Murine Lymphocyte Proliferation Research Articles

Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway.

The immunomodulatory function of mesenchymal stem cells (MSCs) has been considered to be vital for MSC-based therapies. Many works have been devoted to excavate effective strategies for enhancing the immunomodulation effect of MSCs. Nonetheless, canine MSC-mediated immunomodulation is still poorly understood. The inflammatory microenvironment was simulated through the employment of interferon-γ (IFN-γ) in a culture system. Compared with unstimulated cBMSCs, IFN-γ stimulation increased the mRNA levels of Toll-like receptor 3 (TLR3) and indoleamine 2, 3-dioxygenase 1 (IDO-1), and simultaneously enhanced the secretion of immunosuppressive molecules, including interleukin (IL)-10, hepatocyte growth factor (HGF), and kynurenine in cBMSCs. IFN-γ stimulation significantly enhanced the ability of cBMSCs and their supernatant to suppress the proliferation of murine spleen lymphocytes. Lymphocyte subtyping evaluation revealed that cBMSCs and their supernatant diminished the percentage of CD3+CD4+ and CD3+CD8+ lymphocytes compared with the control group, with a decreasing CD4+/CD8+ ratio. Notably, exposure to IFN-γ decreased the CD4+/CD8+ ratio more effectively than unstimulated cells or supernatant. Additionally, IFN-γ-stimulation increased the mRNA levels of the Th1 cytokines TNF-α, and remarkably decreased the mRNA level of the Th2 cytokine IL-4 and IL-10. Our findings substantiate that IFN-γ stimulation can enhance the immunomodulatory properties of cBMSCs by promoting TLR3-dependent activation of the IDO/kynurenine pathway, increasing the secretion of immunoregulatory molecules and strengthening interactions with T lymphocytes, which may provide a meaningful strategy for the clinical application of cBMSCs in immune-related diseases.

Nanobubble-based anti-hepatocellular carcinoma therapy combining immune check inhibitors and sonodynamic therapy.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, posing a global threat to human healthcare, and current approved treatment strategies do not produce satisfactory outcomes. Here, nanobubbles (NBs) were prepared that carried Immune Check Inhibitors (ICIs), PD-L1 antibody (PD-L1 Ab) and sonodynamic agent CHLORIN E6 (Ce6); the anti-cancer properties of these NBs were analyzed from the point of view of immune and sonodynamic therapies. The PD-L1 Ab/Ce6-NBs could inhibit tumor growth through regulating reactive oxygen species (ROS) production, apoptosis, and most importantly, the function of associated immunocytes, including natural killer cells and lymphocytes. The tumor tissues highly expressed markers of immunogenic tumor cell death (ICD) in which the expression of calreticulin (CRT) and ICD-related immune cytokines (CD80, CD86, INF-γ, and IL-2) were increased in PD-L1 Ab/Ce6-NB treated mice. PD-L1 Ab/Ce6-NBs also promoted murine spleen lymphocyte proliferation and cytotoxic activity, as well as CD8+ T cell infiltration in the tumor tissues, and downregulation of the PD-L1 protein and mRNA expression. Furthermore, Bax expression was increased and Bcl-2 was inhibited at the mRNA and protein levels in a murine subcutaneous transplanted tumor model. These results indicate that PD-L1 Ab/Ce6-NBs can induce ROS-dependent ICD to further boost anti-cancer immune responses under the action of targeting the PD-1/PD-L1 immune check point in the tumor microenvironment as a promising therapeutic agent for HCC.

