Melatonin mediates monochromatic light-induced proliferation of T/B lymphocytes in the spleen via the membrane receptor or nuclear receptor.

Our studies found that melatonin mediates the monochromatic light-induced lymphocyte proliferation in chickens. However, melatonin receptor subtypes contain membrane receptor (Mel1a/Mel1b/Mel1c) and nuclear receptor (Retinoic acid receptor-related orphan receptor [ROR] α/RORß/RORγ) and are characteristic with cell specificity. This study compared receptor pathway of melatonin, which mediated the monochromatic light-induced T/B lymphocyte proliferations in chickens. Newly hatched chicks were randomly divided into white light, red light, green light (GL), and blue light groups. Green light promoted the membrane receptor expression in the spleen but decreased the nuclear receptor level compared with that of red light. These changes were accompanied by increase of T/B lymphocyte proliferation and plasma melatonin level under GL. Pinealectomy reversed aforementioned changes and resulted in no differences among the light-treated groups. Supplementation of exogenous melatonin enhanced GL-induced T/B lymphocyte proliferation in the spleen but was reversed by Mel1c antagonist prazosin and RORα agonist SR1078 and enhanced by RORα antagonist SR3335. However, Mel1b antagonist 4P-PDOT and RORγ antagonist GSK increased the stimulation effect of melatonin on GL-induced T lymphocyte proliferation but no effect on the B-lymphocyte proliferation. These results indicate that melatonin promotes the GL-induced T lymphocyte proliferation through Mel1b, Mel1c, and RORα/RORγ; however, the Mel1a, Mel1c, and RORα may be involved in the B lymphocyte proliferation.

Innovative Mind-Body Intervention Day Easy Exercise Increases Peripheral Blood CD34+ Cells in Adults.

Mind-body interventions (MBIs) have many health benefits, such as reducing stress, modulating blood pressure, and improving sleep and life quality. The long-term practice of Tai chi, an MBI, also increases the number of CD34+ cells, which are surface markers of hematopoietic stem cells, so prolonged Tai chi practice may have antiaging effects. We developed the day easy exercise (DEE), an innovative MBI, that is easy to learn and requires only a small exercise area and a short practice time. The aim of this study was to explore whether DEE, like Tai chi, has antiaging effects after short-term practice. Total 44 individuals (25 to 62 years old) with or without 3-month DEE practice were divided into young- and middle-aged groups (≤30 and >30 years old) and peripheral blood was collected at 0, 1, 2, and 3 months for analysis of CD34+ cells. The number of CD34+ cells in peripheral blood remained unchanged in control young- and middle-aged groups. After DEE, the number of CD34+ cells in peripheral blood was increased over time in both young- and middle-aged groups. For young-aged adults, the cell number was markedly increased by threefold at 3 months after DEE, and for middle-aged adults, the increase was significant from the first month. DEE practice indeed increased the number of CD34+ cells in peripheral blood and the increase was more significant for older people in a shorter time. This is the first study to provide evidence that the DEE may have antiaging effects and could be beneficial for older people.

Natural pseurotins and analogs thereof inhibit activation of B-cells and differentiation into the plasma cells.

BACKGROUND: The frequency of allergic diseases is constantly rising. Dysregulated production of isotype E immunoglobulins is one of the key factors behind allergic reactions and its modulation is therefore an important target for pharmacological intervention. Natural products of the pseurotin family were reported to be inhibitors of IgE production in B-cells. Mechanistic details underlying these effects are however not well understood. PURPOSE: In the present study, we synthesized new analogs of natural pseurotins and extensively investigated their inhibitory effects on activation, proliferation and differentiation of B-cells, as well as on the production of IgE. STUDY DESIGN: Effects of two natural pseurotins (pseurotins A and D) and a collection of fully synthetic pseurotin analogs were studied on mouse B-cells stimulated by the combination of IL-4 and E. coli lipopolysaccharide. The IgE production was determined along with cell viability and cell proliferation. The phosphorylation of selected members of the STAT transcription factor family was subsequently investigated. Finally, the in vivo effect of pseurotin D on the ovalbumin-induced delayed type hypersensitivity response was tested in mice. RESULTS: We discovered that several fully synthetic pseurotin analogs were able to decrease the production of IgE in stimulated B-cells with potency comparable to that of pseurotins A and D. We found that the two natural pseurotins and the active synthetic analogs inhibited the phosphorylation of STAT3, STAT5 and STAT6 proteins in stimulated B-cells, resulting in the inhibition of B-cell proliferation and differentiation into the plasma cells. In vivo, pseurotin D decreased ovalbumin-induced foot pad edema. CONCLUSION: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of IgE production in B-cells by linking the effect to STAT signaling, and associated modulation of B-cell proliferation and differentiation. Together with our finding that structurally simpler pseurotin analogs were able to reproduce the effects of natural pseurotins, the presented work has implications for the future research on these secondary metabolites in the context of allergic diseases.

