Reversing the polarization of tumor-associated macrophages inhibits tumor metastasis

ObjectiveThe M2 phenotype is dominant in tumor associated macrophages (TAM), and plays a key role in promoting tumor growth, invasion and metastasis. Converting TAM polarization from M2 to M1 may contribute to eliciting anti-tumor-specific immune responses and inhibiting tumor metastasis. In this study, the effect of reversing the polarization of TAM on tumor metastasis was investigated.MethodsPeritoneal macrophages were obtained from BABL/c mice, and M2 polarization was induced by IL-4. In an in vivo experiment, BABL/c mice were transplanted with 4 T1 tumor cells. In vitro and in vivo experimental studies, M2 macrophage polarization was reversed with CpG-DNA or CpG-DNA combined with anti-IL-10R Ab. CD68, MHCII and FRβ molecular expression in macrophages were examined with immunofluorescence staining. The mRNA expression of IL-2, IL-6, IL-13, VEGF and MMP-9 were detected with RT-PCR. VEGF and MMP-9 protein expression of tumors in situ was measured by western blot assay. Lung-metastasis of the tumor was observed and assessed by micro-CT.ResultsCpG-DNA and CpG-DNA combined with anti-IL-10R Ab could promote MHCII, IL-2, IL-6 and IL-13 molecular expression, and suppress the expression of FRβ, MMP-9 and VEGF, in both freshly isolated peritoneal macrophages and M2 macrophages. In the CpG-DNA combined with anti-IL-10R Ab injecting group, the percentage of CD68⁺ MHCII⁺ cells were significantly higher than that of CD68⁺ FRβ⁺ cells (P < 0.05). This was distinct from the result of the control group, which CD68⁺ FRβ⁺ was higher than CD68⁺ MHCII⁺ cells (P < 0.01). Furthermore, VEGF-A and MMP-9 level in primary tumor tissues in the experimental group was significantly lower (P < 0.01), compared to the control group. Moreover, the number of detectable lung-metastasis foci was significantly lower in the experimental group than in the control group (P < 0.05).ConclusionReversing the polarization of TAM from M2 to M1 phenotype can inhibit tumor metastasis.     

Identification of novel reaction products of methylene-bis-phenylisocyanate (“MDI”) with oxidized glutathione in aqueous solution and also during incubation of MDI with a murine hepatic S9 fraction

Methylene diphenyl diisocyanate (MDI) is an important industrial chemical and asthmagenic respiratory sensitizer, however its metabolism remains unclear. In this study we used LC-MS and LC-MS/MS to identify novel reaction products of MDI with oxidized glutathione (GSSG), including an 837 m/z [M + H]⁺ ion corresponding to GSSG bound (via one of its N-termini) to partially hydrolyzed MDI, and an 863 m/z [M + H]⁺ ion corresponding to GSSG cross-linked by MDI (via its two γ-glutamine N-termini). Further studies with heavy isotope labeled and native reduced glutathione (GSH) identified an [M + H]⁺ ion corresponding to previously described mono(GSH)-MDI, and evidence for “oligomeric” GSH-MDI conjugates. This study also investigated transformational changes in MDI after incubation with an S9 fraction prepared from murine liver. LC-MS analyses of the S9 reaction products revealed the formation of [M + H]⁺ ions with m/z's and retention times identical to the newly described GSSG-MDI (837 and 863) conjugates and the previously described mono(GSH)-MDI conjugate. Together the data identify novel biological transformations of MDI, which could have implications for exposure-related health effects, and may help target future in vivo studies of metabolism.     

In vitro induced CD4+CD25+Foxp3+ Tregs attenuate hepatic ischemia–reperfusion injury

Reperfusion injury causes liver dysfunction after warm or cold ischemia. Emerging data suggest a role of T cells as mediators in this ischemia/reperfusion (I/R) injury. In the T cells, a part of CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) were reported to facilitate recovery from I/R injury. These Tregs can be induced by TGF-β in vitro. Interestingly, rapamycin was reported to selectively expand these Tregs in vitro. In the present study, addition of rapamycin to cultures containing TGF-β further increased the frequency and absolute number of functional CD4⁺ Tregs. Using a partial (70%) hepatic warm ischemia model, we investigated the effects of liver function recovery under the treatment of Tregs induced by rapamycin and TGF-β. The treatment of Tregs significantly reduced serum alanine aminotransferase and aspartate aminotransferase compared to I/R control animals at 24 h after reperfusion (P < 0.05). They also significantly attenuated the up-regulation of IFN-γ and IL-17 compared to the I/R control animals (P < 0.05). In conclusion, Tregs ameliorate the biochemical of hepatic I/R injury by preventing proinflammatory cytokines following a warm I/R insult. These data may pave the way to use Tregs as cell therapy to prevent hepatic I/R injury.     

