# Vol 24, No 1 (2021)

## INTERNATIONAL DOCUMENTS

### Translation into Russian of the Classification of inborn errors of immunity in humans updated by experts from a Committee on Congenital Immunity Errors of International Union of Immunological Societies (Russian version 2019)

Bolkov M.A., Tuzankina I.A., Shinwari K., Cheremokhin D.A.

#### Abstract

We present to Russian-speaking audience a translation and commentary on the classification of inborn errors of immunity presented at the end of 2019 by the Committee on Congenital Immunity Errors at the International Union of Immunological Societies (IUIS). Inborn errors of immunity, or, as they were called earlier, primary immunodeficiencies, is a rapidly expanding class of diseases that includes the most diverse congenital pathologies which can manifest at any age by heterogenous symptomes. Clinical masks characterize these diseases, hence the time from the onset of clinical disorder to the final diagnosis may take many years. A doctor of any specialty encounters these patients, and the molecular mechanisms of pathology concern different organs and systems of the patients. The classification consists of ten tables covering more than 400 syndromes and their corresponding genes, or associated chromosomal abnormalities. This is a tool, which allows navigating a wide variety of different primary immunodeficiencies, autoimmune and autoinflammatory syndromes, complement defects, and bone marrow failure syndromes. We hope that, due to translation, current knowledge about these various diseases will become more close and available to the Russian-speaking audience.

Russian Journal of Immunology. 2021;24(1):7-68
7-68

## ORIGINAL ARTICLES

### Macrophage differentiation in the tissues of myomatous nodes, depending on MRI pattern

Sotnikova N.Y., Malyshkina A.I., Voronin D.N., Nagornyi S.N., Voskresnskaya D.L.

#### Abstract

Macrophage differentiation is known to be regulated by specific microenvironment invaded by these cells. However, despite numerous studies on pathogenesis of uterine leiomyoma, a common benign tumor of reproductive system, the features of macrophage polarization within myoma nodes are still scarcely studied. The aim of our work was to reveal some differentiation features of macrophages which invade the tissues of myomatous nodes in the patients with different types of uterine leiomyoma (UL) dependent on MRI patterns observed. We have performed a study of 42 patients in their reproductive age with intramural UL. All the patients were subjected to MRI of pelvic area. Twelve samples of endometrium have been taken from healthy women without any signs of UL, being used as controls. UL biopsies and endometrium in its projection served as study material. Phenotype of endometrial and UL-invading macrophages was evaluated by means of multi-color flow cytometry. Expression of Activin А and RARα mRNAs was estimated in endometrial and myoma node macrophages. Likewise, collagen type 1 mRNA expression was evaluated by means of reverse-transcription real time PCR. The collagen type 1 concentration in myomatous nodes was assessed by ELISA technique. We have revealed that peripheral blood monocytes, macrophages in endometrium, and UL nodes each consist of three different subpopulations, dependent on expression levels of expression levels of CD14 and CD16 membrane receptors. For endometrium projected onto myomatous node, the ratios of “intermediate” macrophages (CD14++CD16+), and alternatively activated macrophages (CD36+) was increased, thus exerting potentially negative effects upon reproductive functions in women with uterine leiomyoma. Immediately in myomatous tissue, we have found a shift of macrophage differentiation from ‘classic’ forms towards “intermediate” and “non-classical” macrophages associated with alternative activation. However, the percentage of scavenger receptor-expressing macrophages (CD36+, CD206+) was decreased in the myoma nodes. Enhanced expression of RARα mRNA was observed in macrophages invading the myomatous nodes, whereas Activin А synthesis was higher in the macrophages invading leiomyomas with MRI pattern of simple and degenerative nodes. Imbalance between “intermediate” and “non-classical” UL-invading macrophages was associated with fibrosis, or degenerative changes of myomatous tissues, thus, probably, representing an important pathogenetic link in development of different clinical variants of uterine leiomyoma.

Russian Journal of Immunology. 2021;24(1):69-76
69-76

### Changes in cytokines and urothelial factors of urine in interstitial cystitis/painful bladder syndrome

Sholan R.F.