Emerging Roles of Dipeptidyl Peptidase-4 Inhibitors in Delaying the Progression of Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM) results from the immune cell-mediated destruction of functional pancreatic β-cells. In the presymptomatic period, T1DM is characterized by the presence of two or more autoantibodies against the islet cells in patients without glycemic decompensation. Therapeutic strategies that can modify the autoimmune process could slow the progression of T1DM. Dipeptidyl peptidase-4 (DPP-4) or CD26, a multifunctional serine protease with a dual function (regulatory protease and binding protein), can modulate inflammation and immune cell-mediated β-cell destruction. CD26 is involved in T-cell co-stimulation, migration, memory development, thymic maturation, and emigration patterns. DPP-4 degrades the peptide hormones GLP-1 and GIP. In addition to regulating glucose metabolism, DPP-4 exerts anti-apoptotic, regenerative, and proliferative effects to promote β-cell mass expansion. GLP-1 receptor signaling may regulate murine lymphocyte proliferation and maintenance of peripheral regulatory T-cells. In patients with T1DM, the serum DPP-4 activity is upregulated. Several studies have suggested that the upregulated DPP-4 activity is correlated with T1DM pathophysiology. DPP-4, which is preferentially expressed on the Th1 surface, can promote the polarization of Th1 immunity, a prerequisite for T1DM development. CD26 inhibition can suppress T-cell proliferation and Th1 cytokine production and stimulate tumor growth factor beta-1 (TGF-β1) secretion, which plays an important role in the regulation of autoimmunity in T1DM. Studies on humans or animal models of T1DM have suggested that DPP-4 inhibitors can improve β-cell function and attenuate autoimmunity in addition to decreasing insulin dependence. This review summarizes the emerging roles of DPP-4 inhibitors in potentially delaying the progression of T1DM.

Human Adipose Tissue-Derived Stromal Cells Suppress Human, but Not Murine Lymphocyte Proliferation, via Indoleamine 2,3-Dioxygenase Activity.

Over recent years, mesenchymal stromal cells (MSC) have gained immense attraction in immunotherapy, regenerative medicine and tissue engineering. MSC microenvironment modulation occurs through synergy of direct cell–cell contact, and secreted soluble factors and extracellular vesicles (EV). MSC-derived EV have been suggested as cell-free immunomodulatory alternative to MSC; however, previous findings have challenged this. Furthermore, recent data suggest that evaluating the mechanism of action of human MSC (hMSC) in animal models might promote adverse immune reactions or lack of functionality due to xeno-incompatibilities. In this study, we first assessed the immunomodulatory strength of different human MSC sources on in vitro stimulated T cells and compared this to interferon-gamma (IFNγ) primed MSC conditioned medium (CM) and EV. Second, we addressed the main molecular mechanisms, and third, we assessed the MSC in vitro immunosuppressive effect across interspecies barriers. We identified human adipose tissue-derived stromal cells (ASC) with strongest immunomodulatory strength, followed by bone marrow (BM) and cord blood-derived MSC (CB). Whilst CM from primed ASC managed to exert analogous effects as their cellular counterpart, EV derived thereof did not, reproducing previous findings. IFNγ-induced indoleamine 2,3-dioxygenase (IDO) activity was identified as key mechanism to suppress human lymphocyte proliferation, as in the presence of the IDO inhibitor epacadostat (Epac) a stimulation of proliferation was seen. In addition, we revealed MSC immunosuppressive effects to be species-specific, because human cells failed to suppress murine lymphocyte proliferation. In summary, ASC were the strongest immunomodulators with the IDO-kynurenine pathway being key within the human system. Importantly, the in vitro lack of interspecies immunomodulatory strength suggests that preclinical data need to be carefully interpreted especially when considering a possible translation to clinical field.

In vitro antitumor, pro-inflammatory, and pro-coagulant activities of Megalopyge opercularis J.E. Smith hemolymph and spine venom

Contact with stinging spines venom from several Lepidoptera larvae may result in skin lesions. In Mexico, envenomation outbreaks caused by Megalopyge opercularis were reported between 2015 and 2016. The aim of this study was to identify the venomous caterpillars in Nuevo Leon, Mexico and evaluate several biological activities of their hemolymph (HEV) and spine setae (SSV) venoms. M. opercularis was identified by cytochrome oxidase subunit (COI) designed primers. HEV and SSV extracts cytotoxic activity was assessed on the L5178Y-R lymphoma cell line. For apoptotic cells number and apoptosis, cells were stained with acridine orange/ethidium bromide and validated by DNA fragmentation. Human peripheral blood mononuclear cells (hPBMC) cytokine response to the extracts was measured by the cytometric bead array assay. Extracts effect on pro-coagulation activity on human plasma was also evaluated. HEV and SSV extracts significantly inhibited (p < 0.01) up to 63% L5178Y-R tumor cell growth at 125–500 µg/mL, as compared with 43% of Vincristine. About 79% extracts-treated tumor cells death was caused by apoptosis. Extracts stimulated (p < 0.01) up to 60% proliferation of resident murine lymphocytes, upregulated IL-1β, IL-6, IL-8, and TNF-α production by hPBMC, and showed potent pro-coagulant effects. The pharmacological relevance of these venoms is discussed.

