Peripheral blood subpopulations of Bregs producing IL-35 in women with endometriosis.

PROBLEM: Interleukin 35 (IL-35) is involved in the pathogenesis of endometriosis by suppressing immunoreaction and promoting endometrial cell proliferation. It may also be an essential cytokine in forming the immunosuppressive functions of regulatory B lymphocytes (Bregs). The involvement of Bregs in the pathogenesis of endometriosis has not been previously investigated. In this study, we determined the frequencies of different Breg subpopulations, namely, B10, immature B-cells, and plasmablasts, and their abilities to produce IL-35 in women with endometriosis compared to healthy women. METHODS: The frequencies of different subpopulations of Bregs producing IL-35 were measured in the peripheral blood of women with endometriosis (total pool), women with deep infiltration endometriosis (DIE), women with ovarian endometriosis, and healthy women as a control by flow cytometry. RESULTS: We observed a decrease in the percentage of B10 cells and plasmablasts in women with endometriosis and an increase in the percentage of these Breg populations producing IL-35 in the same experimental group. Interestingly, we also revealed that women with DIE had increased percentages of B10 cells and plasmablasts producing IL-35. CONCLUSION: Taken together, our findings are the first to reveal the frequencies of different subpopulations of Bregs producing IL-35 in women with endometriosis. The results suggest that IL-35 expression in B lymphocytes could be used as a peripheral marker of endometriosis; however, further studies are needed.

Homeostasis of the Intestinal Mucosa in Healthy Horses-Correlation between the Fecal Microbiome, Secretory Immunoglobulin A and Fecal Egg Count.

The defensive function of the intestinal mucosa depends both on the ability to secrete immunoglobulin A and communication with the mucus microbiome. In horses, the functioning of this system is also influenced by the presence of nematode eggs. Feces collected from healthy horses were examined to determine the fecal egg count, immunoglobulin A level (ELISA), microbiome composition (Next-Generation Sequencing, NGS, V3−V4 and V7−V9 hypervariable regions of the 16S rRNA gene analysis and short-chain fatty acid (SCFA) production ((high-performance liquid chromatography, HPLC). In the taxonomic analysis within the phylum, the following order of dominance was found: Firmicutes, Bacteroidota, Verrucomicrobiota and Fibrobacterota. The coefficient of phylogenetic diversity of the microbiome positively correlated with both secretory immunoglobulin A (SIgA) [µg/g of feces] (p = 0.0354, r = 0.61) and SIgA [µg/mg of fecal protein] (p = 0.0382, r = 0.6) and with the number of Cyathostomum eggs (p = 0.0023, r = 0.79). Important components of the key microbiome in horses, such as phylum Proteobacteria and species Ruminococcus flavefaciens, were positively correlated with the fecal SIgA (p < 0.05). All the obtained results indicate the existence of significant relationships between the host response (SIgA production) and composition and SCFA production in the microbiome as well as the presence of small strongyles in the digestive tract of horses.

Effectiveness of immunization with multi-component bacterial immunomodulator in foals at 35th day of life.

The aim of the study was to investigate the mechanisms leading to immunization through the use of a multicomponent bacterial immunomodulator and to find out the relationship between the TLR 4 receptor with selected parameters of innate immunity and to acquire immunity. The study was conducted on 18 Polish Pony Horses foals divided into two study groups: control (n = 9) and experimental (n = 9). Foals from the experimental group received intramuscular duplicate injection of 5 ml of multi-component bacterial immunomodular at 35 and 40 days of age. RNA isolated from venous blood was used to evaluate the expression of TLR4 genes using RT-PCR. Concentration of Il-6, IL-10, IgM and IgG2 was determined by the ELISA method in blood plasma. Immunostimulation had a significant impact on the level of genes expression for TLR4 expression and IL-6 concentration. No effect of stimulation on IgM and IgG2 concentrations was found. The expression of TLR4 genes as well as the levels of interleukins could be modulated by stimulation with a pharmacological agent multi-component bacterial immunomodulator. The experiment demonstrated a strong positive correlation between TLR4 gene expression and IL-6 concentration and between TLR4 gene expression and IgM concentration.

Fibronectin Molecular Status in Plasma of Women with Endometriosis and Fertility Disorders.

