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ANTIBODY TITERS TO BACTERIAL ANTIGENS IN COLON DYSBIOSIS
Masharipov Valijon Urinovich
Tashkent Medical Universtitet, Tashkent, Uzbekistan
masharipovvalijon231@ gmail.com
Abstract :
The levels of antibodies to enterobacteria of intestinal microflora in dysbiosis of the
large intestine and in practically healthy individuals were determined. Cellular sensitization to
antigens of intestinal microflora and its significance in extraintestinal manifestations of
dysbiosis (morbidity, susceptibility to infections, allergies) were determined.
The risk groups for secondary immunodeficiency were determined by the magnitude of
antidiv titers to intestinal microflora antigens (antibodies to intestinal microflora as a marker
of immunodeficiency).
The place of the antimicrobial immunity system in the development of immunopathology in
dysbiosis of the large intestine was established.
The presence of antibodies against UPE enterotoxin was revealed in all examined children. A
relationship was established between an increase in the age of children with the detection of
antibodies, as well as an increase in the intensity of formation of antienterotoxic antibodies in
the blood serum of children with dysbiosis of the large intestine.
A relationship was established between dysbiosis of the large intestine and the state of the
antimicrobial immune response of the div of children.
Keywords:
Intestinal Dysbiosis, Intestinal Microflora, IFA, diarrhea, coprofiltrate, protease.
Introduction
Currently, intestinal dysbiosis in various forms is quite common in patients not only with acute
and chronic gastrointestinal diseases, but also with other diseases . Diagnosis of dysbiotic
changs in the intestine presents certain difficulties, since, in addition to microbiological changes,
many clinical features must be taken into account. Despite the fact that in recent years, thanks
to the efforts of many scientists, intestinal dysbiosis has been described in the literature,
previously unknown features of this pathology have been studied, many controversial issues
remain about their clinical significance. In this regard, doctors of many specialties face
difficulties not only in diagnosing this pathology, but also in providing assistance to patients
with intestinal dysbiosis.
A wide range of functions allows us to consider human microflora as one of the most important
factors of homeostasis. At the same time, it is known that normal microflora (mainly its
transient and facultative parts) is not optimal and can acquire the significance of a pathogenic
object for the div under conditions of decreased immunity. This issue is especially acute at
present due to environmental problems and altered immunological reactivity of the majority of
the population.
To date, there is no consensus on the role of microecological changes in the digestive tract in
the etiopathogenesis of most pathological processes. Thus, some authors, when studying the
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intestinal microbiocenosis in individuals with surgical pathology of the biliary tract, revealed a
high (96.6%) frequency of colon dysbiosis. The issues of recognizing the microflora of the
digestive tract as an etiopathogenetic factor in hospital infections and infectious complications
are resolved ambiguously, although it has been established that opportunistic pathogens are
most often gram-negative opportunistic bacteria and cocci. Opportunistic microorganisms
become pathogenic in individuals with microecological and immune disorders, as well as those
with the ability to overcome the main mechanisms of natural immunity and create conditions
for the expression of virulence. The participation of indigenous microflora in the production of
stimulants and activators of phagocytic and enzymatic activity, as well as the ability of
muramyl dipeptides of gram-positive anaerobic and microaerophilic bacteria to stimulate
immunogenesis and activate the system of mononuclear phagocytes, as well as participation in
the regulation of IgA bacterial lipopolysaccharides and stimulation of the synthesis of secretory
antibodies, cytokines and interferon by immunocompetent cells have been well studied.
Normal microflora is a stimulator of plasma cell proliferation. When the integrity of biofilms
normally present on mucous membranes, consisting of cellular mucin, fibronectin and bacterial
exopolysaccharide and microcolonies of bacteria contained within this matrix - representatives
of indigenous microflora, is disrupted, they are replaced by microcolonies consisting of
opportunistic or pathogenic microorganisms. As a result of such a replacement, a local
infectious process is formed, which can subsequently become generalized.