Immunomodulatory effects of pepsin-educed soy protein hydrolysate in rats and murine cells

Background: There has been an increase in demand for soy and its products, with soy protein having a huge market in particular. Several reports have established how soy protein hydrolysates (SPHs) yield better physiological properties and play essential functional roles than the crude soy protein.Objective: To investigate the immunomodulatory and acute toxicity of soy protein hydrolysate (SPH) produced with pepsin protease in mice and rats.Methods: Soy protein hydrolysate was enzymatically produced using pepsin, with E/S (enzyme to substrate ratio) of 0.5% (250 u/mg) and hydrolysis time of 4h. Afterwards, the SPH effects on murine spleen lymphocyte proliferation, and peritoneal macrophage phagocytosis were investigated in vitro. Confirmation studies were explored in rats’ sera IgG and IgA, and the acute toxic effect of SPH was observed in mice subjects.Results: The hydrolysate increased the levels of splenocytes (stimulative index of 1.269 - 1.402) and peritoneal macrophages (phagocytic index of 0.717 – 1.304). Furthermore, the concentrations of sera IgG and IgA obtained from SPH-fed rats ranged from 0.198 – 0.345 mg/ml and 0.0184 – 0.0194 mg/ml, respectively in comparison with the soy protein isolate (SPI) -fed rats (0.208 – 0.322 mg/ml and 0.0188 – 0.0189 mg/ml, respectively). Additionally, 10 mg dose of SPH stably elicited serum IgG in contrast to other doses, while there was a general decrease in the amounts of IgA obtained in the rat subjects. Moreover, there was no acute toxic effect recorded in the mice subjects. Conclusion: In light of the results, it is possible that SPH prepared with pepsin has the potential of improving the immune system, and may therefore be used as immunomodulatory or functional food product.Keywords: Soy protein hydrolysates, splenocyte proliferation, immunomodulatory, phagocytosis and pepsin

Highly oxygenated lanostane-type triterpenoids and their bioactivity from the fruiting body of Ganoderma gibbosum

Eight new highly oxygenated lanostane triterpenes, gibbosic acids A–H (1–8), along with three known ones (9–11), were isolated from the fruiting body of Ganoderma gibbosum. The structures of new isolates were assigned by NMR and HRESIMS experiments. The absolute configurations of 1 were further confirmed by single crystal X-ray diffraction data and computational ECD methods. Immunoregulatory effect and anti-inflammatory activities of these compounds were screened in murine lymphocyte proliferation assay and in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, respectively. Compound 2 exhibited immunostimulatory effect both in lymphocyte proliferation assay without any induction and ConA-induced mitogenic activity of T-lymphocyte, and the proportion of lymphocyte proliferation at the concentration of 0.1μM are 20.01% and 21.40%, respectively.

Lapachol, a compound targeting pyrimidine metabolism, ameliorates experimental autoimmune arthritis

BackgroundThe inhibition of pyrimidine biosynthesis by blocking the dihydroorotate dehydrogenase (DHODH) activity, the prime target of leflunomide (LEF), has been proven to be an effective strategy for rheumatoid arthritis (RA) treatment. However, a considerable proportion of RA patients are refractory to LEF. Here, we investigated lapachol (LAP), a natural naphthoquinone, as a potential DHODH inhibitor and addressed its immunosuppressive properties.MethodsMolecular flexible docking studies and bioactivity assays were performed to determine the ability of LAP to interact and inhibit DHODH. In vitro studies were conducted to assess the antiproliferative effect of LAP using isolated lymphocytes. Finally, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA) models were employed to address the anti-arthritic effects of LAP.ResultsWe found that LAP is a potent DHODH inhibitor which had a remarkable ability to inhibit both human and murine lymphocyte proliferation in vitro. Importantly, uridine supplementation abrogated the antiproliferative effect of LAP, supporting that the pyrimidine metabolic pathway is the target of LAP. In vivo, LAP treatment markedly reduced CIA and AIA progression as evidenced by the reduction in clinical score, articular tissue damage, and inflammation.ConclusionsOur findings propose a binding model of interaction and support the ability of LAP to inhibit DHODH, decreasing lymphocyte proliferation and attenuating the severity of experimental autoimmune arthritis. Therefore, LAP could be considered as a potential immunosuppressive lead candidate with potential therapeutic implications for RA.