The diagnosis of endometriosis and fertility disorders is difficult; therefore, it is necessary to look for reliable biomarkers. Analysis of the molecular status of fibronectin as a key player in repair and wound healing processes, as well as in coagulation and fibrinolysis pathways, is justified. ELISA and SDS-agarose immunoblotting were applied to determine the fibronectin concentration and presence and occurrence of soluble FN-fibrin complexes in the blood plasma of women with endometriosis (n = 38), fertility disorders (n = 28) and the healthy group (n = 25). The concentration of fibronectin in the blood plasma of women with endometriosis (292.61 ± 96.17 mg/L) and fertility disorders (287.53 ± 122.68 mg/L) was significantly higher than in the normal group (226.55 ± 91.98 mg/L). The presence of FN-fibrin complexes of 750, 1000, 1300, 1600 and 1900 kDa in the plasma of women with endometriosis and fertility disorders was shown. The presence of FN-fibrin complexes with a molecular mass of more than 1300 kDa in women with endometriosis and infertility and the complete absence of these complexes in healthy women may indicate an increased and chronic activation of coagulation mechanisms in these patients. The presence of complexes of high molecular mass may be one of the biomarkers of fertility disorders in women.

CD91 Derived Treg Epitope Modulates Regulatory T Lymphocyte Response, Regulates Expression of Costimulatory Molecules on Antigen-Presenting Cells, and Rescues Pregnancy in Mouse Pregnancy Loss Model.

The loss of immune tolerance to fetal antigens may result in reproductive failure. The downregulated number and activity of T regulatory lymphocytes, which are critical for the establishment of immune tolerance to fetal antigens, during pregnancy may lead to miscarriage. The adoptive transfer of Tregs prevents fetal loss in abortion-prone mice. Recently, we demonstrated that the administration of tregitopes, which are short peptides found in human and mouse immunoglobulins (IgGs), decreased the incidence of abortions in female CBA/J mice mated with DBA/2J mice. Here, two non-IgG source peptides (SGS and LKD) that can potentially bind to the major histocompatibility complex II (MHC II) with high affinity and induce Treg expansion were designed in silico. The immune dysregulation-induced pregnancy failure mouse model was used to evaluate the effect of SGS and LKD on immune response and pregnancy outcome. The fetal death rate in the SGS-treated group was lower than that in the phosphate-buffered saline-treated group. SGS and LKD upregulated the splenic pool of Tregs and modulated the T-helper cell (Th1)/Th2-related cytokine response at the preimplantation stage. Additionally, SGS and LKD downregulated the expression of CD80 and MHC class II molecules in splenic CD11c+ antigen-presenting cells. Thus, SGS treatment can result in beneficial pregnancy outcomes. Additionally, SGS peptide-mediated immunomodulation can be a potential therapeutic strategy for immune dysregulation-induced pregnancy failure.

Differential Signals From TNFα-Treated and Untreated Embryos in Uterine Tissues and Splenic CD4+ T Lymphocytes During Preimplantation Pregnancy in Mice.

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT2 Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4+ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4+ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

Influence of Age and Immunostimulation on the Level of Toll-Like Receptor Gene (TLR3, 4, and 7) Expression in Foals.

The aim of this study was to investigate the molecular mechanisms leading to the identification of pathogens by congenital immune receptors in foals up to 60 days of age. The study was conducted on 16 foal Polish Pony Horses (Polish Konik) divided into two study groups: control (n = 9) and experimental (n = 7). Foals from the experimental group received an intramuscular duplicate injection of 5 mL of Biotropina (Biowet) at 35 and 40 days of age. The RNA isolated from venous blood was used to evaluate the expression of theTLR3, TLR4, and TLR7 genes using RT-PCR. The results of the experiment demonstrated a statistically significant increase in the level of TLR3 gene expression and a decrease in the level ofTLR4 gene expression with foal aging. The level of TLR7 gene expression did not show age dependence. Immunostimulation with Biotropina had a significant impact on the level of the genes' expression for Toll-like receptors. It increased the level of TLR4 expression and decreased TLR3 expression. Thus, it was concluded that the expression of theTLR3 and TLR4genes in peripheral blood cells is dependent on age. This experiment demonstrated a strong negative correlation between TLR3 and TLR4 gene expression.

Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings.