#### Abstract

Interstitial cystitis/bladder pain syndrome (IC/BPS) is thought to have an autoimmune component due to increased prevalence of certain autoimmune conditions in the patients. Recent studies have revealed abnormalities in cytokine signaling in cultured urothelial cells of the urinary bladder. However, there is only scarce information on concentration of cytokines and some biomarkers, e.g., heparin-binding growth factor (HB-EGF) and epidermal growth factor (EGF), in the patients with IC/BPS. The purpose of present study was to determine concentrations of cytokines, HB-EGF and EGF in urine and their interrelations in patients with IC/BPS. 68 women with IC/BPS and 20 women without this disease (control group) were examined. The average age of women with IC/BPS was 54.2±12.4 years, the control group was 35.3±9.7 years. The morning urine samples were tested for interleukin contents (IL-1β, IL-6, IL-8), tumor necrosis factor-α (TNFα), as well as HB-EGF and EGF concentrations measured by ELISA technique. Statistical analysis of the obtained results was performed using Statistica software in Microsoft Excel. A relationship between the indexes was calculated with Pearson correlation quotient. The ratios for Th1/Th2 cytokines were calculated. Urination frequency, clinical symptoms including pollakiuria, nocturia and its urgency, were significantly more often in patients with IC/BPS compared to the control group (p < 0.05-0.001). In patients with IC/BPS, the average levels of IL-1β, IL-8, IL-6 and TNFα exhibited, respectively, 2.4-, 2.3- and 2.0-fold increase over control values (p < 0.05). The HB-EGF and EGF excretion in the urine was 2.3 times (p < 0.05) over control levels. The IL-1β/IL-6 ratio averaged 1.16 in the group of patients with IC/BPS versus 0.95 in the control group, and the TNFα/IL-6 ratio was 1.0, respectively. There was a positive correlation between the levels of IL-1β and IL-6, TNFα (p < 0.05), HB-EGF and EGF, and a negative correlation with IL-8 (p < 0.01). In women with IC/BPS, there was a weak multidirectional correlation between IL-6 and other cytokines, as well as with HB-EGF and EGF contents. IL-8 levels in both groups showed weak correlation with other indexes. The TNFα amount weakly correlated with HB-EGF and EGF in IC/BPS. Based on the results obtained, one may state that, in IC/BPS, the excretion of pro-inflammatory cytokines was significantly increased, the balance of Th1/Th2 cytokines and contents of growth factors are disturbed. Expression of cytokines in combination with growth factors (HB-EGF and EGF) can be used to explain the pathophysiology of the clinical features in IC/BPS. Analysis of cytokine profiles may be used for differential diagnosis of IC/BPS and other disorders of urinary bladder.

Russian Journal of Immunology. 2021;24(1):77-84
77-84

### Polymorphism of genes controlling phase I and II detoxification in phenol-exposed women with spontaneous miscarriage diagnosis

Kazakova O.A., Dolgikh O.V.

#### Abstract

The environmental issues in contemporary megapolis require studying of multiple candidate genes that may contribute to occurrence of reproductive disorders. Exogenous phenol compounds are contaminants that produce negative effects upon female reproductive system. Detoxification genes from CYP450 and GSTs family belong to the I and II detoxification phases of xenobiotics including phenols. Our goal was to examine some features of polymorphism in I and II phase detoxication genes in women with diagnosed miscarriage who were subjected to excessive phenol exposure. The test group consisted of 37 women who had miscarriage; the reference group included 41 conditionally healthy women. All the examined women lived under airborn exposure to phenol (an average of > 1.0 daily minimal acceptable concentration). The following parameters were examined in both groups: phenol contents in blood were detected with capillary gas chromatography; polymorphisms of CYP1A1 rs1048943 Ile462Val, CYP1A1_3 rs4646421 C6310T, GSTA4 rs3756980 T/C, GSTP1 rs1695 Ile105Val, GSTP1 rs1138272, and Ala114Val genes were revealed with polymerase chain reaction. There were significant discrepancies between the examined groups, both for phenol contents in blood, compared to upper standardized limits (p < 0.05). The examined gene polymorphisms fit the Hardy–Weinberg rule. Statistical analysis in multiplicative inheritance model allowed us to show that A allele in CYP1A1 rs1048943 Ile462Val gene, and A allele in GSTP1 rs1695 Ile105Val gene could be the factors associated with probable miscarriage risk in case of excessive contamination of biological media with exogenous estrogens (e.g., phenol). The examined gene polymorphisms may be suggested as marker genes for early prediction of miscarriage risk, when excessive contents of exogenous estrogen imitator (phenol) are present in biological media.