YY1 control of mitochondrial-related genes does not account for regulation of immunoglobulin class switch recombination in mice.

Immunoglobulin class switch recombination (CSR) occurs in activated B cells with increased mitochondrial mass and membrane potential. Transcription factor Yin Yang 1 (YY1) is critical for CSR and for formation of the DNA loops involved in this process. We therefore sought to determine if YY1 knockout impacts mitochondrial gene expression and mitochondrial function in murine splenic B cells, providing a potential mechanism for regulating CSR. We identified numerous genes in splenic B cells differentially regulated when cells are induced to undergo CSR. YY1 conditional knockout caused differential expression of 1129 genes, with 59 being mitochondrial-related genes. ChIP-seq analyses showed YY1 was directly bound to nearly half of these mitochondrial-related genes. Surprisingly, at the time when YY1 knockout dramatically reduces DNA loop formation and CSR, mitochondrial mass and membrane potential were not significantly impacted, nor was there a significant change in mitochondrial oxygen consumption, extracellular acidification rate, or mitochondrial complex I or IV activities. Our results indicate that YY1 regulates numerous mitochondrial-related genes in splenic B cells, but this does not account for the impact of YY1 on CSR or long-distance DNA loop formation.

A Mixture of Functional Complex Extracts from Lycium barbarum and Grape Seed Enhances Immunity Synergistically In Vitro and In Vivo.

A mixture of multiple ingredients is often more effective than the individual ingredients. The functions of Lycium barbarum polysaccharide (LBP) glycoconjugate and grape seed procyanidins (GSP) are widely known. Here, we investigated the synergistic immune-enhancing activity of LBP and GSP. Atomic force microscopy results suggested that the mixture of LBP and GSP exhibited circular structure unlike LBP alone, and the addition of polyphenols may change the spatial conformation of the sugar chain. The changes in the structure were related to the synergistic effect of the two functional agents on immune recovery. In vitro, the proliferation rate of splenocytes was higher in LBP + GSP group (64.16%), rather than the sum of LBP group (13.01%) and GSP group (43.61%) individually used. This synergistical proliferation of splenocytes may be correlated to the increasing intracellular free calcium levels. Furthermore, the mixture significantly enhanced the immunity in vivo, as evident from the recovery of peripheral white blood cell counts in LBP + GSP group (18.535 × 109 /L) to normal group levels (18.115 × 109 /L) and higher B cell proliferation than normal group (P < 0.05). These results highlight the immune-enhancing activity of the combination of LBP and GSP associated with the structural changes, which may facilitate the development of functional foods with fewer resources but enhanced activities. PRACTICAL APPLICATION: The synergistic effects of LBP and GSP on immunomodulatory were better than the sum of the effects of the individual agents both in vitro and in vivo. Our results may provide a research-based support for the development of related functional products and an insight into the production of food resources with a fewer but more effective functional agents for better results.

Btk Inhibitors as First Oral Atherothrombosis-Selective Antiplatelet Drugs?

Bruton's tyrosine kinase (Btk) is essential for B cell differentiation and proliferation, but also platelets express Btk. Patients with X-linked agammaglobulinemia due to hereditary Btk deficiency do not show bleeding, but a mild bleeding tendency is observed in high dose therapy of B-cell malignancies with ibrutinib and novel second-generation irreversible Btk inhibitors (acalabrutinib and ONO/GS-4059). This review discusses recent studies that may explain this apparent paradox and gives mechanistic insights that suggest a unique potential of low dose irreversible Btk inhibitors as atherothrombosis-focused antiplatelet drugs.