The imbalance in immune tolerance may cause the variety of reproductive failures. An intravenous immunoglobulin infusion (IVIg) therapy is used to improve the live birth rate in women suffering from recurrent pregnancy loss, recurrent spontaneous abortions and recurrent implantation failures. However, the results of IVIg studies are still inconclusive as IVIg infusion in women suffering from pregnancy loss is sometimes ineffective. One of the mechanisms of action of this treatment is inhibition of B cells differentiation and expansion of Tregs and secretion of interleukin 10. It was proposed that immunomodulatory effects of IVIg may be attributed to tregitopes - self-IgG-derived epitopes present in the structure of immunoglobulins. Similarly to IVIg, tregitopes cause the expansion of Tregs and secretion of antigen-specific effector cytokine response. Here, we studied whether the administration of mouse tregitope 167 and/or 289 can prevent abortions in mouse abortion-prone mouse matings. We revealed that tregitopes reduce the foetal death rate. This may be driven by observed higher pool of peripheral Tregs, increased production of IL-10 by Tregs and Bregs and/or maintaining the tolerogenic phenotype of antigen-presenting cells. We believe that our findings may indicate a potential alternative to IVIg for therapeutic intervention in case of pregnancy failures.

Iridophoroma associated with the Lemon Frost colour morph of the leopard gecko (Eublepharis macularius).

The Lemon Frost is a new colour morph of the leopard gecko, which emerged in ca. 2015 as a result of selective breeding and spontaneous mutation. According to multiple breeders observation of Lemon Frost inbreeding with wild-type leopard geckos, Lemon Frost seems to be a codominant trait. Additionally breeders observed another, presumably associated trait - tumour-like skin lesions. Three private-owned Lemon Frost morph leopard geckos with tumour-like skin lesions were admitted to our clinic for examination, which included histopathology, X-ray and ultrasonography. The histopathological investigation of the biopsies indicated malignant iridophoroma; however, no changes were observed in diagnostic imaging. This research is the first report of clinical and histopathological findings of iridophoroma in leopard geckos.

Regulatory B cells with IL-35 and IL-10 expression in a normal and abortion-prone murine pregnancy model.

PROBLEM: Interleukin 35 is a relatively newly discovered cytokine that is produced by regulatory B cells (Bregs) and contributes to their suppressive function, which may contribute to fetal tolerance development and pregnancy maintenance. Therefore, the aim of this study was to determine the frequency of Bregs and expression of IL-35 and IL-10 in these cells in a normal and abortion-prone murine pregnancy model. METHODS OF STUDY: The frequency of Bregs and expression level of IL-35 and IL-10 in these cells were measured in peripheral blood, uterine draining lymph nodes, uterus, and decidua using flow cytometry. The analysis was performed on days 3 and 14 of pregnancy in normal mice (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy model. RESULTS: A decreased percentage of Breg cells expressing IL-35 on day 3 of pregnancy in the uterine draining lymph nodes and in peripheral blood in mice from the abortion group compared with the normal pregnancy group was observed. A similar decrease was also observed in the Breg cells population producing IL-10 in peripheral blood. In the uterus (3 dpc) and decidua (14 dpc), a lower percentage of CD19+ IL-35+ was also noted in the abortion-prone model. CONCLUSION: We indicated that the early stages of abortion-prone pregnancy (3 dpc) in mice were characterized by diminished frequency of B cells producing IL-35 at both local and peripheral levels. These results and the observed lower level of IL-35 in women suffering from recurrent spontaneous abortion suggest that IL-35 may be involved in the maintenance of pregnancy.

Impact of tissue enzymatic digestion on analysis of immune cells in mouse reproductive mucosa with a focus on γδ T cells.

Mucosal tissues are enriched in γδ T lymphocytes, which maintain epithelial homeostasis, however, the homeostatic mechanisms are still incompletely understood. To elucidate their role in the tissue integrity governance within the female genital mucosa we employed flow cytometry, which is a powerful tool used for the characterization of tissue-resident immune cells, however, often requiring cell release upon tissue enzymatic disaggregation. Here, we analyzed the impact of various proteolytic enzymes in their ability to effectively isolate viable immune cells from the reproductive system of non-pregnant mice. Murine vaginas and uteri were digested using commercially available enzyme blends (liberases) and single enzymes (dispase II and collagenase IV). Among tested enzymes, liberases released the highest number of cells from digested tissues while dispase II and collagenase IV led to a significant decrease in the number of isolated live cells. Also, liberases had only minor detrimental effects on cell viability and detection of CD45, CD3ε, γδ TCR and CD11c positive cells. We found that a single liberase blend called Liberase TL was the most suited for the analysis of γδ T cells in the reproductive tract. By examining two distinct phases of the estrous cycle - estrus and diestrus, characterized by high and low epithelial stratification, respectively, we showed that higher numbers of γδ T lymphocytes were present in the latter cycle phase in vagina and uterus. Interestingly, the diestrus-associated increase in γδ T lymphocyte number was also observed in reproductive tract draining lumbar lymph nodes but not in more distant, inguinal lymph nodes. Our data indicate that enzymes used for reproductive mucosa digestion have profound effects on the cell viability and isolation efficiency, which consequently influence the phenotypic and quantitative analysis of immune cells.