Russian Journal of Immunology. 2021;24(1):85-90
85-90

### Variability study of immune status modified by polymorphic genetic profile in women with pregnancy loss under hydroxybenzene exposure

Kazakova O.A., Dolgikh O.V.

#### Abstract

The study was performed in Perm, an industrially developed Russian city where hydroxybenzene concentrations are excessive due to dust and gaseous emissions from non-ferrous metallurgic plants. Their concentrations exceed average daily and single maximally permissible amounts. Hydroxybenzene and its derivatives are hormone-like substances, thus being hazardous to female reproductive system. The goal of our study was to analyze variability of immune state influenced by polymorphic genetic profile of women with pregnancy loss under hydroxybenzene exposure. Chemical analysis of ambient air quality was performed according to MG 4.1.617-96 regulations. Biological samples were taken from 129 women with reproductive disorders at their fertile age during the five-year observation period. Hydroxybenzene contents in blood were detected with gas chromatography with inductively coupled plasma. The test group 1 included women with reproductive disorders and phenol contents in blood exceeding the reference level (> 0.016 mg/cm3 ). Test group 2 consisted of women with reproductive disorders and phenol contents in blood within standard ranges. The control group 1 consisted of conditionally healthy women with phenol contents in their blood being higher than 0.016 mg/cm3 . Control group 2 included conditionally healthy women with phenol contents in their blood corresponding to the standard values. Serotonin content in blood serum considered catecholamine regulation marker was estimated with ELISA technique; phenol-specific IgG contents were measured by means of modified competitive ELISA method. PCR technique was used for detection of ACTN3 rs1815739 gene polymorphism associated with catecholamine metabolism. We found that a half of the total study group were exposed to phenol and cresols at concentrations > 1.0 MPC average daily and single maximal dose. Incidence of endometriosis among them tended to increase over the examined 5-year periodL this disorder is known to contribute to pregnancy loss. Chemical analysis of blood allowed us to reveal phenol contamination in all the women to varying degrees. Regression analysis allowed to reveal a significant dependence between phenol contents in blood and phenol-specific IgG, as well as significant correlation between blood serotonin contents and cresols concentrations (p < 0.05). Kruskal–Wallis test revealed significant intergroup differences by serotonin levels. There were significant differences between the test groups 1 and 2 by serotonin contents (at p < 0.0083 with Bonferroni correction). Frequencies of ACTN3 rs1815739 genotypes, using a multiplicative model, allowed us to assign T allele to the factors contributing to risk of reproductive losses induced by biological media contaminated with phenol in excess. Cross-classification analysis revealed a causal relationship between serotonin contents and ACTN3 rs1815739 genotype.

Russian Journal of Immunology. 2021;24(1):91-100
91-100

### Immune reactions to clinical carcinogens and steroid hormones in breast pre-cancer and cancer patients

Glushkov A.N., Polenok E.G., Gordeeva L.A., Mun S.A., Kostyanko M.V., Kolpinckiy G.I., Vafin I.A., Antonov A.V., Verzhbitskaja N.E.