Helminths-based bi-functional molecule, tuftsin-phosphorylcholine (TPC), ameliorates an established murine arthritis.

A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-ß, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.

YY1 binds to the E3' enhancer and inhibits the expression of the immunoglobulin κ gene via epigenetic modifications.

The rearrangement and expression of immunoglobulin genes are regulated by enhancers and their binding transcriptional factors that activate or suppress the activities of the enhancers. The immunoglobulin κ (Igκ) gene locus has three important enhancers: the intrinsic enhancer (Ei), 3' enhancer (E3'), and distal enhancer (Ed). Ei and E3' are both required for Igκ gene rearrangement during early stages of B-cell development, whereas optimal expression of the rearranged Igκ gene relies on both E3' and Ed. The transcription factor YY1 affects the expression of many genes involved in B-cell development, probably by mediating interactions between their enhancers and promoters. Herein, we found that YY1 binds to the E3' enhancer and suppresses Igκ expression in B lymphoma cells by epigenetically modifying the enhancer. Knocking down YY1 enhanced Igκ expression, which was associated with increased levels of E2A (encoded by the TCF3 gene) and its binding to the E3' enhancer. Moreover, in germinal centre B cells and plasma cells, YY1 expression was reversely associated with Igκ levels, implying that YY1 might facilitate antibody affinity maturation in germinal centre B cells through the transient attenuation of Igκ expression.

Nonclinical Safety Assessment of CFZ533, a Fc-Silent Anti-CD40 Antibody, in Cynomolgus Monkeys.

CFZ533 is a pathway blocking, nondepleting anti-CD40 antibody that is in clinical development for inhibition of transplant organ rejection and therapy for autoimmune diseases. A 26-week GLP toxicity study in sexually mature Cynomolgus monkeys was conducted in order to support chronic application of CFZ533. CFZ533 was subcutaneously administered at doses up to 150 mg/kg/week and was safe and generally well tolerated. CFZ533 showed no adverse effects for cardiovascular, respiratory, and neurobehavioral endpoints, and no changes were observed for blood lymphocyte and platelet counts or blood coagulation markers. In line with the nondepleting nature of CFZ533, CD20+ B cells in the blood were only marginally reduced. A complete suppression of germinal center (GC) development in lymph nodes and spleen was the most prominent result of post-mortem histological investigations. This was corroborated by an abrogated T-dependent antibody response (TDAR) to the antigen Keyhole Limpet Hemocyanin (KLH) as well as an absence of anti-drug antibodies (ADAs) in the absence of B cell depletion as seen with immunophenotyping and histology. When serum levels of CFZ533 in recovery animals dropped levels necessary for full CD40 occupancy on B cells, all animals were able to mount a TDAR to KLH. All histological changes also reverted to normal appearance after recovery. In summary, CFZ533 was shown to be well tolerated and safe in the 26-week toxicity study with a distinct pharmacodynamic profile in histology and immune function.

Beneficial Effects of Bushen Formula Combined with Enticavir on Chronic Hepatitis B Patients with Suboptimal Response to Enticavir by Regulating B-Cell Differentiation.

BACKGROUND/AIMS: To investigate the clinical effects of the combination therapy with Bushen Formula (BSF) plus enticavir (ETV) on chronic hepatitis B (CHB) patients with suboptimal response to ETV and explore the regulatory mechanisms of BSF on B cells-mediated humoral immunity. METHODS: Sixty-four HBeAg-positive CHB patients with suboptimal response to ETV were enrolled, and were randomly assigned into control group (C-Group, placebo combined with ETV for 12 months) or treatment group (T-Group, BSF combined with ETV for 12 months). Serum samples from 57 treatment-naïve CHB patients and 15 healthy controls were collected. Serum HBV DNA levels were evaluated by real-time PCR. Characteristics of peripheral blood B-cell subtypes were analyzed by flow cytometry. Serum HBV markers and B cell-activating factor (BAFF) levels were detected by ELISA. Chinese medicine symptom complex score was evaluated and recorded. RESULTS: After treatment, the rates of patients with a reduction of HBsAg > 0.5 log10 IU/ml or 1.0 log10 IU/ml and the rates of HBeAg clearance in T-Group were all higher than those in C-group, with no significant intergroup difference. Only in T-Group, Chinese medicine symptom complex score and the frequency of total B cells were significantly decreased, and the frequencies of Bm1, CD24+CD27-switched B cells and plasma cells were markedly increased after treatment compared with those before treatment. Compared with healthy controls, serum BAFF levels in treatment-naïve CHB patients were increased, and there was a significant positive correlation between serum BAFF and HBsAg levels. However, serum BAFF levels did not differ after treatment in T-Group and C-Group. CONCLUSIONS: The combination therapy with BSF plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg in CHB patients with partial response to ETV through regulating the differentiation of B-cell subsets.