P-glycoprotein function in peripheral T lymphocyte subsets of myasthenia gravis patients: Clinical implications and influence of glucocorticoid administration

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder with a chronic clinical course that requires long-term glucocorticoid (GC) therapy. A drug efflux pump, P-glycoprotein (P-gp), actively transports GC out of target cells, thereby reducing its efficacy. We evaluated the P-gp function of peripheral-blood mononuclear cells in 59 MG patients. P-gp function was estimated from a decrease in fluorescent P-gp substrate Rhodamine 123 and its inhibition by the conformation-sensitive UIC2 monoclonal antibody. P-gp function on CD8⁺ T cells in 21 MG patients having experienced GC therapy was higher than that in 19 MG patients having no history of GC therapy (p = 0.026). There was a significant correlation between P-gp function in CD3⁺ (r = 0.55, p = 0.014) or CD4⁺ (r = 0.48, p = 0.034) T cells and the total dose of prednisolone for treatment. P-gp function on CD4⁺ T cells in MG patients who showed low responses to prednisolone therapy (n = 8) was higher than that in patients who showed relatively high responses to prednisolone therapy (n = 10) (p = 0.045). These results suggest that higher P-glycoprotein activity on CD3⁺ or CD4⁺ cells necessitated treatment with higher steroid doses in order to achieve a clinical response. The measurement of P-gp function on CD4⁺ T cells is useful in the assessment of clinical response to GC therapy.     

Chicken egg yolk antibodies (IgY) modulate the intestinal mucosal immune response in a mouse model of Salmonella typhimurium infection

This study determined the effects of chicken egg yolk antibodies (IgY) on immune responses in the intestinal mucosal of mice infected with Salmonella typhimurium. Sixty, 28-day-old mice were divided into 4 groups and treated with streptomycin or sterile water for 2 days followed by 1 day without treatment. The control group was unchallenged whereas the mice in the other three groups were treated twice with 10⁹ CFU mL^− 1 S. typhimurium. For the next 3 days, control mice continued to receive no treatment whereas the mice in the remaining three groups were orally administered with 20 mg mL^− 1 of specific IgY, 20 mg mL^− 1 of nonspecific IgY or PBS. S. typhimurium activated gut-associated lymphoid tissue, increasing the release of IFN-γ and TNF-α in the mucosa and increased the number of activated T-lymphocytes and cytotoxic T-γδ. Specific IgY attenuated the increase in IFN-γ and TNF-α and the decrease in IL-10. S. typhimurium induced mobilization of CD8⁺ and CD8⁺ TCRγδ T cells in the epithelium and CD4⁺ and CD8⁺ T cells in the lamina propria reflecting an inflammatory process that was attenuated by IgY. These results suggest that specific IgY modulates intestinal mucosal immune responses during a S. typhimurium infection.     

Infliximab reduces CD147, MMP-3, and MMP-9 expression in peripheral blood monocytes in patients with active rheumatoid arthritis

Recent studies have reported elevated expression levels in active rheumatoid arthritis patients of the cluster of differentiation (CD) 147 on CD14⁺ peripheral blood monocytes and as a result, CD147 may be a target for the development of a novel rheumatoid arthritis therapy. This report describes the inhibitory effects of infliximab on CD147 and metalloproteinases (MMP)-3 and MMP-9 overexpression in peripheral blood monocytes obtained from patients with active rheumatoid arthritis. Thirty patients with active rheumatoid arthritis that were refractory to methotrexate therapy were randomized at a 4:1 ratio into groups A and B, respectively. Group A received three to four infusions of infliximab (3 mg/kg) and group B participants received four infusions of placebo. Both groups were also treated with a stable background dose of methotrexate. The CD147 expression levels on CD14⁺ peripheral blood monocytes of rheumatoid arthritis patients was detected by flow cytometry. The expression of CD147, MMP-3, and, MMP-9 mRNA in peripheral blood mononuclear cells was assayed by real-time quantitative PCR, and the expression of MMP-3 and MMP-9 in serum was measured by a multiplexed microsphere-based flow assay. Results showed that the expression of CD147 and MMP-9 mRNA in group A decreased compared to group B. Expression of CD147 on CD14⁺ monocytes was reduced (P<0.05), and serum MMP-3 and -9 levels in group A were decreased by week 18. These data suggested that infliximab could inhibit CD147 expression on CD14⁺ monocytes as well as reduce the levels of MMP-3 and MMP-9 in peripheral blood monocytes.     