#### Abstract

Detection of postmenopausal women at high risk for breast pre-cancer and cancer is a key condition to prevent these diseases. Aim of our research was to study possible usage of immunoassay for antibodies specific to benzo[a]pyrene, estradiol, and progesterone (IgA-Bp, IgA-Es, IgA-Pg) in determination of personal risks for fibrocystic disease and breast cancer at the early stage, with respect to hormone receptor status in tumor tissues. Blood serum IgA-Bp, IgA-Es, IgA-Pg were studied by ELISA in postmenopausal women: healthy controls (n = 401), patients with fibrocystic breast disease (n = 50), and breast cancer (stage I, n = 575, stages II-IV, n = 861). High individual IgA-Bp/IgA-Pg ratios of > 1.5 were found in 19.7% of healthy women, and in 50.0% of fibrocystic breast disease patients (p < 0.0001; OR = 4.1). IgA-Es/IgA-Pg ratios of > 1.0 were revealed in 48.4% healthy women and in 68.0% fibrocystic breast disease patients (p < 0.01; OR = 2.3). IgA-Bp/IgAPg values > 1.0 were found in 41.9% of healthy women, and, at higher rates, in the patients with breast cancer stage I: 68.3% ER- tumors (p < 0.0001; OR = 3.0) and 75.9% ER+ tumors (p < 0.0001; OR = 4.4). IgA-Es/ IgA-Pg ratios > 1.0 were revealed in 48.4% of healthy women, and in patients with breast cancer stage I: 65.3% ER- tumors (p < 0.003; OR = 2.0), and 76.8% ER+ tumors (p < 0.0001; OR = 3.5). Some associations of studied antibodies with cancer progression were revealed. Frequency of individual cases with IgA-Bp/IgA-Pg > 1.0 in patients with ER- tumors increased from 12.0% at stage I to 19.9% at stage II. Frequency of cases with IgA-Bp/IgA-Pg > 1.0 in the patients with ER+ tumors decreased from 62.0% at stage I to 57.3% at stage II (p = 0.002). Frequency of cases with IgA-Es/IgA-Pg > 1.0 in the patients with ER- tumors increased from 11.5% at stage I to 21.4% at stage II. Frequency of cases with IgA-Es/IgA-Pg > 1.0 in patients with ER+ tumors decreased from 63.3% at stage I to 56.1% at stage II (p < 0.001). The cases with individual excessive IgA-Bp and IgA-Es levels are associated with fibrocystic breast disease and ER+ breast cancer at the onset of the disease. Breast cancer progression was associated with the relative decrease of ER in tumor tissues, along with higher individual levels of IgA-Bp and IgА-Es and lower IgA-Pg levels. ELISA testing of IgА-Bp, IgА-Es, IgA-Pg could be recommended for detection of individual risk for fibrocystic breast disease and stage I of breast cancer, as well as for more efficient prevention and therapy by selective modulators of estrogen receptor (raloxifene, arzoxifene and lasofoxifine) and aromatase inhibitors (exemestane, anastrozole).

Russian Journal of Immunology. 2021;24(1):101-108
101-108

### Features of sensitization to molds and its role in development of respiratory allergic diseases

Gervazieva V.B., Samoylikov P.V., Vasilyeva G.V.

#### Abstract

Over recent decades, a steady increase in the number of allergic diseases has been shown. Current evidence demonstrate a close association between their emergence and exposure to fungal allergens. In this regard, the aim of the present study was to identify frequency and structure of sensitization to the most clinically significant molds in the patients with respiratory allergic diseases. In blood serum of 283 patients with allergic rhinitis and bronchial asthma, we determined total IgE and sIgE to the mold allergens: Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus and Alternaria alternata by the ImmunoCAP method (Phadia, Sweden). Statistical analysis was carried out by nonparametric methods. The total IgE levels (420 (225.5-641) kU/l) were higher (p < 0.05) in patients with sensitization to fungal allergens than in general group (296 (129- 530) kU/l). Multiple sensitization to respiratory allergens was revealed in the patients with allergic rhinitis and bronchial asthma, and sensitization to fungal allergens was associated with increasingly severe manifestations of the disease. In the patients with fungal allergies, sIgE to Alternaria alternata was most often detected (92.5%), with average level of 3.52 (0.635-19.525) kUA/l. Sixteen patients (40%) were sensitized to Aspergillus fumigatus (0.14 (0.06-0.63) kUA/l). In 19 patients (47.5%), we found increased levels of sIgE to Cladosporium herbarum (0.29 (0.045-1.005) kUA/l). Sensitization to Penicillium notatum was detected in 12 patients (30%), the sIgE levels were 0.125 (0.01-0.5) kUA/l. Detection rates in the total group of fungus-allergic patients with respiratory allergies were as follows: Penicillium notatum, 4.2%; Cladosporium herbarum, 6.7%; Aspergillus fumigatus, 5.6%; Alternaria alternata, 13.07%. We found a significant correlation (p < 0.05) between the sIgE contents to different fungal allergens. The levels of IgE antibodies to Alternaria alternata correlated with the levels of sIgE to other fungi (Aspergillus fumigates, r = 0.45; Cladosporium herbarum, r = 0.39; Penicillium notatum, r = 0.39). These findings allow us to suggest that sensitization to Alternaria alternata (13.07%) and Cladosporium herbarum (6.7%) is most common among the patients with allergic rhinitis and bronchial asthma, whereas fungal sensitization aggravates clinical course of these diseases. Determination of sIgE to Alternaria alternata can serve as a marker for the presence of potential cross-sensitization to other fungal allergens, i.e., Aspergillus fumigatus, Cladosporium herbarum, and Penicillium notatum.