The ion channel TRPM7 is required for B cell lymphopoiesis.

The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+ It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

Argemone oil, an edible oil adulterant, induces systemic immunosuppression in Balb/c mice in an oral 28 days repeated dose toxicity study.

Consumption of edible oils contaminated with Argemone oil (AO) leads to a clinical condition called "Epidemic dropsy". Earlier studies have reported that metabolism and oxidative stress primarily contributes to AO toxicity, however, the involvement of immune system has not been assessed so far. Therefore, the present study was undertaken to systematically assess the effect of AO exposure on the function of immune system in Balb/c mice. The repeated exposure of AO for 28 days caused prominent regression of spleen and thymus; severe inflammatory changes in spleen depicted by the loss of distinct follicles, increased megakaryocyte infiltration, and enhanced expression levels of inflammatory markers (iNOS & COX-2). At the functional level, AO exposure significantly abrogated the mixed lymphocyte reaction and mitogen-stimulated lymphoproliferative activity of T and B cells, which is reflective of profound lymphocyte dysfunction upon antigen exposure. In concordance with the loss in functional activity of lymphocytes in AO exposed animals, it was found the AO altered the relative percentage of CD3+, CD4+, and CD28 + T cells. Further, there was a marked decrease in the relative distribution of cells with prominent MHC I and CD1d expression in AO exposed splenocytes. Moreover, reduced levels of immune stimulatory cytokines (TNF-α, IFN-γ, IL-2, IL-4, and IL-6), and increased levels of immunosuppressive cytokine IL-10 were detected in the serum of AO treated mice. Along with T and B cells, AO exposure also affected the phenotype and activation status of macrophages suggesting the inclination towards "alternative activation of macrophages". Altogether, these functional changes in the immune cells are contributing factors in AO induced immunosuppression.

Nonclinical Safety Assessment of the γ-Secretase Inhibitor Avagacestat.

The toxicity of avagacestat, a sulfonamide-based gamma (γ)-secretase inhibitor that was in development as a treatment for Alzheimer's disease, was evaluated in a comprehensive nonclinical toxicology program that included 6-month and 1-year repeat-dose toxicity studies in rats and dogs, respectively. There was a spectrum of mechanism-based changes attributed to inhibition of Notch signaling that regulates the differentiation and proliferation of cells throughout development and in adult tissues. In both rats and dogs, ovarian follicular degeneration and atrophy and a low incidence of granulosa cell hyperplasia and benign granulosa-thecal cell tumors were observed. Gastrointestinal (GI) findings, including goblet cell metaplasia, dilatation of intestinal crypts/glands, mucosal epithelial necrosis and regeneration, and villous atrophy, were limited to dogs that had clinical evidence of GI toxicity. Other avagacestat-related findings attributed to interference with Notch signaling included decreases in peripheral lymphocytes (T and/or B cells) and lymphoid depletion in lymph nodes and the spleen in both species, as well as epiphyseal cartilage and trabecular bone changes in rats. Pharmacologically mediated decreases in brain and cerebrospinal fluid levels of ß-amyloid (Aß) peptides Aß40 and Aß42 and decreased expression of white blood cell mRNA levels of the Notch-regulated gene hairy and enhancer of split-1 confirmed target engagement at all doses. Reductions in brain Aß peptide levels (22 to 34%) in dogs after 1 year at exposures up to the no-observed-effect level for GI toxicity of 1.1× the human plasma exposure, and reversible GI changes at a 3.2× multiple, indicated that a sustained pharmacodynamic effect was attained at exposures without dose-limiting toxicity.

