; normal values for aPTT is 25 to 35 seconds) were detected according to Abilgaard[23] and Caen[24], respectively.
Transmissive microscopy of atheroma samples was done according to Brown[16] with some modifications. Material was fixed with 2.5% glutaric aldehyde on phosphate mixture (pH 7.2), then, treated with 1% solution of osmium oxide during 4 h with addition of 2.5 mM saccharose. After dehydration in spirit of ascending concentration, 100% acetone and oxidopropylene, material was perfused by epon-812. Thin slices obtained using ultramicrotome LKB-Ⅲ (Sweden) were contrasted with the saturated water solution of uranyl acetate and then with the solution of zinc citrate.
Statistical analysis of the obtained data was performed using Statistica program. The data were presented as mean ± standard deviation. Data between three groups (Table 1) were analysed by ANOVA[25]; a P value of <0.05 was considered to indicate significance.
Table 1 Some Hemostatic,Immunologic Parameters and Content of Macroelements and Microelements in the Blood Serum of People under Study
Abbreviation: aPTT=activated partial thromboplastin time; SPA=spontaneous platelet aggregation; IPA=induced platelet aggregation; PR= prothrombin ratio; CIC=circulating immune complexes
RESULTS
As a result of system investigations, mycoplasma infections were revealed in 52% of patients. In the control group, 5% of patients were infected with mycoplasmas. All patients with AMI also had antibodies to some microorganisms associated persistent infections. In the control group,antibodies to cytomegalic virus and rubella were detected (12.5% and 2.5%, respectively).
In the mycoplasma-infected patients, in comparison with non-infected ones, antibodies to cytomegalic virus, toxoplasma and rubella were detected more often,while to chlamidia more rarely (38.5% and 27.3%; 26.9% and 13.6%; 15.4% and 9.3%, 3.8% and 4.6%, respectively;P<0.05). In the mycoplasma-infected group,pathologies of urogenital system were more frequent in comparison with the non-infected cohort (31.8% and 15%, respectively).
As a result of ultracytostructural investigation of coronary artery atheromas in the mycoplasma-infected patients, microbodies with sizes of 0.2～0.6 μm and their structures corresponding to vegetative form of mycoplasmas cells as well as ultramicroforms (nanocells) of mycoplasmas[26] were detected among villi at outer membrane of endotheliocytes and in cytoplasm of macrophages localized in the subendothelial stratum. In this case, statistically significant increased levels of strontium and zinc were detected in the atheromas of the mycoplasma-infected patients (12.28±2.53 μg/ml*; 44.6±2.02 μg/ml**;*P<0.05;**P<0.01, respectively) in comparison with the non-infected ones (2.49±0.29 μg/ml; 24.7±2.71 μg/ml, respectively). In blood serum of the mycoplasma-infected patients, the increased level of strontium was observed while the level of zinc was decreased (Table 1).
The increased levels of IgG, IgM and circulating immune complexes as well as the decreased content of total complement were found in serum of the mycoplasma-infected patients (Table 1).
In the mycoplasma-infected patients, thrombophilia was observed as evidenced by the decreased activated partial thromboplastin time, the increased spontaneous and induced platelet aggretability as well as by tendency to the increased level of fibrinogen (Table 1).
As a result of the investigation of frequency of postinfarction complications in the tested groups of patients a low rate of ventricular tachycardia, ventricular fibrillation, atrioventricular block, premature postinfarction stenocardia as well as a low hospital lethality were observed in the mycoplasma-infected patients(Table 2).
Table 2 Frequency of Postinfarction Complications in AMI Patients (in %)
DISCUSSION
Recently, a lot of reports on high level of distribution of persistent infections with mycoplasmas in humans have been published. The data found in our study that mycoplasmas were detected in 5% of the control group reflect the fact of a high level of distribution of the persistent infection in general but the difference with the control group-52% of mycoplasma infected patients with AMI allowed to implicate uncausal relations of the microbes with the disease. As to AB, the immunologic parameter unlike PCR is known to be not a perfect evidence of presence of infections in patients at the moment of testing. Thus, we may conclude only that prevalence of mycoplasma infections in patients with AMI was found.