Mesenchymal stem cells upregulate Treg cells via sHLA-G in SLE patients

BackgroundSoluble human leukocyte antigen-G (sHLA-G) is a non-classical HLA class I molecule, exhibiting strong immunosuppressive properties by inducing the differentiation of T regulatory cells (Treg). Mesenchymal stem cells (MSCs) transplantation alleviates disease progression in systemic lupus erythematosus (SLE) patients. However, the underlying mechanisms are largely unknown.ObjectivesTo explore whether sHLA-G is involved in upregulating effects of MSCs on Treg, which contributes to therapeutic effects of MSCs transplantation in SLE.MethodsThe serum sHLA-G levels of SLE patients and healthy controls were detected by ELISA. The percentages of peripheral blood CD4 + ILT2 +, CD8 + ILT2 +, CD19 + ILT2 + cells and Treg cells were examined by flow cytometry. Ten patients with active SLE, refractory to conventional therapies, were infused with umbilical cord derived MSCs (UC-MSCs) and serum sHLA-G was measured 24 h and 1 month after infusion. The mice were divided into three groups: C57BL/6 mice, B6.MRL-Fas^lpr mice infused with phosphate buffer saline (PBS), and B6.MRL-Fas^lpr mice infused with bone marrow MSCs (BM-MSCs). Then, the concentrations of serum Qa-2 were detected. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and co-cultured with UC-MSCs for 3 days at different ratios (50:1, 10:1, and 2:1) with or without HLA-G antibody, and the frequencies of CD4 + CD25 + Foxp3 + T cells were then determined by flow cytometry.ResultsThe concentrations of serum sHLA-G were comparable between SLE patients and healthy controls. However, there was a negative correlation between sHLA-G levels and SLE disease activity index (SLEDAI) scores in active SLE patients (SLEDAI > 4). We found that serum sHLA-G levels were negatively correlated with blood urea nitrogen, serum creatinine and 24-hour urine protein in SLE patients. The sHLA-G levels were significantly lower in SLE patients with renal involvement than those without renal involvement. The expression of ILT2 on CD4 + T cells from SLE patients decreased significantly compared to that of healthy controls. A positive correlation between the frequencies of Treg and CD4 + ILT2 + T cells was found in SLE patients. The levels of sHLA-G increased 24 h post UC-MSCs transplantation. The concentrations of Qa-2 in BM-MSCs transplanted mice were significantly higher than those of control group. In vitro studies showed that MSCs increased the frequency of Treg cells in SLE patients in a dose-dependent manner, which was partly abrogated by the anti-HLA-G antibody.ConclusionsOur results suggested that MSCs may alleviate SLE through upregulating Treg cells, which was partly dependent on sHLA-G.     

A higher frequency of CD4+ CXCR5+ T follicular helper cells in patients with newly diagnosed Henoch–Schönlein purpura nephritis

T follicular helper (TFH) cells play an important role in the humoral immune responses. The aim of this study was to examine the frequency of different subsets of CD4⁺ CXCR5⁺ TFH cells and B cells in patients with new-onset Henoch–Schönlein purpura nephritis (HSPN). The numbers of different subsets of CD4⁺ CXCR5⁺ TFH cells, B cells and the constituents of serum cytokines were detected in a total of 25 patients with newly diagnosed HSPN before and after treatment, and in 14 healthy controls (HC). The potential connection of these cells with the clinical characteristics in HSPN patients was analyzed. The numbers of circulating CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ ICOS⁺ and CD4⁺ CXCR5⁺ PD-1⁺ TFH cells, CD86⁺ CD19⁺, CD38⁺ CD19⁺ B cells and serum IL-2, IL-4, IL-17A, IL-21 and IFN-γ were significantly higher in HSPN patients (p < 0.05) than in HC. Before and after treatment the numbers of CD4⁺ CXCR5⁺ TFH cells were negatively correlated with the values of eGFR (r = − 0.7162, p < 0.05; r = − 0.732, p < 0.05, respectively). Similarly the numbers of CD4⁺ CXCR5⁺ PD-1⁺ TFH cells were negatively correlated with 24-h urinary proteins (r = − 0.4013, p < 0.05; r = − 0.7857, p < 0.05, respectively), and the numbers of CD4⁺ CXCR5⁺ ICOS⁺ TFH cells were positively correlated with the levels of serum IL-21 (r = 0.5186, p < 0.05; r = 0.8503, p < 0.05, respectively) and 24-h urinary protein (r = 0.6045, p < 0.05; r = 0.833, p < 0.05, respectively) in these patients, regardless of treatment. Following treatment the numbers of CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ PD-1⁺, and CD4⁺ CXCR5⁺ ICOS⁺ TFH cells, as well as serum levels of IL-21 were significantly reduced, however IL-4 levels were noticeably increased (p < 0.05). A higher frequency of circulating CD4⁺ CXCR5⁺ TFH cells existed in patients with HSPN and may be a viable therapeutic target.     