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

Sequestration of 2,3,7,8-tetrachlorodibenzo-p-dioxin by activated carbon eliminates bioavailability and the suppression of immune function in mice.

The effectiveness of activated carbon in reducing the bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined from the context of using in situ sorbent amendments to remediate soils/sediments contaminated with polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). This technology has gained rapid acceptance based on observations that activated carbon amendments predictably lower PCDD/F concentrations in water and bioaccumulation by simple aquatic organisms and earthworms; it has been assumed that bioavailability to mammals is similarly reduced, although this has been disproven for other sorbent materials. In the present study TCDD was absorbed to a microporous activated carbon (TCDD-AC) using the incipient wetness method. An aqueous suspension of TCDD-AC and an equivalent dosage of TCDD in corn oil were administered by oral gavage to B6C3F1 mice. The relative bioavailability of TCDD-AC was determined by quantifying and comparing the hepatic induction of cyp1A1 (messenger ribonucleic acid) and suppression of the immunoglobulin M antibody-forming cell immune response by the 2 forms of TCDD. A concentration-dependent response was observed for both assays when TCDD in corn oil was administered to mice. However, when equivalent masses of TCDD were administered as TCDD-AC, no induction of cyp1A1 or suppression of the immunoglobulin M antibody-forming cell response was observed. The absence of these 2 sensitive aryl hydrocarbon receptor-mediated responses in mice provides the first direct evidence that activated carbon can sequester TCDD in a form that eliminates its bioavailability to mammals. These results support the premise that activated carbon can be used to reduce the bioeffective dose of TCDD delivered to mammals and that activated carbon amendments may provide a low-cost alternative to traditional remediation technologies. Environ Toxicol Chem 2017;36:2671-2678. © 2017 SETAC.

Camel Whey Protein Protects B and T Cells from Apoptosis by Suppressing Activating Transcription Factor-3 (ATF-3)-Mediated Oxidative Stress and Enhancing Phosphorylation of AKT and IκB-α in Type I Diabetic Mice.

BACKGROUND: Diabetes mellitus (DM) is associated with severe immune system complications. Camel whey protein (CWP) decreases free radicals (ROS) and modulates immune functions, but its effect on DM-impaired immune systems has not been studied. We investigated the impact of CWP on the immune system in a Type 1 diabetes mouse model. METHODS: Three experimental groups were used: (1) non-diabetic control; (2) diabetic; and (3) CWP-treated diabetic mice. RESULTS: Induction of diabetes by streptozotocin was associated with reduction of body weight and insulin level, increase in glucose level and pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), and reduction in IL-2 and IL-4 levels. Upregulated ATF-3 expression was followed by a marked elevation in ROS levels. Lymphocytes from diabetic mice exhibited increased apoptosis through decreased phosphorylation of AKT and IκB-α, increased infiltration of T cells in the spleen and thymus, and decreased B cell numbers in the spleen. Supplementation with CWP decreased the levels of proinflammatory cytokines, ROS, and ATF-3 expression, and increased the levels of IL-4. Treatment with CWP decreased apoptosis by enhancing the phosphorylation of AKT and IκB-α as well as T-cell and B-cell distribution in the spleen and thymus. CONCLUSIONS: Our findings suggest the beneficial effects of CWP supplementation during diabetes on decreasing and orchestrating the redox status and subsequently rescuing the immune cells from exhaustion.

Anti-Fatigue Effects of the Unique Polysaccharide Marker of Dendrobium officinale on BALB/c Mice.

