The Experience of Using Multipotent Mesenchymal Stromal Cells in the Treatment of Severe Recurrent Cholangitis in Children with Biliary Atresia after Kasai Surgery.

This article describes the experience of application of multipotent mesenchymal stromal cells in the complex therapy of severe recurrent cholangitis in 2 children with biliary atresia after Kasai surgery. In both children, hepatic cellular insufficiency and portal hypertension developed against the background of long-term inflammatory process poorly controlled by standard therapy, which was the indication for liver transplantation. During the course of mesenchymal stromal cells therapy, the relief of the inflammatory process and functional recovery of the liver were achieved. At the time of preparing the article, the follow-up of two children since the start of multipotent mesenchymal stromal cell therapy was 3 years 9 months and 2 years 6 months. No recurrence of cholangitis was observed in the patients during the follow-up period, the liver function was preserved. There are no indications for liver transplantation at this moment. Thus, despite the fact that the mechanisms of therapeutic action of multipotent mesenchymal stromal cells in biliary atresia require further investigation, we obtained promising results suggesting the possibility of using mesenchymal stromal cells in the treatment of postoperative complications in children with biliary atresia.

[The use of a complex of natural antimicrobial peptides and cytokines for treatment of male infertility and chronic prostatitis].

INTRODUCTION: Chronic prostatitis (CP) has long been considered one of the common causes of male infertility. The influence of therapy for CP on spermatogenesis is well studied. However, recently, especially in the era of the new coronavirus infection (COVID-19), much attention has been paid to the effects of cytokines on the pathogenesis of the inflammatory process, and their impact on male fertility. AIM: To study the effect of a complex of natural antimicrobial peptides and cytokines (Superlymph) on ejaculate, MAR test, oxidative stress (OS), and sperm DNA fragmentation (SDF) in men with infertility and a history of CP. In some patients, the level of cytokines before and after treatment was determined. MATERIALS AND METHODS: A total of 583 infertile men with a history of CP, aged 18-75 years, were screened according to the inclusion/ exclusion criteria. In group I, patients received antioxidant stimulation of spermatogenesis (triovit, trental, mexidol) in combination with a complex of natural antimicrobial peptides and cytokines (Superlymph) one suppository 25 IU at night for 20 days for 1-3 weeks and for 6-8 weeks for 2 months. In group II, antioxidant stimulation of spermatogenesis was prescribed along with suppositories for the prevention of CP for 2 months. The evaluated criteria included sperm analysis, MAR test, oxidative stress in spermatozoa (reactive oxygen species (ROS)) and SDF. The levels of seminal plasma cytokines were also measured before and after treatment in 32 patients using the Bio-Plex Pro panel for the simultaneous determination of 17 human cytokines, the Human Cytokine 17-plex Assay. Statistica-10 software was used for statistical analysis (p<0.05). RESULTS: Of 135 eligible patients, results were collected from 125 men (mean age 31.7 +/- 13.4 years). In group I, there was a more pronounced increase in motility by 42.3%, a decrease in the MAR-test level by 64.7%, oxidative stress in spermatozoa by 56.1%, and SDF by 25.6%, compared to group II (30.4 %, 10.5%, 45.7%, 21.9%), respectively. All these changes were significant with the exception of a decrease in the level of SDF. In group I, there was a predominant increase in the activity of anti-inflammatory cytokines (IL-4, 10, 13), a decrease in the level of pro-inflammatory cytokines ((IL-2, 12). In group II, there was a significant decrease in both pro-inflammatory (IL-2, 6, 8), and anti-inflammatory cytokines (IL-4). The remaining changes were insignificant. Among mild side effects, there were a change in the color of the sclera in 4 (3.2%), anal pain in 3 (2.4%), a decrease in libido in 2 (1.6%) patients. No differences between groups in the frequency of side effects were recorded (p>0.05). CONCLUSION: Antioxidant stimulation of spermatogenesis in infertile men and CP is an effective and safe. The addition of a complex of natural antimicrobial peptides and cytokines (Superlymph) leads to a more profound increase in sperm motility, a decrease in the MAR-test, oxidative stress in sperm cells, which potentiates the effects of therapy. The use of Superlymph is accompanied by a more pronounced positive effect on pathospermia, an increase in anti-inflammatory and a decrease in pro-inflammatory cytokines. In the contrary, in control group, where patients received antioxidant stimulation of spermatogenesis, only a decrease in the level of main cytokines was observed. The effect of a complex of natural antimicrobial peptides and cytokines (Superlymph) on the level of SDF, as well as the relationship with fertility and childbirth rates, requires further study.

[Evaluation of aqueous cytokine levels in eyes with Fuchs endothelial corneal dystrophy and bullous keratopathy]. / Opredelenie urovnya tsitokinov vo vnutriglaznoi zhidkosti pri endotelial'noi distrofii rogovitsy Fuksa i bulleznoi keratopatii.