In addition to changes in the organs and systems of the div associated with the development
of the infectious process, especially in generalized forms of dysbiosis, clinical signs of delayed-
type hypersensitivity are noteworthy, which manifest themselves in the form of damage to
internal organs of allergic genesis, especially the myocardium, as well as the intestines, liver,
and others. It is from these positions that the origin of allergic myocarditis and
pseudomembranous colitis in patients with generalized forms of staphylococcal dysbacteriosis
is currently considered.
To date, the participation of endotoxins of gram-negative representatives of the microbiota in
the stimulation of lymphoid tissue and effector cells of the liver, the enhancement of the
mitogenic activity of B- and T-lymphocytes, the implementation of antitumor immunity (in
particular, the secretion of tumor necrosis factor - TNF), the activation of the
polymorphonuclear leukocyte system (neutrophils), which under physiological conditions are
important elements of the antibacterial defense of the div, has been identified. However, the
most important function of endotoxin for the host, apparently, is its ability to carry out antiviral
protection, in particular against human immunodeficiency viruses. According to Dubo, one of
the reasons why the div does not reject the microflora despite multiple interactions is its weak
immunogenicity for the host. This phenomenon may be based on molecular mimicry. Fou and
Lee were among the first to draw attention to this phenomenon, having identified the presence
of common antigens in one of the Basteroi strains.
Materials and Methodology
For the set tasks we examined children aged from 3 to 14 years. Among the examined children,
intestinal dysbiosis of III and IV degree was detected in 66 children. They were included in the
main group. The control group consisted of 20 practically healthy children of the same gender
and age composition. For comparison with the main group, children with various acute
intestinal infections were also studied for comparison: bacterial dysentery - 20 children;
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salmonellosis - 27 children; gastroenteritis caused by opportunistic flora (E. coli, S. aureus,
Proteus sp., Klebsiellaе sp.) - 42 children. The gender and age composition of these examined
children were identical to the main and control groups. The subject of the study was the study
of normal microflora of the colon, the study of antidiv titers in blood serum and coprofiltrates,
and antiendotoxic antibodies in blood serum. In the course of the scientific work,
bacteriological, bacterioscopic, serological, immunological, IFA, and statistical methods were
used.
Results
The criterion of the etiological significance of UPE should be considered a set of indicators,
among which one of the most important is the detection of specific serum antibodies to the
antigens of the suspected pathogen. False positive results due to cross-reacting antibodies in the
agglutination reaction with the autostrain are observed in no more than 5-10% of healthy
individuals and carriers. It should be especially emphasized that in healthy young children,
positive and false positive reactions are not observed due to the imperfection of the immune
system and a short period of antigen stimulation. Based on the above, the next stage of our
research was to study the level and evaluate circulating antibodies to UPE in children with
intestinal dysbiosis. To determine the intensity of antidiv formation to various UPE antigens,
the average geometric titers of antibodies were calculated, expressed as negative logarithms
with a base of 2 (- log2). When assessing the reliability of the difference between the indicators,
when the number of one compared group exceeded the number of the other by at least 25%, the
error in the difference of relative indicators in percent was calculated using the formula for
unequal samples
Specific antibodies to UPE antigens were found in the blood serum of most of the examined
children. A positive result was obtained in 80.0±2.6%, and the immune response of the div
was found with a high frequency in children with the UPE association - mixed autostrains of all
the listed representatives of the colon microflora, than in children from whom mainly
monocultures were isolated.
The results of determining antibodies to UPE representatives showed comparable results for 6
representatives of the Enterobacteriaceae family - seronegative results were 19.7%-30.3% of
sera, respectively, seropositive 69.7-80.3%. For P.aeruginosaе, the seropositive sera were
slightly higher, but statistically insignificant, compared to other antigens (seronegative - 15.2%,
seropositive - 84.8%).
However, for all 7 antigens, the frequency of seropositive sera was significantly higher than
seronegative (p < 0.001). This is especially true for E. coli (80.0 ± 8.9%), C. freindii (80.0 ±
8.9%) and P.aeruginosaе (85.0 ± 7.9%). The percentage of detection of which was higher than
other microorganisms. Apparently, these pathogens colonized the intestine and aggravated
dysbiosis of the large intestine in children.
