Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response.

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4+ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4+ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.

A Role of NKR-P1A (CD161) and Lectin-like Transcript 1 in Natural Cytotoxicity against Human Articular Chondrocytes.

Normal cartilage cells are susceptible to lysis by NK cells. This phenomenon may play a role in immune cartilage destruction; however, the mechanisms of chondrocyte recognition by NK cells remain poorly understood. Therefore, the aim of this study was to reveal a possible role of NKR-P1A/lectin-like transcript 1 (LLT1) interaction in NK cell-mediated cytotoxicity against normal human articular chondrocytes. Chondrocytes were isolated from articular cartilage obtained during talonavicular joint surgery. PBMC or polyclonal NK cells isolated from normal donors served as effector cells. Cell-mediated cytotoxicity against chondrocytes was evaluated by means of 18-h 51Cr-release assay. Specific mRNA expression was evaluated by classical and quantitative RT-PCR, and proteins were detected by Western blot analysis. We found that lysis of articular chondrocytes by PBMC or polyclonal NK cells was potentiated by stimulation with IL-2. Stimulation of effector cells with IL-2 downregulated mRNA expression of inhibitory NKR-P1A NK cell receptor, and blocking of NKR-P1A with specific mAbs resulted in increased chondrocyte killing. Chondrocytes constitutively expressed LLT1, a ligand of NKR-P1A. LLT1 expression by chondrocytes could be upregulated by IL-1α and TNF. Chondrocyte treatment with IL-1α resulted in their increased resistance to killing by natural cytotoxic cells. This could be reversed by blocking of NKR-P1A. These results show that susceptibility of normal articular chondrocytes to lysis by NK cells is modulated by NKR-P1A/LLT1 interactions. Thus, NKR-P1A/LLT1 interaction might provide some novel target for therapeutic interventions in the course of pathological cartilage injury.

Pentosidine, advanced glycation end product, in acute ischaemic stroke patients with and without atrial rhythm disturbances.

Atrial fibrillation (AF) and atherosclerotic disease are independent risk factors for acute ischaemic stroke (AIS). The optimal biological marker which could allow differentiation between AF and non-AF AIS patients is still not available. AIM OF THE STUDY: Aim of the present study was to investigate the role of pentosidine as a potential biological marker for AF in an AIS patient group. MATERIALS AND METHODS: Sixty-three acute ischaemic hemispheric stroke patients were recruited and divided into two groups according to the presumed underlying mechanism: with or without atrial rhythm disorders. Ten healthy volunteers were a reference group for serum level of pentosidine. Carotid artery ultrasound was performed, and common carotid artery stiffness and intima-media thickness were measured. Serum levels of pentosidine and selected routine biochemical risk factors for atherosclerosis (cholesterol and its lipoprotein fractions, homocysteine) were examined. RESULTS: A higher serum level of pentosidine was observed in patients without atrial fibrillation (1,509 ± 485.13pmol/ml); a statistically significant difference was observed compared to the reference group (1,041.52 ± 411.17pmol/ml; p = 0.01), but not the AF patients (1,438.19 ± 495.97pmol/ml; p = 0.59). No significant difference in the non-AF group compared to the AF group for carotid intima-media thickness (IMT)/stiffness and pentosidine serum level was recorded. CONCLUSIONS AND CLINICAL IMPLICATIONS: A higher serum level of pentosidine was observed in AIS patients without atrial fibrillation compared to the healthy volunteers. According to the results of the present study, no difference between these patients in the selected risk factors of atherosclerosis were observed. Further studies are needed to identify a reliable marker of AF that would bring added value to the standard diagnostic workup after acute ischaemic stroke.

Inflammatory response during slow- and fast-twitch muscle regeneration.

INTRODUCTION: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so-called fast-twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow-twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation. METHODS: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury. RESULTS: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles. CONCLUSIONS: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow- and fast-twitch muscles. Muscle Nerve 55: 400-409, 2017.

Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.

CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis.