Expression of Toll-like receptors and costimulatory molecules in splenic B cells in a normal and abortion-prone murine pregnancy model.

PROBLEM: The regulatory role of B lymphocytes in the pregnancy-induced maternal immune response is not well recognized. B lymphocytes function as antigen-presenting cells (APCs) and regulate Toll-like receptors and costimulatory molecule expression in response to intrinsic and extrinsic signals. Therefore, the aim of this study was to determine the expression of TLR2, TLR4, TLR9, and MHC class II and the costimulatory molecules CD80, CD86, and CD40 in splenic B cells in a normal and abortion-prone murine pregnancy model. METHODS OF STUDY: The expression level of these molecules on female splenic B cells was investigated using real-time PCR and flow cytometry. The analysis was performed on the 3rd and 14th day of normal (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy. RESULTS: The expression of Tlr9, Cd86, and H2-Ab1 in splenic B cells on the 3rd day after mating was upregulated, whereas Tlr2 was downregulated in abortion-prone females. On day 14, we observed lower expression levels of Tlr4 and Cd80 and higher expression levels of Cd86 in CBA/J females mated with DBA/2J males. At the protein level, the differences were observed only on day 3 of pregnancy. TLR4 and CD40 molecules were upregulated in splenic B cells, while TLR9 and CD86 were downregulated in abortion-prone mice. CONCLUSION: Differential expression of TLRs and costimulatory molecules in splenic B cells in abortion-prone and normal pregnancies suggests the involvement of these cells in the regulation of the immune response at the periphery in pregnant females.

Fluctuations in Pharmacokinetics Profiles of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are a group of drugs with predicted slow linear and target-mediated distribution and elimination. Visual inspection of published pharmacokinetic profiles of mAbs frequently reveals plateaus in the distribution phase or an increasing concentration many days after a single intravenous dose. A question which has been left unanswered until now is whether mAbs undergo recirculation mechanisms. If so, then which mechanisms are crucial for the fluctuation in their pharmacokinetics profiles? What is the impact of such mechanisms on mAb absorption, distribution and elimination? Current commentary accounts for the fluctuation of mAbs concentrations based on different mechanisms, as well in different phases of their in vivo disposition. Current knowledge shows significant impact of mAbs lymphatic recirculation on characteristics of their pharmacokinetics profiles. Fluctuating or plateau phases in pharmacokinetic profiles of mAbs are a consequence of multiple simultaneously occurring recirculatory as well as adsorption/desorption processes rather than only slow, continuous elimination. Lymphatic recirculation as well as other mechanisms appears to be an obvious element of the mAbs disposition. Periodic changes in the key factors affecting mAbs disposition can be responsible for the unpredictable concentration peaks in absorption, distribution and the elimination phase.

Differentiated all-trans retinoic acid response of naive CD4+CD25- cells isolated from rats with collagen-induced arthritis and healthy ones under in vitro conditions.

AIM O THE STUDY: To compare the potential of CD4+CD25- cells, isolated from both healthy rats and rats with CIA (Collagen-Induced Arthritis), for differentiation into regulatory T cells in the presence of all-trans retinoic acid in order to learn more about the activation mechanisms and therapeutic potential of regulatory T cells. MATERIAL AND METHODS: Sorted CD4+CD25- cells were cultured in vitro with/without ATRA, and then the frequency of regulatory T cells and their ability to secrete IL-10 by CD4+ FOXP3+ cells was examined. Gene expression of the foxp3, rarα, rarß, rxrß, and ppar ß/δ and protein expression of the Rarα, Rarß, and Rxrß in cells after stimulation with ATRA were also investigated. RESULTS: CD4+CD25- cells isolated from healthy animals or from animals with CIA are characterised by different potential of the differentiation into CD4+CD25+ FOXP3+ cells. Retinoic acid receptor Rxrß is present in the CD4+CD25- cells isolated from rats with CIA. CONCLUSIONS: We showed that although ATRA did not increase the frequency of Treg in culture, it significantly increased expression of rarß and rxrß only in lymphocytes taken from diseased animals and foxp3 expression only in healthy animals. Moreover, after ATRA stimulation, the frequency of Treg-produced IL-10 tended to be lower in diseased animals than in the healthy group. The results imply that the potential of naïve cell CD4 lymphocytes to differentiate into Tregs and their putative suppressive function is dependent on the donor's health status.