Poly(3-hydroxybutyrate)/caffeic acid electrospun fibrous materials coated with polyelectrolyte complex and their antibacterial activity and in vitro antitumor effect against HeLa cells

The purpose of this work was to investigate the possibility for the preparation of new poly(3-hydroxybutyrate) (PHB)/poly(ethylene glycol) (PEG)-based fibrous materials containing natural phenolic compound caffeic acid (CA) of diverse architectures, as well as to study the impact of the fiber composition on the in vitro CA release profile and on the biological properties of the fibrous materials. The application of the one-pot electrospinning enabled the fabrication of nanofibrous materials from PHB and PEG loaded with the CA. Materials with targeted design were obtained by coating with polyelectrolyte complex of alginate (Alg) and N,N,N-trimethylchitosan (TMCh). Three different processing paths were used to obtain coated mats: (i) with CA incorporated in the PHB/PEG core; (ii) with CA embedded in the Alg layer; and (iii) with CA included in the TMCh layer. The in vitro release of CA was modulated by controlling the composition and the architecture of the nanofibrous mats. The performed microbiological screening and MTT cell viability studies revealed that in contrast to the bare mats, the CA-containing nanofibrous materials were effective in suppressing the growth of the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli and displayed good cytotoxicity against human cervical HeLa tumor cells. In addition, the proliferation of murine spleen lymphocytes and peritoneal macrophages was increased by the prepared CA-containing nanofibrous materials. The obtained materials are promising for antibacterial wound dressing applications as well as for application in local treatment of cervical tumors.

Subcutaneous Injections of the Mannose-Sensitive Hemagglutination Pilus Strain of Pseudomonas aeruginosa Stimulate Host Immunity, Reduce Bladder Cancer Size and Improve Tumor Survival in Mice.

We wished to evaluate the effects of Pseudomonas aeruginosa (mannose-sensitive hemagglutination pilus strain, PA-MSHA) as an immunostimulating and anti-tumor agent for treatment of bladder cancer. Immunostimulating effects were assessed by the in vitro proliferation assay of murine splenic lymphocytes. Anti-tumor effects were studied in a subcutaneous tumor model established in female C57BL/6 mice using the MB49 bladder cell line. These mice received subcutaneous injections of normal saline (control group) or PA-MSHA (high, medium, or low dose, respectively, 1.6-2.0×10(9), 3.2- .0×10(8), 6.4-8.0×10(7) CFU/ml) twice a week for 3weeks. Mice survival, tumor volume, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD), serum levels of TNF-α and IFN-γ, and blood CD4(+) /CD8(+) counts were the study outcomes. We observed that PA-MSHA promoted the growth of splenic lymphocytes in vitro. In the murine tumor model, PA-MSHA prolonged mice survival and reduced tumor growth. Furthermore, VEGF and MVD were also diminished by PA-MSHA. Mice that received high and medium dose of PA-MSHA had significantly higher serum levels of IFN-γ and TNF-α (days 21 and 28), and higher levels of CD4(+) /CD8(+) cells (days 21 and 28). In conclusion, PA-MSHA exerts beneficial effects on increasing proliferation of murine splenic lymphocytes in vitro and inhibits the growth of bladder tumor in a murine model. Therefore, PA-MSHA may be useful an immunostimulating and anti-tumor agent for bladder cancer therapy.