Russian Journal of Immunology. 2021;24(1):109-114
109-114

### New opportunities of immunocorrection in complex treatment of cervical cancer patients

Menshenina A.P., Zlatnik E.Y., Sagakyants A.B., Moiseenko T.I., Ushakova N.D., Frantsiyants E.M., Verenikina E.V., Chalabova T.G.

#### Abstract

Etiologic role of human papilloma virus in cervical carcinoma is well known so this might be the base of application of interferonogenicimmunomodulators in their complex treatment. Evaluation of the role and place of immunotherapy and immunocorrection in combination treatment is one of the urgent problems in both clinical immunology and oncology. In this study we used one of them – allokin-alpha (alloferon) – together with plasmapheresis which contributes to detoxication and enhancement of the tumor cells sensitivity to cytostatics in complex treatment of locally advanced cervical cancer patients receiving neoadjuvantpolychemotherapy (NCT). Clinical effect of such treatment was described in our previous studies. The purpose of the present research is to study the effect of the application of immunomodulatorallokin-alpha (A) and plasmapheresis (PP) in complex treatment of cervical cancer patients on their cell-mediated immunity and cytokines composition in serum. Cervical cancer patients with locally-advanced tumors were divided into two groups. The control one received NCT, the basic one consisted of two subgroups – the 1st one in the course of NCT additionally received procedures of gravitational PP, the 2nd one received NCT, PP and 6 injections of allokin-alpha. Immunological parameters was tested in dynamics by flow cytometry and ELISA. Dynamics of cell-mediated immunity parameters revealed the decrease of CD19+ cells in patients of all the groups, of Treg in both subgroups of the basic group; of lymphocytes, CD3+ and CD8+ cells in patients after NCT + PP; activated CD3+HLA-DR+ cells in patients after NCT + PP + A though the amount of CD8+HLA-DR+ was elevated. When PP was applied CD3+ cells levels was found to be lower than after additional administration of A. Analysis of the cytokines levels showed the increase of IL-8 after NCT + PP, but administration of A caused its decrease. IL-10 level was minimal in patients having received NCT + PP + A. Application of NCT + PP and NCT + PP + A prevented the elevation of inflammatory cytokines levels (IL-1β and TNFα) which was observed in patients of the control group after the course of neoadjuvant treatment. Such a dynamics of cytokines levels suggests that application of allokin-alpha together with plasmapheresis and neoadjuvant chemotherapy might be useful in complex treatment of cervical cancer patients with locally advanced tumors.

Russian Journal of Immunology. 2021;24(1):115-122
115-122

### Effect of antiplague vaccination on phagocytic activity of human blood granulocytes

Kravtsov A.L., Klyueva S.N., Kozhevnikov V.A., Kudryavtseva O.M., Bugorkova S.A.