Mycoplasmas are believed to be causative agents of many diseases in humans, animals and plants. They are the main contaminants of cell cultures and occur particularly in cells used in biotechnology for producing viral vaccines. It was suggested that mycoplasmas are associated with AIDS, malignancy, chronic fatigue syndrome, Gulf War Syndrome and a variety of other illness as either the primary agent or cofactor[27]. These microorganisms possess unique adaptive abilities allowing to overcome various defense systems of higher eukaryots and to persist in host tissues affecting homeostasis, immune state and hemocoagulation reactions[13].
It was shown in our studies that shift of some parameters for hemostasis and immune system, and content of some micro-and macroelements follow persistence of mycoplasmas in humans[14]. Chronic mycoplasma infections are usually accompanied by increasing vascular wall permeability, disturbance of microcirculation and thrombosis[28]. Indeed, an obvious trend to thrombophilia was observed in the mycoplasma-infected patients. These facts could not be explained by the anticoagulant therapy since it was equal in the groups of patients.
The more frequent cases of inflammatory processes of urogenital system in the mycoplasma-infected patients with AMI may be consequence of the microbe influence on the immune system, microbiocenosis and urogenital tract. It was marked that abnormalities in microbiocenosia[28], respiratory and urogenital tract[13,27] arouse due to mycoplasma persistence, which was followed by coinfections. In these connection the tendency of specific AB presents to some persistent infectious agents found in the examined groups of patients is of a separate interest.
The increased levels of IgG, IgM and circulating immune complexes as well as the decreased content of total complement found in the mycoplasma-infected patients may be an evidence of the immunoreactivity towards infection agents and of destroy its unspecific humoral mechanisms. The content of strontium in the blood serum and atherosclerotic plaques features in the mycoplasma-infected patients with AMI seems to be an evidence of the involvement of the element into pathogenic reactions induced by these microbes, but the fine mechanisms of the process remain to be clarified.
The limited biochemical abilities determine state of dependence of these microbes on higher organisms. Mycoplasmas may reside outside or inside of the host cell but always in the closest contact with the host cell membrane. Due to this fact mycoplasmas are obviously called the “membrane parasites”. It is the “membrane parasitism” that may determine the peculiarities of the pathogenesis in humans with persistence of mycoplasmas[26].
Mycoplasmas are cell-wall-free bacteria. For synthesis of own membrane, mycoplasmas need cholesterol. Extracting of the component from the host cell membranes promotes to destroy of the membrane structure, its antigenic profile and, as a consequence, arising immune response to the infected cell. In this line, the presence of mycoplasmas in the atherosclerosis plaques (also found in our study) may be a risk factor for destabilizing the plaques. Mycoplasmas may trigger destabilization of atheroma and development of AMI according to the known mechanisms of reactivity of nonspecific signal systems towards persistent agents[4,29] (Figure 1).
There are some findings allowed to propose active participation of mycoplasmas in vulnerable plaques, by entering the subendothelial space and creating conditions that favor fat accumulation, dysfunction of the immunological and endothelial response, inflammation and increasing apoptosis, which are fundamental ingredients for plaque rupture[11,12, 30,31]. The bacteria induce depression in the immune response[32, 33], mainly of T cells through possible mechanisms of apoptosis[34,35] with a reduction in the CD4 T cells, which may be a facilitating mechanism for other infections. Chronic oxidative stress is a characteristic of mycoplasma infections[36]. Epitopes of oxidazed LDL being antigenic promote to produce inflammatory reaction in artery wall suggesting a possible autoimmune reaction[37,38]. In this connection, antioxidant agents may be reasonable for preventing complications of atherosclerosis[39,40].