STUDY QUESTION: Is endometriosis associated with changes in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg cells)? SUMMARY ANSWER: Endometriosis is associated with disturbed compartmentalization of CD25(high) FOXP3⁺ Treg cells. WHAT IS KNOWN ALREADY: Endometriosis is associated with an abrogated immune response and displays some features of an autoimmune disorder. Treg cells play a part in the development of autoimmune reactions; however, their role in pathogenesis of endometriosis is still poorly recognized. STUDY DESIGN, SIZE AND DURATION: Case-control study comparing 17 women with laparoscopically and histopathologically confirmed ovarian endometriosis with 15 control women without visible endometriosis foci, pelvic inflammation or related pathology who were subjected to laparoscopic surgery between 2010 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Peripheral blood and peritoneal fluid were collected during laparoscopy and T cell subpopulations were analysed by flow cytometry using specific monoclonal antibodies recognizing CD4⁺, CD25⁺ and FOXP3⁺ markers. MAIN RESULTS: The percentage of CD25(high) FOXP3⁺ Treg cells was significantly decreased in the peripheral blood of women with ovarian endometriosis compared with control women. On the other hand, the proportion of these cells was significantly increased in the peritoneal fluid of women with endometriosis. LIMITATIONS, REASONS FOR CAUTION: The present study is limited to patients with ovarian endometrioma and further investigations are needed, including patients with lower grade of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The present results suggest that Treg cells may play a part in immunopathogenesis of endometriosis being responsible for abrogated local cellular immune responses and facilitation and development of autoimmune reactions. Treg cells may be thus a potential target in the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 1M15/N/2011 and NK1W grants from the I Faculty of Medicine, Warsaw Medical University. None of the authors has any competing interests to declare.

The controversy over anti-Helicobacter pylori therapy.

Helicobacter pylori is a Gram-negative spiral-shaped bacterium, member of epsilon-Proteobacteria specifically colonizing the gastric epithelium of humans. It causes one of the most common infections worldwide, affecting about half of the world's population. However, it should be noted that the prevalence of H. pylori, particularly in the Western world, has significantly decreased coinciding with an increase of some autoimmune and allergic diseases, such as asthma. Various epidemiological studies have also documented a negative association between H. pylori colonization and the presence of GERD (gastroesophageal reflux disease) and risk of esophageal cancer. Additionally, an upward trend of obesity recently observed in inhabitants of developed countries raised a question about the relationship between H. pylori infection and the human body mass index. The first part of this review describes common, recommended anti-H. pylori treatments. The second part, presents the results of recent experiments aimed at evaluating the association between H. pylori infections and gastro-esophageal diseases, the level of stomach hormones, the human body mass index and allergic diseases. Although some studies suggest an inverse association of H. pylori infection with some health problems of the modern world such as asthma, obesity or GERD, H. pylori should be considered as a harmful human pathogen responsible for serious and sometimes lethal diseases. Thus, many scientists advocate the eradication of H. pylori.

Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms.

Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-ß), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs.

Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells.

Endometriosis is a common gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus. The disease is associated with disturbed local and systemic immunity. It has been reported that the proportion of CD4+CD25highFOXP3+ Treg cells may be significantly increased in the peritoneal fluid of patients with endometriosis. Therefore, the aim of our study was to investigate whether the proportions of Treg cells in the peritoneal cavity of patients with endometriosis are related to the chemotactic and stimulatory activity of the local peritoneal milieu. The peritoneal fluid was collected from 13 women with ovarian endometriosis and 12 control women without the disease. T cell populations were analyzed by flow cytometry, cytokines and chemokines were evaluated using the cytometric bead kit, and cell chemotaxis was studied by cell migration assay. We confirmed that the proportions of Treg cells are increased in the peritoneal fluid of women with endometriosis as compared to the control women. Endometriosis was also associated with elevated concentrations of IL-6, IL-10, and TGF-ß1/2 as well as CCL20, CXCL8, CXCL9, and CXCL10. We did not reveal any changes in the proportion of peritoneal Th17 cells and concentrations of IL-17A. Peritoneal Treg cells positively correlated with concentrations of TGF-ß, IL-10, and CCL20. Endometriotic peritoneal fluid stimulated chemotaxis of both CD4+ and Treg cells. This chemotactic activity positively correlated with concentrations of CCL20. CCL20 stimulated the migration of Treg cells, and the chemotactic activity of the endometriotic peritoneal fluid was inhibited by neutralizing anti-CCL20 antibodies. These results imply that increased proportions of the peritoneal Treg cells in women with endometriosis may result from attraction and activation by local chemokines and cytokines, especially CCL20 and TGF-ß. Since Treg cells contribute to the immunopathogenesis of endometriosis, their chemotaxis and activation may be considered as a target for therapeutic intervention.

Banking of AT-MSC and its Influence on Their Application to Clinical Procedures.