Effects of atrazine on the proliferation and cytotoxicity of murine lymphocytes with the use of carboxyfluorescein succinimidyl ester-based flow cytometric approaches

Atrazine (ATZ) is one of the most commonly applied herbicides worldwide. ATZ has been associated with adverse effects on the immune system; however, the mechanism of its immunotoxicity has not been completely elucidated. In this study, the immunotoxic effects of ATZ on murine splenic lymphocytes and magnetic bead-enriched NK cells were investigated in vitro with the use of carboxyfluorescein succinimidyl ester (CFDA-SE)-based flow cytometric approaches. Proliferation responses, NK cell activity, and T-cell early activation were determined with CFDA-SE loading, CFDA-SE/propidium iodide (PI) staining, and CD69+ expression, respectively. Cell apoptosis/cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated using PI, 2′,7′-dichlorodihydrofluorescein diacetate, and rhodamine 123, respectively. The intracellular expressions of apoptosis-related Bcl-2 and caspase-3 were analyzed through intracellular staining and flow cytometry. Results showed that proliferation and NK cell activity were suppressed by ATZ treatment. Such suppression might be associated with the cell apoptosis induced by increased ROS and declined MMP. The underlying mechanism might be the induced caspase-3 expression and decreased Bcl-2 expression. ATZ could elicit immunotoxic effects on murine lymphocytes; its presence in the environment might compromise immune function in organisms. The flow cytometric methods presented in this study should be further investigated in immunotoxicology.

Immunological characterization of Neospora caninum Cyclophilin

Neosporosis is caused by an intracellular protozoan,Neospora caninum, which is an important pathogen of dogs and cattle. In this study, we analyzed Neospora caninum cyclophilin 18 protein (NcCyp18), which regulates proliferation and migration of murine macrophages and lymphocytes. From our study, NcCyp18 showed the activity of macrophage proliferation in a CCR5-independent manner and activity of spleen cell proliferation in a CCR5-dependent manner. NcCyp18 showed the activity of macrophage migration in a CCR5-independent manner and activity of spleen cell migration in a CCR5-dependent manner. Moreover, recombinant NcCyp18 induced the production of the nitric oxide and IL-12 p40. The data suggest that NcCyp18 recruits host cells and enhances the growth of host cells at the site of infection for maintenance of interaction between the parasite and host. DOI: http://dx.doi.org/10.5564/mjas.v11i2.209 Mongolian Journal of Agricultural Sciences Vol.11(2) 2013 pp.17-23

Increased antitumor activity of tumor-specific peptide modified thymopentin.

Thymopoietin pentapeptide (thymopentin, TP5), an immunomodulatory peptide, has been successfully used as an immune system enhancer for treating immune deficiency, cancer, and infectious diseases. However, poor penetration into tumors remains a key limitation to the efficacy and application of TP5. iRGD (CRGDK/RGPD/EC) has been introduced to certain anticancer agents, and increased specific tumor penetrability of drugs and cell internalization have been observed. In the present study, we fused this iRGD fragment with the C-terminal of TP5 to yield a new product, TP5-iRGD. Cell attachment assay showed that TP5-iRGD exhibits more extensive attachment to the melanoma cell line B16F10 than wild-type TP5. Tumor cell viability assay showed that iRGD conjugation with the TP5 C-terminus increases the basal antiproliferative activity of the pentapeptide against the melanoma cell line B16F10, the human lung cancer cell line H460, and the human breast cancer cell line MCF-7. Subsequent injections of TP5-iRGD inhibited invivo melanoma progression more efficiently than the native TP5. Murine spleen lymphocyte proliferation assay also showed that TP5-iRGD and the parent pentapeptide feature nearly identical spleen lymphocyte proliferation activities. We built an integrin αvβ3 and TP5-iRGD computational binding model to investigate the mechanism by which TP5-iRGD promotes increased activity further. Conjugation with iRGD promotes binding to integrin αvβ3, thereby increasing the tumor-homing efficiency of the resultant peptide. These experimental and computational observations of increased TP5-iRGD activity help broaden the usage of TP5 and reflect the great application potential of the peptide as an anticancer agent.

Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

Alkylglycerols (AKGs) are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg). Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL) transcription of IgG (γ1) mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1)/GATA-3 (Th2) flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2). It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1) and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ) upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10) were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response

The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5–200 μg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose–response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150 μg/ml, increased the LPS-induced production of IL-1β and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.