Global decrease in the expression of signalling pathways' genes in murine uterus during preimplantation pregnancy.

Early preimplantation embryo-maternal communication is crucial for the establishment and development of pregnancy. Though the involvement of several candidate genes and proteins in this complex event has been described, the hierarchy of molecular networks governing this communication remains unknown. The primary objective of this study was to determine whether the presence of embryos in the uterine lumen stimulated or inhibited gene expression in the uterine tissue on day 3.5 post coitum. To answer this question, we investigated the gene expression of dedicated signal transduction pathways in the uterus of CD-1 mice during the preimplantation stage of pregnancy and compared this expression to mice with induced pseudopregnancy. The expression levels of 84 genes assigned to nine intracellular signalling pathways were investigated by real-time PCR. The results demonstrated down-regulation of the uterine gene expression in the majority of pathways. Among target genes, 27 were significantly (p<0.05) down-regulated, and only three were significantly up-regulated. A majority of the down-regulated genes were found to be regulated by the TGFB and NFKB pathways, which suggests that the presence of the embryo selectively regulates signalling within signal transduction pathways. One of the up-regulated genes crucial for early pregnancy was Ptgs2 (p<0.05). The increased amount of both Ptgs2 gene and protein products indicates that Ptgs2 expression may be the earliest positive embryo signal for implantation and pregnancy recognition in mice. In conclusion, our results not only underline which signalling pathways are regulated in embryo-maternal communication before implantation but also support "the quiet state hypothesis" of silencing gene expression.

Adoptively transferred Tregs accumulate in a site-specific manner and ameliorate signs of less advanced collagen-induced arthritis progress in rats.

AIM: The aim of the study was to assess the therapeutic effect and migration of adoptively transferred Tregs in the course of collagen-induced arthritis (CIA) in rats. METHODS: Sorted CD4(+)CD25(+) cells were cultured in the presence of 17-ß-estradiol, stained with CellTracker and then administered into the articular capsule of ankle joint of animals in different stages of CIA progression. RESULTS: Tregs diminished CIA signs only in animals with less advanced disease progress. Moreover, migration of transferred cells into the LN in the near proximity of the injection site and with distal location was almost completely stopped in animals with fully developed CIA. CONCLUSION: Disease progression-related differences in migratory potential of in vitro induced Tregs may be responsible for the failure of cellular therapy during the advanced stages of CIA.

CD80 and CD86 costimulatory molecules differentially regulate OT-II CD4⁺ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice.

Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs) derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c(+) dendritic cells and macrophages F4/80(+) and CD11b(+) presenting ovalbumin (OVA) were cocultured with CD4(+) T cells derived from OT-II mice's (C57BL6/J-Tg(TcraTcrb)1100Mjb/J) spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU), activation of these cells (flow cytometry), cytokine profile (ELISA), and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4(+) T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear.

Diagnostic accuracy of interleukin-6 levels in peritoneal fluid for detection of endometriosis.

PURPOSE: To determine, with extended receiver operating characteristic (ROC) curve analysis, the diagnostic value of cytokines showing significantly different peritoneal concentrations between women with and without endometriosis. METHODS: Multiplex cytokine concentration measurement of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ levels in peritoneal fluid of women with minimal to mild (n = 10) and moderate to severe (n = 26) endometriosis, and 42 controls. RESULTS: Only IL-6 and IL-10 concentrations were significantly higher in endometriosis patients than in controls. Specifically, significantly higher IL-6 and IL-10 levels were found in moderate to severe but not in minimal to mild endometriosis as compared to controls. For evaluation of diagnostic significance, ROC analysis determined discriminating parameters for IL-6, while those calculated for IL-10 were useless. Importantly, ROC analysis for IL-6 levels limited to women with moderate to severe endometriosis showed the highest area under the curve with the sample size sufficient to achieve 90 % power of the test. Finally, extended ROC including cost of analysis for this group of patients determined the optimal cut-off leading to high specificity and positive likelihood ratio resulting in 79 % effectiveness of the test. CONCLUSIONS: While our outcomes show moderate usefulness of peritoneal IL-6 levels in discrimination of moderate to severe endometriosis, further studies might be needed to determine the usefulness of peritoneal IL-6 levels in detection of early stages of endometriosis, as such a finding would be more relevant in clinical decision making.