Dendrobium officinale extract shows potent anti-fatigue effects; however, the active substance responsible for these effects remains undetermined. A glucomannan with a huge molecular size of 730 kDa, called DOP, was identified as the unique authentication marker of this expensive herb. DOP exhibited immunomodulating effects on macrophages and lymphocytes in our previous study. Clinical reports also showed that people with fatigue syndrome have a disturbed immune system. Because DOP is the unique and dominant component of D. officinale, we hypothesize that DOP may also have anti-fatigue activity. The present study aims to evaluate the anti-fatigue activity of DOP on BALB/c mice, with Rhodiola rosea extract as a positive control. DOP and Rhodiola rosea extract were orally administered at doses of 50 mg/kg and 100 mg/kg, respectively, for four weeks, and the anti-fatigue activity of DOP on BALB/c mice was evaluated using the weight-loaded swimming test. The contents of lactic dehydrogenase (LDH), creatine phosphokinase (CK), triglyceride (TG), blood urea nitrogen (BUN), superoxide dismutase (SOD), malondialdehyde (MDA), lactic acid (LD), and glutathione peroxidase (GSH-Px) in serum, glycogen of liver and gastrocnemius muscle were also determined. Their effects on variability of T cells and B cells were determined by using tetrazolium compound (MTS) method. The weight-loaded swimming exercise caused fatigue syndrome, mainly including the decreases of serum SOD/GSH-Px and gastrocnemius glycogen, as well as the increases of LDH, BUN, MDA, CK, TG, and LD in serum. All of these indicators of fatigue were inhibited to a certain extent by both DOP and Rhodiola rosea extract; however, the effects of DOP were much stronger than those of Rhodiola rosea extract. Compared to the positive control, mice dosed with DOP showed increases in endurance, body weight, and food intake. Furthermore, DOP-feeding mice significantly increased the cell variability of T lymphocytes and B lymphocytes, compared with that of mice in control group. This study indicates that the unique and dominant polysaccharide DOP of D. officinale has stronger anti-fatigue activity than Rhodiola rosea extract. As such, DOP has promising potential for pharmaceutical development into health products to reduce fatigue.

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6ß, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

Synthesis and immunostimulatory activity of substituted TLR7 agonists.

Fifteen new substituted adenines were synthesized as potential TLR7 agonists. These compounds, along with 9 previously reported compounds, were analyzed for TLR7 activity and for the selective stimulation of B cell proliferation. Several functionalized derivatives exhibit significant activity, suggesting their potential for use as vaccine adjuvants.

[THE EXCESS OF PALMITIC FATTY ACID IN FOOD AS MAIN CAUSE OF LIPOIDOSIS OF INSULIN-DEPENDENT CELLS: SKELETAL MYOCYTES, CARDIO-MYOCYTES, PERIPORTAL HEPATOCYTES, KUPFFER MACROPHAGES AND B-CELLS OF PANCREAS].

In phylogenesis, becoming of biologicalfunctions and biological reactions proceeds with the purpose ofpermanent increasing of "kinetic perfection ". The main role belongs to factors ofphysical, chemical and biological kinetics, their evaluation using systemic approach technique under permanent effect of natural selection. The late-in-phylogenesis insulin, proceeded with, in development of biological function of locomotion, specialization of insulin-dependent cells: skeletal myocytes, syncytium of cardiomyocytes, subcutaneous adipocytes, periportal hepatocytes, Kupffer's macrophages and ß-cells of islets of pancreas. The insulin initiated formation of new, late in phylogenesis, large pool of fatty cells-subcutaneous adipocytes that increased kinetic parameters of biological function of locomotion. In realization of biological function of locomotion only adipocytes absorb exogenous mono unsaturated and saturated fatty acids in the form of triglycerides in composition of oleic and palmitic lipoproteins of very low density using apoE/B-100 endocytosis. The rest of insulin-dependent cells absorb fatty acids in the form of unesterified fatty acids from associates with albumin and under effect of CD36 of translocase offatty acids. The insulin in all insulin-depended cells inhibits biological reaction of lipolysis enhancing contributing into development of lipoidosis. The insulin expresses transfer offatty acids in the form of unsaturated fatty acids from adipocytes into matrix of mitochondria. The insulin supplies insulin-dependent cells with substrates for acquiring energy subject to that in pool of unsaturated fatty acids in adipocytes prevails hydrophobic palmitic unsaturated fatiy acid that slowly passes into matrix through external membrane ofmitochondria; oxidases of mitochondria so slowly implement its ß-oxidation that content of exogenous palmitic unsaturatedfatty acid can't be higher than phylogenetic, physiological level - 15% of all amount offatty acids transferring to insulin-dependent cells. The insulin can't both to decrease content of exogenous palmitic fatty acid and inhibit lipolysis in visceral fatty cells of omentum.