It was reported[41] that despite long-term treatment with a bactericidal antibiotic effective against C. pneumoniae, no reduction in the rate of cardiovascular events was observed in the microbe-infected patients with AMI. In this line, the authors insisted that efforts toward secondary prevention in patients with coronary syndromes should be aimed at proven therapies, including antiplatelet therapy, treatment with beta-blockers and angiotensin-converting enzyme inhibitors, and intensive lowering of lipid levels with the use of statins[41]. Meanwhile recently it was found that C. pneumoniae the most suspected bacterium into atherogenesis and AMI, presents in the plaques mainly with mycoplasmas[11,12]. In this line, coinfection with mycoplasma may be involved into non-efficacy of the antibiotic therapy for the patients with C. pneumoniae. Suppression of mycoplasma infection is a serious problem[13].
Mycoplasmas are able to adapt to unfavorable growth conditions-through transformation of the vegetative cell forms into ultramicroforms (UMF) which are resistant to stress factors and able to revert[26]. The dormant state of the bacterium is followed by shift of its virulence. Our data on transmissive microscopy showed that in the plaques part of the cell population of mycoplasmas were presented mainly as UMF. Unfavorable growth conditions (including antibiotic therapy) may result in prevalence of dormant state of bacteria (UMF) able to revert into active forms (vegetative cell forms) while disappearing stress factors.
Figure 1 Scheme of the possible mechanisms of development of acute myocardial infarction during persistence of mycoplasmas in humans
Abbreviations: EC-endotheliocyte; pM-persistent mycoplasmas; L-arg-L-arginine; NO-nitric oxide; PAF-platelet activating factor; АA-arachidonic acid; COG-cyclo-oxygenase; LOG-lipoxygenase; TO-thromboxan; LT-leukotriene; TC / аTC-thrombocytes / activated thrombocytes; PC / аPC-phagocytes / activated phagocytes; RO-reactive oxygen; ED-endothelial destruction; HCC / аHCC-hemocoagulation cascade/activation of hemocoagulation cascade; FL-fibrinolysis; AC-anticoagulation; AG-changed antigens of own cells; HSP-heat shock proteins; CD-collagen destruction; SMCP-proliferation of smooth muscle cells; AP-atheromatous plaque; TG-thrombogenesis; M-macrophages; Bc-B-cells; Tc-Т-cells (CD4+) whose proliferation (Th1 and Th2 producing proinflammatory and anti-inflammatory cytokines) may be induced or inhibited by mycoplasmas; I/APD-inflammation/atheromatous plaque destabilization; AMI-acute myocardial infarction. Solid line corresponds to activation (→) or inhibition (—<) events
Mycoplasmas are called the “elite” parasites due to their ability to modulate reactivity of specific and nonspecific signal pathways of the host organism. That may probably cause the suppression of pathological processes connected with the activation of superoxide dismutase (SOD) and NO-synthesizing systems producing forms of active oxygen[13,42]. The mycoplasma features provide the phenomenon revealed in our study-a tend to “mild AMI pattern” observed in the mycoplasma-infected patients, but other factors connected with peculiarities of interrelating the host-parasite signal systems due to unique biology of the microbe and individual immunoreactivity, can not be excluded. In this line, an “AMI pattern” arising on the background of other (non mycoplasma) persistent infections is of a special interest.
As it was marked above, the tendency of shifting hemocoagulation reactions and immune state as well as content of strontium in blood serum being similar in mycoplasma infected patients with or without AMI proved to be similar, but quite differ in comparison with the mycoplasma free patients. The data may reflect peculiarities of the mycoplasma influence on the respective systems, and the cardiovascular problems. Taking into account the distribution and ability of mycoplasma infections to transferring by both horizontal and vertical (from mother-to-child) ways[43], an additional aspects of the cardiovascular disease prophylaxis, presuming the detection of the persistent infections (as a possible risk factor for atherosclerosis, coronary disease and AMI) at the beginning seem to require a careful consideration.
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1 Kazan Medical Academy, Kazan, Russia
2 Kazan Institute of Biochemistry and Biophysics, Kazan, Russia
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