PURPOSE: To evaluate cytokine levels in the aqueous humor (AH) of patients with Fuchs endothelial corneal dystrophy (FECD) and bullous keratopathy (BK). MATERIAL AND METHODS: The study included 74 patients (74 eyes). The first group consisted of 31 patients (72.7±9.2 years) with FECD; the second group included 35 patients (72.4±9.1 years) with BK. The control group comprised 8 patients (74.3±4.1 years) with immature cataract. Before surgery, patients underwent pachymetry of the central cornea (RTvue-100 OCT, Optovue, USA). Patients of groups 1 and 2 underwent endothelial keratoplasty (DSAEK or DMEK), or penetrating corneal transplantation. Patients of the control group underwent phacoemulsification with implantation of intraocular lens. The initial stage of the surgery involved AH sample collection for evaluation of cytokine levels (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, MCP-1, MIP-1ß and TNF-α) by fluorescent flow cytometry using the Bio-Plex Pro Human Cytokine Panel, 17-plex (Bio-Rad, USA). RESULTS: Multiplex analysis of the AH content did not show any statistically significant differences in cytokine levels between decompensated FECD and BK eyes. The levels of IL-6, IL-8, GM-CSF, IFNγ, MCP-1, MIP-1ß were significantly elevated in FECD and BK eyes compared with healthy control. An insignificant deviation of IL-4 and IL-13 levels was detected in FECD and BK eyes compared with healthy controls. There were no significant differences in IL-1ß and TNF-α (indicators of acute inflammation) between the study groups. CONCLUSION: The obtained data confirm that FECD and BK are associated with disruption of ocular immune privilege that leads to chronic local inflammation, which in turn causes remodeling of the corneal tissues resulting in fibrosis.

[Significance of evaluation of CD69 expression by peripheral blood lymphocytes for predicting pregnancy outcome in women with recurrent pregnancy loss]. / Znachimost' otsenki ékspressii CD69 limfotsitami perifericheskoi krovi dlia prognoza iskhodov beremennosti u zhenshchin s privychnym vykidyshem.

The aim of this work was to characterize phenotypically peripheral blood T- and NK lymphocytes expressing an early marker of activation, CD69, and assess the significance of CD69 expression for predicting pregnancy outcome in women with idiopathic reccurent pregnancy loss (IRP) before and after immunocytotherapy (ICT). The study group consisted of 36 patients with IRP who became pregnant after pre-gestational allimmunization, in 30 patients the pregnancy was prolonged to the full term and ended with the birth of a viable baby, in 6 - it was terminated before 12 weeks of gestation. In the control group, 15 fertile women outside pregnancy and 11 women at 12 weeks of physiological pregnancy were examined. Assessment of the CD69 expression in women with prolonged pregnancy revealed the absence of significant differences with the control group in the content and proportion of activated lymphocytes (CD69+). In women with aborted pregnancy after pre-gestational ICT, an increase in the number of almost all analyzed lymphocyte subpopulations responding to the activation stimulus, with a clear tendency to increase the proportion of activated T- but not NK-lymphocytes was found. At 5-6 weeks, the proportion of activated lymphocytes among a subpopulation of cytotoxic T-lymphocytes (CD3+CD8+/CD3+CD8+CD69+) in these women was significantly higher than in women with prolonged pregnancy, which confirms the leading role of effector cytotoxic T-lymphocytes in rejection reactions. Thus, the studies showed the promise of evaluating the expression of the early activation marker CD69 as an additional laboratory criterion for the personable appointment of immunocytotherapy to women with a common reccurent pregnancy loss.

Human Umbilical Cord Tissue-Derived Multipotent Mesenchymal Stromal Cells Exhibit Maximum Secretory Activity in the Presence of Umbilical Cord Blood Serum.

Using multiplex analysis, we performed a comparative study of cytokine and growth factor production by human umbilical cord tissue-derived multipotent mesenchymal stromal cells (UC-MSC) cultured under standard conditions and in the presence of human umbilical cord blood serum (UCBS). It was found that the secretion of most studied molecules, including well-known inductors of regeneration HGF, G-CSF, GM-CSF, and VEGF by UCMSC considerably increased in the presence of 5% UCBS. The use of UCBS allows not only obtaining xenogenic-free cellular and cell-free therapeutic products, but also increasing the secretion of most biologically active molecules capable of stimulating repair processes.

Lymphocyte Activation in the Development of Immune Tolerance in Women with Recurrent Pregnancy Loss.

Association between lymphocyte activation and formation of immune tolerance, as well as pregnancy outcome, in the case of immunocytotherapy (ICT) was studied in women with idiopathic recurrent pregnancy loss (IRPL). The content and phenotypic characteristics of activated T lymphocytes and NK cells were investigated in the peripheral blood of IRPL patients with different pregnancy outcomes (pregnancy prolongation to the full term and habitual miscarriage). The fraction of activated cells in the subpopulation of cytotoxic T lymphocytes (CD3+CD8+/CD3+CD8+CD69+) before ICT was significantly lower in women who lost the pregnancy. After ICT, the fraction of these cells during weeks 5-6 of pregnancy in woman with miscarriage was higher than in women with pregnancy prolonged to the full-term. Excessive content of activated cytotoxic lymphocytes can be a mechanism underlying impaired maternal immunotolerance to fetal alloantigens, which is a leading factor of early pregnancy loss. The obtained data confirm the involvement of activated Th17 cells and FOXP3+ Treg cells in the formation of tolerance to paternal antigens of the fetus. Comparison of the decrease in the fraction of CD4+CD25highRORγt+ lymphocytes at the early gestation period (5-6 weeks) and significant upregulation of the IL-17 production by in vitro stimulated whole blood cells in women with miscarriage with the same parameters in women with prolonged pregnancy suggested an imbalance between pro-inflammatory Th17 cells and Treg cells. No such imbalance in the content effector T lymphocytes was observed in women with the full-term pregnancy. Taken together, our data indicate an important role of gestational activation of lymphocytes in the formation of maternal immune response to fetal alloantigens necessary for the prolongation of pregnancy.