Processing of MSCs to obtain a therapeutic product consists of two main steps: 1) the in vitro expansion of the cells until an appropriate number of them is obtained, and 2) freezing and storage of the expanded cells. The last step is critical and must be optimized so that after thawing the cells retain all their physiological properties including the secretory function. In this paper, we evaluated physiological parameters of AT-MSC's after a full cycle of their processing, particularly freezing and storing at the liquid nitrogen vapor temperature. Based on the recovered proliferative and secretory capacities of the thawed cells, we have designed the optimal technique for processing of MSCs for clinical applications. In our work, we tried to select the best DMSO-based cryoprotectant mixture on the base of post thawing fully retain their properties. We have demonstrated the effectiveness of the use of DMSO in various configurations of the constituent cryoprotective fluids. We have also shown that AT-MSCs that show control levels in most standard tests (viability, shape, culture behaviour, and proliferative properties) after thawing, may show transient variations in some important physiological properties, such as the level of secreted growth factors. Obtained results let us to indicate how to optimize the AT-MSC preparation process for clinical applications. We suggest that before their clinical application the cells should be cultured for at least one passage to recover their physiological stability and thus assure their optimal therapeutic potential.

Mesenchymal Stromal Cells from Different Parts of Umbilical Cord: Approach to Comparison & Characteristics.

Mesenchymal stromal/stem cells (MSCs) are a unique population of cells that play an important role in the regeneration potential of the body. MSCs exhibit a characteristic phenotype and are capable of modulating the immune response. MSCs can be isolated from various tissues such as: bone marrow, adipose tissue, placenta, umbilical cord and others. The umbilical cord as a source of MSCs, has strong advantages, such as no-risk procedure of tissue retrieval after birth and easiness of the MSCs isolation. As the umbilical cord (UC) is a complex organ and we decided to evaluate, whether the cells derived from different regions of umbilical cord show similar or distinct properties. In this study we characterized and compared MSCs from three regions of the umbilical cord: Wharton's Jelly (WJ), the perivascular space (PRV) and the umbilical membrane (UCM). The analysis was carried out in terms of morphology, phenotype, immunomodulation potential and secretome. Based on the obtained results, we were able to conclude, that MSCs derived from distinct UC regions differ in their properties. According to our result WJ-MSCs have high and stabile proliferation potential and phenotype, when compare with other MSCs and can be treated as a preferable source of cells for medical application.

Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs.

BACKGROUND: A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro. METHODS: CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS: Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Treg's suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1. CONCLUSIONS: Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.

The anti-inflammatory potential of cefazolin as common gamma chain cytokine inhibitor.

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γc). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rß subunit and two within the γc subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γc receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

The Effects of Intestinal Nematode L4 Stage on Mouse Experimental Autoimmune Encephalomyelitis.

Helminths use various immunomodulatory and anti-inflammatory strategies to evade immune attack by the host. During pathological conditions, these strategies alter the course of disease by reducing immune-mediated pathology. The study examines the therapeutic effect of the nematode L4 stage based on an in vivo model of multiple sclerosis, monophasic encephalomyelitis (EAE), induced by sensitization with MOG35-55 peptide in C57BL/6 female mice infected with the intestinal nematode Heligmosomoides polygyrus. The EAE remission was correlated with altered leukocyte number identified in the central nervous system (CNS), and temporary permeability of the blood-brain barrier at the histotrophic phase of infection. At 6 days post-infection, when the L4 stage had almost completely attenuated the clinical severity and pathological signs of EAE, CD25+ cell numbers expanded significantly, with parallel growth of CD8+ and CD4+, both CD25+Foxp3+ and CD25+Foxp3- subsets and alternatively activated macrophages. The phenotypic changes in distinct subsets of cerebrospinal fluid cells were correlated with an inhibited proliferative response of encephalitogenic T cells and elevated levels of nerve growth factor and TGF-ß. These results enhance our understanding of mechanisms involved in the inhibition of immune responses in the CNS during nematode infection.

Expanding Diversity and Common Goal of Regulatory T and B Cells. II: In Allergy, Malignancy, and Transplantation.