#### Abstract

Anti-plague vaccination stimulates phagocytosis of Yersinia pestis cells by blood leukocytes of laboratory animals. However its effect on the phagocytic activity of human white blood cells towards plague microbes has not been studied. In this work, flow cytometry was used to study phagocytic activity of blood granulocytes towards Y. pestis, Escherichia coli and Staphylococcus aureus in the people vaccinated, or not vaccinated against plague. These persons inhabited the territories of natural plague foci in Russian Federation. The results of phagocytic activity were evaluated in microvolumes of whole blood. The induced IFNγ production was evaluated in blood by enzyme immunoassay technique, and the titers of specific antibodies to plague-specific F1 antigen were determined in blood serum samples. It was found, that the subjects who have never been vaccinated against plague had 3-fold lower granulocyte phagocytic activity towards Y. pestis than the persons repeatedly vaccinated with live plague vaccine, and 5-fold lower than phagocytosis of E. coli and S. aureus. The next annual revaccination caused additional stimulation of in vitro phagocytic capacity of blood leukocytes with respect to plague microbes. Individual values of phagocytic indices correlated with serum antibody titers (r = 0.65, p = 0.0004), and with levels of IFNγ production (r = 0.73, p = 0.0004). Thus, the obtained data confirmed dependence between phagocytic activity of human blood leukocytes and the pathogen type, thus allowing to demonstrate a connection between activated phagocytic response to Y. pestis cells in live plague-vaccinated individuals with other immune indexes of vaccine efficiency (specific antibodies to F1, induced IFNγ production). Further research in this direction will bring us closer to development of an informative test for assessing the intensity of antiplague immunity.

Russian Journal of Immunology. 2021;24(1):123-132
123-132

## SHORT COMMUNICATIONS

### Pathogenetic rationale for usage of recombinant interleukins in the patients with mandibular fractures to prevent post-traumatic osteomyelitis

Paskova E.V., Markelova E.V., Golitsyna A.A., Rusakova E.Y., Romanchuk A.L., Nevezhkin D.A., Knysh S.V.

#### Abstract

The paper presents the results on spontaneous and induced production of IFNγ, IL-4, IL-13 cytokines in blood cells of patients with mandibular fractures and post-traumatic osteomyelitis. Osteomyelitis of the jaw represents one of the urgent challenges in modern medicine. There are many reasons for development of purulent necrotic processes of the jaw bones, including disorders of innate and adaptive immune response. Currently, the immunological aspects of post-traumatic complications of maxillofacial region remain poorly understood. There are no unambiguous and systematic studies of immune mechanisms in pathogenesis of post-traumatic osteomyelitis of the lower jaw. The aim of our study was to theoretically confirm application of recombinant interleukins (IL-1β, IL-2, IFNγ) in the patients with jaw fractures, in order to prevent osteomyelitis, as based on the studies of spontaneous and induced cytokine production (IFNγ, IL-4, IL-13). Spontaneous and stimulated production of IFNγ, IL-4, and IL-13 cytokines by blood cells was determined using specific reagent kits from R&D Diagnostic Inc. (USA). The results were recorded using an ELISA Multiscan analyzer (Finland). Statistical significance of intergroup differences was determined by the Mann–Whitney method. The level of statistical significance at which the null hypotheses were rejected was < 0.05. To stratify the cases of post-traumatic osteomyelitis and uncomplicated mandibular fracture, the models were developed with a singlelayer neural network, using the nnet R-studio package. To assess quality of the models, the areas under the ROC curves (AUC), Akaike criterion (AIC), and the relative classification accuracy (OTC) were used, which was determined as the ratio of correctly established model diagnosis numbers to the total number of patients. The study results demonstrate that the patients with mandibular fractures exhibit a moderate impairment of LPS-induced IL-4 and IFNγ production by leukocytes with IL-13 activation. In presence of osteomyelitis, this imbalance is promoted, thus suggesting an impaired ability of the cells to produce IL-4 and IFNγ. An opportunity for usage immunomodulation when treating the patients with mandibular fractures is represented by P = 1/(1+e-z) where z is defined via IL-1β, IL-2 and IFNγ levels on the first day of observation. In vitro supplementation with recombinant IL-1β and IFNγ modulates cell function, improving the cytokine profile. The prognostic models, imitating binary logistic regression, were used to demonstrate that recombinant IL-1β could be used to prevent patients with mandibular fractures from developing post-traumatic osteomyelitis (AIC = 13.2, AUC = 0.96, р = 0.026).

Russian Journal of Immunology. 2021;24(1):133-140
133-140