Human Umbilical Cord Blood Serum/Plasma: Cytokine Profile and Prospective Application in Regenerative Medicine.

The concentrations of cytokines and growth factors in human umbilical cord blood serum and plasma samples were measured by multiplex analysis. It was found that in comparison with peripheral blood serum of adult donors, umbilical cord blood serum and plasma contain significantly higher concentrations of the most studied molecules including IL-4, 5, 6, 7, 10 and 15, MCP-1, SCF, and SDF, as well as growth factors directly involved in the processes of regeneration (G-CSF, HGF, PDGF-BB, and VEGF). Thus, umbilical cord blood plasma and especially serum are a rich source of cytokines and growth factors with anti-inflammatory, anti-apoptotic, and angiogenic effects and can be used in various fields of regenerative medicine.

[The content of cytokines IL-6, IL-8, TNF-α, IL-4 and the level of expression in macrophages CD86 and CD163 in peritoneal fluid has a reverse correlation with the degree of severity of external genital endometriosis]. / Soderzhanie tsitokinovIL-6, IL-8, TNF-α, IL-4 i uroven' ékspressii makrofagami CD86 i CD163 v peritoneal'noi zhidkosti imeet obratnuiu korreliatsiiu so stepen'iu tiazhesti naruzhnogo genital'nogo éndometrioza.

Concentrations of eight different cytokines and the level of expression of CD86 and CD163 macrophages were studied in peritoneal fluid in women with endometriosis. It was found that the concentration of both inflammatory (IL-6, IL-8, TNF-α) and anti-inflammatory cytokines (IL-4) as well as the level of macrophage expression of the proinflammatory marker CD86 and anti-inflammatory marker CD163 increased in women with mild external genital endometriosis (1-2 stage), and did not differ from the control group in women with severe endometriosis (3-4 stage). The content of IL-2, IL-10, CM-CSF and IFN-γ in the peritoneal fluid of women with endometriosis did not differ significantly from the control group. The results of the study indicate that the development of external genital endometriosis may be based on insufficient both inflammatory and anti-inflammatory activity of macrophages in the peritoneal fluid.

Comparative Analysis of Secretome of Human Umbilical Cord- and Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells.

Production of cytokines and growth factors by cultured human umbilical cord tissue- and bone marrow-derived multipotent mesenchymal stromal cells was measured by multiplex analysis. In most cases, the concentrations of bioactive factors in the culture medium conditioned by umbilical cord-derived cells was ten- to hundred-times higher than in the medium conditioned by bone marrow-derived cells. These results suggest that both multipotent mesenchymal stromal cells from the umbilical cord and cell-free products can have more pronounced therapeutic effect in comparison with mesenchymal stromal cells obtained from "adult" sources.

Complex Analysis of Total and Fetal DNA and Cytokines in Blood Plasma of Pregnant Women with Preeclampsia.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10-6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

Expression of Early Activation Marker CD69 on Peripheral Blood Lymphocytes from Pregnant Women after First Trimester Alloimmunization.

We studied the expression of an early activation marker CD69 in peripheral blood lymphocytes of pregnant women with a history of recurrent pregnancy loss after immunization with paternal lymphocytes. Spontaneous and phytohemagglutinin-stimulated expression of CD69 on the surface of T cells and NK cells isolated from the peripheral blood was analyzed. On gestation week 5-6, the number of T cells expressing CD69 spontaneously and after stimulation was significantly higher in women with miscarriage than in woman with prolonged pregnancy. However, the number of cells with CD56(+) phenotype expressing CD69 did not differ in these groups. No differences were found in the number of cells of all subpopulations expressing CD69 after stimulation on gestation week 12 in woman with full-term current pregnancy and in woman with physiological pregnancy.

Detection of Antileukocytic Antibodies in Blood Serum using Lymphocytes and Latex Microspheres Carrying HLA-Antigens upon Alloimmunization of Women with Recurrent Pregnancy Loss.

Anti-HLA-antibodies were detected using cross-reaction of blood serum with allogenic T and B cells and latex microspheres coated with HLA-I and HLA-II antigens. HLA+ and HLA-sera obtained from women before and after allogeneic immunization were tested. The results obtained by these methods significantly differed. The test with latex microspheres detected antibodies to HLA-I and HLA-II antigens with high sensitivity and specificity and can be used for assessment of clinical significance of alloantibody detection when using alloimmunization in the therapy of gestation disorders.