Differentiation and modulation of phagocyte activity in murine granuloma after treatment with thymulin 5cH

In previous studies, it was found that thymulin (thymic hormone), when prepared in homeopathic 5CH dilution, can modulate diseases progress and immune processes, such as virus infection in poultry and experimental tumor development. This project aims to study the immune modulatory mechanisms of thymulin 5CH in granulomatous experimental model induced by BCG in mice, with special focus on the phagocytes differentiation from peritoneal B1 stem cells and on the activity of these cells and lymphocyte cooperation at the injury site. Male adult Balb/c mice were inoculated with BCG into the footpad to induce granuloma and its cells were evaluated by histomorphometric methods (Ziehl-Neelsen staining of macrophages) and flow cytometry. The phenotypic characterization of phagocytes, T and B lymphocytes in the peritoneum and regional lymph node was done. Thus, CD11b (activated phagocytes and B1 cells), CD19 (B1 cells and B2), CD23 (B2 cells), CD5 (B1 cell subtypes and T cells), CD4, CD8 (effective T lymphocytes) and CD25 (regulatory lymphocytes) positive cells were quantified. After 21 days of infection, the differentiation of B1 peritoneal stem cells into phagocytes reached the peak, being higher in thymulin-treated mice (X2, p=0.0001). Simultaneously, the number of infected phagocytes in the lesion decreased (KW, p=0.001), indicating remission of the infection. The number of B1 derived phagocytes, CD4+ and CD8+ T lymphocytes in the local lymph node increased in thymulin-treated mice (X2, p=0.0001), at the same time. No difference was seen regarding to CD25+ cells. The results show that thymulin 5CH treatment is able to improve the granuloma inflammatory process by modulating local and systemic phagocyte differentiation, as well as T cell migration into the local lymph node.

Chronic inflammatory response modulation against Leishmania (L.) amazonensis by homeopathic thymulin and antimonium crudum inBalb/c mice

In previous studies it was found that thymulin 5cH (thymic hormone) can modulate immune processes in several diseases. Additionally, the Antimonium crudum has used in dogs bearing leishmaniosis, according to the similia principle. We studied the inflammatory and immune modulation by thymulin 5CH and Antimonium crudum 30CH treatment in mice experimentally inoculated with Leishmania (L.) amazonensis. Male adult Balb/c mice were inoculated with Leishmania (2x105 promastigotes) into the footpad to induce inflammatory response and peritoneum and spleen cells were evaluated by flow cytometry after 60 days. Animals were divided in 3 groups (n=10): thymulin 5cH, Antimonium crudum 30cH and vehicle /control. Treatment was made in blind, daily, in water/alcohol 30% diluted 1:2500 in drinking water, during all experimental period. CD11b (activated phagocytes and B1 cells), CD19 (B1 cells and B2), CD4 and CD8 (effective T lymphocytes) markers were used to identify immune cells subsets in peritoneal washing fluid and spleen cell suspension. Mice treated with thymulin 5cH presented increase in peritoneal and spleen B1 stem cells (X2=0.0001) and higher CD8+/CD4+ ratio in spleen, regarding to the control. Also, Antimonium crudum 30CH induced a mild increase in B1 cells in peritoneum and spleen ( both X2, p=0.0001). Further histological analysis of the primary lesion will be done in the next step, to elucidate the impact of these findings in the disease evolution. The results show that both treatments stimulate B1 stem cell proliferation and suggest the cooperation of T spleen lymphocytes in the process.

CXCR4+ Treg cells control serum IgM levels and natural IgM autoantibody production by B1 cells in the bone marrow

Regulatory T (Treg) cells represent a specialized lineage of suppressive CD4+ T cells, whose functionality is critically dependent on their ability to migrate to, and dwell in the proximity of cells they control. Here we show that continuous expression of the chemokine receptor CXCR4 in Treg cells is required for their ability to accumulate in the bone marrow (BM). Induced CXCR4 ablation in Treg cells led to their rapid depletion and consequent increase in mature B cells, foremost B-1 subset, observed exclusively in the BM without detectable changes in plasma cells or hematopoietic stem cells or any signs of systemic or local immune activation elsewhere. Dysregulation of BM B-1 B cells was associated with a highly specific increase in IgM autoantibodies and the total serum IgM levels. Thus, Treg cells control autoreactive B-1 B cells in a CXCR4-dependent manner. These findings have significant implications for understanding of regulation of B cell autoreactivity and malignancies.

Critical role of IL-25-ILC2-IL-5 axis in the production of anti-Francisella LPS IgM by B1 B cells

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of “natural” IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG3 by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb-/- mice rescued these B1 cells-mediated responses. Il17rb-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens.