Study of the intensity of antidiv formation to the studied UPE and P.aeruginosaе, all children
under 2 years of age were divided into the following groups: 1 - group children under 6 months;
2 - group children aged 7-9 months, 3 - group aged 10-13 months and 4 - group aged 13-24
months.
The obtained results show that the level of specific immunity in the form of the appearance of
antimicrobial antibodies increases regularly from group 1 to group 4. It is noteworthy that in
groups 3 and 4 a higher antidiv titer was established (- log2 7.5 to - log2 4.3) than in groups 1
and 2 - 4.0-4.2 (in - log2) - p < 0.05 (Fig. 1.)
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Fig. 1. Comparative indices of intensity of antidiv formation against UPE antigens
Discussion
The levels of antibodies to intestinal microflora bacteria in colon dysbiosis and in healthy
individuals were determined. Cellular sensitization to intestinal microflora antigens and its
significance in extraintestinal manifestations of dysbiosis (morbidity, susceptibility to infections,
allergies) were revealed. Risk groups for secondary immunodeficiency were determined based
on the titers of antibodies to intestinal microflora antigens (antibodies to intestinal microflora as
a marker of immunodeficiency). The role of antimicrobial immunity systems in the
development of immunopathology in dysbiosis was established. An IFA method adapted for
screening tests was developed to determine antibodies to enterobacteria antigens. A scheme for
correcting intestinal dysbiosis was developed to reduce cellular sensitization to intestinal
bacterial antigens. A relationship was established between intestinal dysbiosis and the state of
the antimicrobial immune response.
Conclusions
1. Differences in the frequency of serum antibodies in the blood of the examined practically
healthy children were established. The detected titers of antibodies to UPE antigens (E. coli, P.
vulgaris, C. freundii, K. pneumoniae, E. aerogenes, E. cloacae, P. aeruginosaе) had a wide
range of variation, on average from 13 to 29%. The conducted division of the groups of
examined subjects into 5 indicators (sharply positive, positive, weakly positive, doubtful,
negative), depending on the values of the antidiv titer in the blood of healthy people
allows for relative normalization. 2. Specific antibodies to UPE antigens were detected in
80.3% of the studied children with grade III-IV intestinal dysbiosis, the div's immune
response was found with a high frequency in children with an association with UPE. It was
found that the number of seronegative indicators was 2.5-3 times lower than that of seropositive
sera with all the studied UPE antigens, and for P.aeroginosa, the number of seropositive sera
was slightly higher in relation to UPE antigens (p<0.05). With increasing age of the studied
children, the level of specific immunity in the form of antimicrobial antibodies significantly
increased (p<0.05).
3. The proposed experimental test system from antigens of collection E.coli strains for the IFA
method is distinguished by sensitivity and specificity with various opportunistic enterobacteria.
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4. All the examined children - with dysbiosis of the large intestine, gastroenteritis caused by
UPE and practically healthy children - were found to have antibodies against UPE enterotoxin
(except for healthy children under 3 years old). With increasing age of children, the detection of
antibodies increases, the indicators of questionable and negative results decrease. The intensity
of formation of antienterotoxic antibodies in the blood serum was significantly higher in
children with colon dysbiosis (p<0.05) than in healthy children and children with gastroenteritis
caused by UPE.
5. In children with colon dysbiosis, strains of colon microflora appear that produce proteases of
immunoglobulin-destroying activity. This is especially characteristic of the total and thiol
activity of proteases. The method for determining the immunoglobulin protease activity of
coprofiltrates can be used as an additional diagnostic test for the diagnosis of colon dysbiosis in
children. 6. Introduction of a biological preparation into the course of treatment normalizes the
composition of normal microflora of the large intestine; a positive effect of biocorrection is also
observed when studying sIgA in blood serum and coprofiltrates (p<0.05), as well as on the
concentration of serum immunoglobulins - IgM, IgG and IgM (p<0.005).
7. When analyzing the results of IFA, it was found that the percentage of seropositive sera with
antigens from the UPE decreased from 1.9 times to 2.7 times. Along with the percentage of
seropositive sera, the intensity of antidiv formation against antigens from the UPE also
decreased.
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