In vivo stimulatory effect of multi-component herbal remedy PADMA 28 on mitogen-induced proliferation of mice splenic lymphocytes and their chemokinetic activity

PADMA 28, a natural herbal multi-compound remedy originates from traditional Tibetan medicine and possesses a variety of beneficial effects on experimental and clinical models of inflammation and atherosclerosis, as well as angioprotecive, antioxidative and wound-healing properties. The aim of the present study was to evaluate the in vivo influence of this remedy on the in vitro mitogen-induced proliferation of murine splenic lymphocytes and their chemokinetic activity in cell culture.The study was performed on 6-8 weeks old inbred Balb/c mice. PADMA28 was administered to mice per os in daily doses 5.8 mg (calculated from the highest dose recommended for human) or 0.085 mg (dose from the range of active doses of other herbal extracts containing polyphenolic substances used previously by us in experiments with mice), for 7 days. Control groups received water. No substantial differences were observed between groups of mice fed with low and high PADMA doses. In both of them, response of splenic lymphocztes to mitogen PHA (p < 0.001) and their in vitro chemokinetic activity (p < 0.001 for low dose and p < 0.01 for high dose) were highly significantly increased as compared to the controls. The results of our investigations suggest that PADMA 28 can stimulate cell-mediated immunity in mice and might be used for this purpose in the wide spectrum of doses.

Pivotal Role of ADP-ribosylation Factor 6 in Toll-like Receptor 9-mediated Immune Signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.

English

The water-soluble polysaccharide (GLP) was obtained from the spores of Ganoderma lucidum by hot water extraction and gel chromatography. The molecular weight was estimated to be 143 KD determined by using HPGPC. Structure features of GLP were investigated by a combination of chemical and instrumental analysis. The results indicated that GLP consisted of a backbone composed of (1→6)- linked-α-d-glucopyranosyl, (1→3)-linked-β-d-glucopyranosyl and (1→3,6)-linked-β-d-glucopyranosyl residues in the ratio of 1:2:1, and terminated with one single terminal (1→)-β-d-glucopyranosyl at the O- 6 position of (1→3,6)-linked-β-d-glucopyranosyl, along the main chain. Preliminary tests in vitro showed that GLP has stimulating effects on murine lymphocyte proliferation induced by concanavalin A (ConA) and lipopolysaccharide (LPS) in a dose-dependent manner. It is a possible potential immunopotentiating agent for use in functional foods or medicine against both pathogens and cancer. Key words: Ganoderma lucidum, polysaccharide, structure, lymphocyte proliferation.

Dimethoxycurcumin, a metabolically stable analogue of curcumin, exhibits anti-inflammatory activities in murine and human lymphocytes

The aim of this study was to investigate whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue having higher metabolic stability over curcumin, could exhibit anti-inflammatory activity in murine and human lymphocytes. Both curcumin and DiMC suppressed mitogen as well as antigen driven proliferation of murine splenic lymphocytes. Further, mitogen and antigen-stimulated cytokine (IL-2, IL-4, IL-6 and IFN-γ) secretion by T cells was also abrogated by curcumin and DiMC. Interestingly, curcumin and DiMC suppressed B cell proliferation induced by lipopolysaccharide. Curcumin and DiMC also inhibited Con A-induced activation of early and late T cell activation markers. They scavenged basal reactive oxygen species and depleted GSH levels in lymphocytes. The suppression of mitogen-induced T cell proliferation and cytokine secretion by curcumin and DiMC was significantly abrogated by thiol containing antioxidants suggesting a role for redox in their anti-inflammatory activity. Further, the possibility of curcumin and DiMC directly interacting with thiol-containing antioxidant GSH was monitored by changes in absorbance. Both curcumin and DiMC inhibited Con A induced activation of NF-κB and MAPK. More importantly, curcumin and DiMC inhibited phytohaemagglutinin induced proliferation and cytokine secretion by human peripheral blood mononuclear cells. To explore their therapeutic efficacy, they were added to lymphocytes post-Con A stimulation and we observed a significant suppression of IL-2, IL-6 and IFN-γ. The present study for the first time demonstrates the potent anti-inflammatory activity of DiMC. Further, DiMC could find application as an alternative to curcumin, which is currently used in several clinical studies, due to its superior bioavailability and comparable efficacy.

Codon optimization of recombinant staphylococcal enterotoxin O enhances the expression level in Escherichia coli

To enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons. The gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined. After the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro. Optimization of rare codons can enhance the expression of SEO effectively.