Changes in Sphingolipid Profile of Benzo[a]Pyrene-Transformed Human Bronchial Epithelial Cells Are Reflected in the Altered Composition of Sphingolipids in Their Exosomes

Sphingolipids (SLs), glycosphingolipids (GSLs), and eicosanoids are bioactive lipids, which play important roles in the etiology of various diseases, including cancer. However, their content and roles in cancer cells, and in particular in the exosomes derived from tumor cells, remain insufficiently characterized. In this study, we evaluated alterations of SL and GSL levels in transformed cells and their exosomes, using comparative HPLC-MS/MS analysis of parental human bronchial epithelial cells HBEC-12KT and their derivative, benzo[a]pyrene-transformed HBEC-12KT-B1 cells with the acquired mesenchymal phenotype. We examined in parallel SL/GSL contents in the exosomes released from both cell lines. We found significant alterations of the SL/GSL profile in the transformed cell line, which corresponded well with alterations of the SL/GSL profile in exosomes derived from these cells. This suggested that a majority of SLs and GSLs were transported by exosomes in the same relative pattern as in the cells of origin. The only exceptions included decreased contents of sphingosin, sphingosin-1-phosphate, and lactosylceramide in exosomes derived from the transformed cells, as compared with the exosomes derived from the parental cell line. Importantly, we found increased levels of ceramide phosphate, globoside Gb3, and ganglioside GD3 in the exosomes derived from the transformed cells. These positive modulators of epithelial–mesenchymal transition and other pro-carcinogenic processes might thus also contribute to cancer progression in recipient cells. In addition, the transformed HBEC-12KT-B1 cells also produced increased amounts of eicosanoids, in particular prostaglandin E2. Taken together, the exosomes derived from the transformed cells with specifically upregulated SL and GSL species, and increased levels of eicosanoids, might contribute to changes within the cancer microenvironment and in recipient cells, which could in turn participate in cancer development. Future studies should address specific roles of individual SL and GSL species identified in the present study.

Character of β-lymphocytes differentiation in women with hypertensive disorders during pregnancy

The aim of the work was to identify the features of B-lymphocyte differentiation in women with hypertensive disorders of various origins, to establish their relationship with indicators of the elastic properties of the vascular wall, and to develop additional diagnostic criteria for the severity of preeclampsia. We examined 193 women at 24-40 weeks of gestation. Of these, 39 women with chronic arterial hypertension, 35 women with preeclampsia that developed against the background of chronic arterial hypertension, 55 with preeclampsia. The control group consisted of 64 women without hypertensive disorders. To assess the elastic properties of the vascular wall, a sphygmographic attachment of the “Poly-Spectrum-8” hardware-software complex (“Neurosoft” Ivanovo) was used. The relative content of B-lymphocytes (CD19 + and CD20 +), regulatory B-cells (CD20 + IL-10 +), switched (CD19 + CD27 + IgD-) and non-switched (CD19 + CD27 + IgD +) memory cells, plasma cells (CD19 + CD20-CD38 +) in the general population of B-lymphocytes was assessed by flow cytometry on a FACSCantoII flow cytometer using the FACS Diva program. Statistical analysis was carried out using the programs “Statistica for Windows 6.0”, “Microsoft Excel 2010” and “MedCals”. All subjects with hypertensive disorders showed an increase in the stiffness of the arteries of the muscular and elastic types, the most pronounced in the groups of patients with PE, the maximum in women with CAH and associated PE. An increased level of B1-lymphocytes in the peripheral blood is also noted in all hypertensive disorders. There were revealed positive correlations of high strength between: the level of CD20 + cells and the velocity of pulse wave propagation through the arteries of the muscular type (PWVm) in all groups with hypertensive disorders; the content of B1 cells and PWVM in moderate preeclampsia; level emory B-cells and PWV in elastic-type arteries in women with CAH and associated PE. The ROC analysis of the relative content of B1 cells in the general population of B lymphocytes and the content of IL-10-producing cells in the population of CD20 + lymphocytes (Breg) in women with moderate and severe PE revealed criteria for the differential diagnosis of preeclampsia of varying severity. The presence of hypertensive disorders of various origins is accompanied by a decrease in the elasticity of the arterial vascular wall, which is most pronounced in patients with CAH and associated PE. These changes are largely correlated with the level of B cells. As additional criteria for determining the severity of PE, the relative content of B1 cells and IL-10-producing cells in the population of Breg CD20 + lymphocytes can be used.