Regulation of immune response was found to play an important role in the course of many diseases such as autoimmune diseases, allergy, malignancy, organ transplantation. The studies on immune regulation focus on the role of regulatory cells (Tregs, Bregs, regulatory myeloid cells) in these disorders. The number and function of Tregs may serve as a marker of disease activity. As in allergy, the depletion of Tregs is observed and the results of allergen-specific immunotherapy could be measured by an increase in the population of IL-10+ regulatory cells. On the basis of the knowledge of anti-cancer immune response regulation, new directions in therapy of tumors are introduced. As the proportion of regulatory cells is increased in the course of neoplasm, the therapeutic action is directed at their inhibition. The depletion of Tregs may be also achieved by an anti-check-point blockade, anti-CD25 agents, and inhibition of regulatory cell recruitment to the tumor site by affecting chemokine pathways. However, the possible favorable role of Tregs in cancer development is considered and the plasticity of immune regulation should be taken into account. The new promising direction of the treatment based on regulatory cells is the prevention of transplant rejection. A different way of production and implementation of classic Tregs as well as other cell types such as double-negative cells, Bregs, CD4+ Tr1 cells are tested in ongoing trials. On the basis of the results of current studies, we could show in this review the significance of therapies based on regulatory cells in different disorders.

Expanding Diversity and Common Goal of Regulatory T and B Cells. I: Origin, Phenotype, Mechanisms.

Immunosuppressive activity of regulatory T and B cells is critical to limit autoimmunity, excessive inflammation, and pathological immune response to conventional antigens or allergens. Both types of regulatory cells are intensively investigated, however, their development and mechanisms of action are still not completely understood. Both T and B regulatory cells represent highly differentiated populations in terms of phenotypes and origin, however, they use similar mechanisms of action. The most investigated CD4+CD25+ regulatory T cells are characterized by the expression of Foxp3+ transcription factor, which is not sufficient to maintain their lineage stability and suppressive function. Currently, it is considered that specific epigenetic changes are critical for defining regulatory T cell stability in the context of their suppressive function. It is not yet known if similar epigenetic regulation determines development, lineage stability, and function of regulatory B cells. Phenotype diversity, confirmed or hypothetical developmental pathways, multiple mechanisms of action, and role of epigenetic changes in these processes are the subject of this review.

Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas.

Glioblastoma (GBM) is the most aggressive primary brain tumor, with ineffective anti-tumor responses and a poor prognosis despite aggressive treatments. GBM immune microenvironment is heterogenous  and activation of specific immune populations in GBM is not fully characterized. Reliable animal models are critical for defining mechanisms of anti-tumor immunity. First we analyzed the immune subpopulations present in rat C6 gliomas. Using flow cytometry we determined kinetics of infiltration of myeloid cells and T lymphocytes into glioma-bearing brains. We found significant increases of the amoeboid, pro-tumorigenic microglia/macrophages, T helper (Th) and T regulatory (Treg) cells in tumor-bearing brains, and rare infiltrating T cytotoxic (Tc) cells. Transcriptomic analyses of glioma-bearing hemispheres revealed overexpression of invasion and immunosuppression-related genes, reflecting the immunosuppressive microenvironment. Microglia, sorted as CD11b+CD45low cells from gliomas, displayed the pro-invasive and immunosuppressive type of activation. Accumulation of Th and Treg cells combined with the reduced presence of Tc lymphocytes in rat gliomas may result in the lack of effective anti-tumor responses. Transcriptional profiles of CD11b+ cells and composition of immune infiltrates in C6 gliomas indicate that rat C6 gliomas employ similar immune system evasion strategies as human GBMs.

Correction to: Expanding Diversity and Common Goal of Regulatory T and B Cells. I: Origin, Phenotype, Mechanisms.

The original article has been published without acknowledgment section. The acknowledgement section is given below for your reading.

Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens.

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis.

Suppressor Properties of Human CD8(+)CD28(-) T Cells in Mixed Leukocyte Reaction are not Affected by CsA and RAPA.

Human CD8(+)CD28(-) T suppressor cells were previously shown to be involved in the control of the immune response to transplanted allografts. It seems essential to examine how immunosuppressive drugs influence these cells. However, the CD8(+)CD28(-) population contains both suppressor (Ts) and cytotoxic (Tc) T cells, and the phenotype of the Ts subpopulation has not been identified explicitly. It is proposed that the transcription factor FOXP3 may be helpful in distinguishing the Ts and Tc subpopulations. The aim of this study was to evaluate the influence of the immunosuppressive drugs cyclosporine A (CsA) and rapamycin (RAPA) on the level, suppressor properties, and phenotype of human CD8(+)CD28(-) T cells in vitro. The model used was the mixed leukocyte reaction performed with peripheral blood mononuclear cells from healthy volunteers. It was observed that CD8(+)CD28(-) T cells from cultures with CsA or RAPA had similar suppressor properties to cells from control cultures, although the drugs influenced the expression of FOXP3. CsA and RAPA did not interfere with the suppressor properties of human CD8(+)CD28(-) T cells in vitro, although they affected the expression of the FOXP3 molecule.