Study of the effects of Silicea terra and Zincum metallicum in various dilutions on the oxidative activity of macrophages in vitro

Silica (silicon dioxide) is found in nature, being composed of 2 chemical elements abundant on the Earth's crust, oxygen and silicon. As homeopathic medicine, silica is used for treatment of chronic ulcers due to its ability to modulate the macrophage activity. Zinc is a cofactor of several immune mediators, especially thymulin, which is also capable of modulating the macrophage activity and recruitment of B1 cells in mice. In the present study, we assessed the homeopathic medicines Silicea terra and Zincum in an in vitro experimental model to determine their effects on the interaction between RAW 264.7 macrophages and BCG (Bacillus Calmette-Guérin). In this step of the study, we assessed oxidative activity through measurement of hydrogen peroxide and nitrite/nitrate in the cell culture supernatant after 24 hours of treatment. Several homeopathic potencies were used for both drugs (6cH, 30cH, 200cH). The tests were performed in duplicate, and the data were analyzed by means one-way ANOVA. The results show no effect on nitric oxide (NO) production, but reduction of hydrogen peroxide production (p≤ 0.001) after treatment with the vehicle (0.03% alcohol). Such reduction was reversed with treatments Silicea terra30cH and Zincum metallicum 30cH (p≤ 0.001). Treatment with Silicea terra 200cH induced significant reduction of hydrogen peroxide production, even when compared to the vehicle (p≤ 0.005). Taken together, the results indicate that only treatment of BCG-challenged macrophages with Silicea terra 200cH was able to significantly reduce the oxidative activity of these cells after 24 hours of incubation. Since NO production in vitro usually occurs after 96 to 120 hours of incubation, one might infer that the negative result obtained in the present study might be associated with the time-point of assessment. Other aspects of the macrophage-BCG interaction are still under evaluation.

The Abnormal Distribution of Peripheral B1 Cells and Transition B Cells in Patients with Idiopathic Dilated Cardiomyopathy

Background The aberrant distribution of peripheral B cell subsets is associated with the pathogenesis of a variety of inflammatory and autoimmune diseases. However, the distribution of peripheral B cell subsets in patients with idiopathic dilated cardiomyopathy (DCM) has yet to elucidated. Methods Twenty-seven idiopathic DCM patients (DCM group), 18 heart failure controls (HF group) and 21 healthy individuals (HC group) were included in this study. Peripheral B cell subsets were analyzed by multi-color flow cytometry. Level of plasma β1 cholinergic receptor (β1-AR) autoantibody was assayed by ELISA. Additionally, clinical features were also collected. Results Compared with HF and HC groups, the percentage of B1 cells were significantly decreased, whereas the percentage of transitional B cells (Tr) were significantly increased in DCM group. Notably, the percentage of B1 cells was significantly lower in patients with β1-AR autoantibody positive DCM compared with β1-AR autoantibody negative patients. Correlation analysis showed that the percentage of B1 cells was negatively correlated with N-terminal pro-brain natriuretic peptide (NT-proBNP) and positively correlated with left ventricular ejection fraction (LVEF) in patients with DCM.Conclusions The study showed that B1 cells in peripheral blood of patients with idiopathic DCM were abnormally decreased, especially in those β1-AR autoantibody positive patients, while Tr cells are significantly increased, indicating that B1 cells and Tr cells may be implicated in the pathogenesis of idiopathic DCM.

Dermatan Sulfate Is a Potential Regulator of IgH via Interactions With Pre-BCR, GTF2I, and BiP ER Complex in Pre-B Lymphoblasts

Dermatan sulfate (DS) and autoantigen (autoAg) complexes are capable of stimulating autoreactive CD5+ B1 cells. We examined the activity of DS on CD5+ pre-B lymphoblast NFS-25 cells. CD19, CD5, CD72, PI3K, and Fas possess varying degrees of DS affinity. The three pre-BCR components, Ig heavy chain mu (IgH), VpreB, and lambda 5, display differential DS affinities, with IgH having the strongest affinity. DS attaches to NFS-25 cells, gradually accumulates in the ER, and eventually localizes to the nucleus. DS and IgH co-localize on the cell surface and in the ER. DS associates strongly with 17 ER proteins (e.g., BiP/Grp78, Grp94, Hsp90ab1, Ganab, Vcp, Canx, Kpnb1, Prkcsh, Pdia3), which points to an IgH-associated multiprotein complex in the ER. In addition, DS interacts with nuclear proteins (Ncl, Xrcc6, Prmt5, Eftud2, Supt16h) and Lck. We also discovered that DS binds GTF2I, a required gene transcription factor at the IgH locus. These findings support DS as a potential regulator of IgH in pre-B cells at protein and gene levels. We propose a (DS•autoAg)-autoBCR dual signal model in which an autoBCR is engaged by both autoAg and DS, and, once internalized, DS recruits a cascade of molecules that may help avert apoptosis and steer autoreactive B cell fate. Through its affinity with autoAgs and its control of IgH, DS emerges as a potential key player in the development of autoreactive B cells and autoimmunity.