Expression of miRNA-155 in carotid atherosclerotic plaques of apolipoprotein E knockout (ApoE−/−) mice and the interventional effect of rapamycin

Carotid atherosclerosis (AS) is an inflammatory process and is the primary pathogenesis of cerebrovascular disease. Many factors are responsible for development of atherosclerosis such as inflammation and autophagy. It is reported that microRNAs (miRNAs) could regulate the development of atherosclerosis through targeting autophagy-related genes. Many studies have demonstrated that miRNA-155 could regulate autophagy in macrophages or tumor cells. However, the role of miRNA-155 on autophagy in carotid plaques is not yet known. In this study, we explore the expression of miRNA-155 and autophagy-related proteins in carotid plaques of ApoE^−/− mice and the interventional effect of rapamycin. We compared the expression of miRNA-155 and autophagy-related proteins between the control, model and rapamycin groups using qRT-PCR and western blot. Compared to the control group, we found the miRNA-155 and LC3-II expression was up-regulated (P < 0.05), expression ratio of phosphorylated mammalian target of rapamycin to total mammalian target of rapamycin (p-mTOR/mTOR) was down-regulated in model group (P < 0.05), but atherosclerotic lesions were still aggravated. These results following rapamycin group indicated that miRNA-155 and LC3-II expression was significantly up-regulated (P < 0.05), the expression ratio of p-mTOR/mTOR was significantly down-regulated (P < 0.05), and atherosclerotic lesions were reduced. Our results showed in the early stages of atherosclerotic plaques development, effective autophagy could attenuate atherosclerosis in ApoE^−/− mice. Furthermore, our results also demonstrated that rapamycin might promote the activation of the autophagy by enhancing the expression of miRNA-155, which delays the development of atherosclerotic plaques.     

Inosine Acedoben Dimepranol promotes an early and sustained increase in the natural killer cell component of circulating lymphocytes: A clinical trial supporting anti-viral indications

Inosine Acedoben Dimepranol (IAD), licensed for the treatment of cell-mediated immune deficiencies associated with viral infections, has been reported to impact a variety of immune parameters both in vitro and in vivo. Here we report the results from a clinical trial where multiple lymphocyte subsets – CD19 + B cells, CD3 + T cells, CD4 + T-helper cells, FoxP3^hi/CD25^hi/CD127^lo regulatory T cells (Tregs), CD3 −/CD56 + NK cells, and CD3 +/CD56 + NKT cells – were, together with serum immunoglobulins and IgG subclasses, followed during 14 days of IAD administration to ten healthy volunteers; these selected from 27 individuals pre-screened in vitro for their capacity to respond to IAD as gauged by increases in the percentage of Treg and/or NKT cells arising in PHA-stimulated cultures. While a transient spike and dip in Treg and T-helper fractions, respectively, was noted, the outstanding consequence of IAD administration (1 g po, qds) was an early and durable rise in NK cells. For half the cohort, NK cells increased as a percentage of total peripheral blood lymphocytes within 1.5 h of receiving drug. By Day 5, all but one of the volunteers displayed higher NK cell percentages, such elevation – effectively a doubling or greater – being maintained at termination of study. The IAD-induced populations were as replete in Granzyme A and Perforin as basal NK cells. The novel finding of IAD boosting phenotypically competent NK numbers in healthy individuals supports the drug's indicated benefit in conditions associated with viral infection and reinforces the potential for uplift where immune performance may be compromised.     

CXCR5+ CD8+ T cells could induce the death of tumor cells in HBV-related hepatocellular carcinoma

The follicular CXCR5⁺ CD8⁺ T cells have recently emerged as a critical cell type in mediating peripheral tolerance as well as antiviral immune responses during chronic infections. In this study, we investigated the function of CXCR5⁺ CD8⁺ T cells in HBV-related hepatocellular carcinoma patients. Compared to CXCR5⁻ CD8⁺ T cells, CXCR5⁺ CD8⁺ T cells presented elevated PD-1 expression but reduced Tim-3 and CTLA-4 expression. Upon anti-CD3/CD28 stimulation, CXCR5⁺ CD8⁺ T cells demonstrated higher proliferation potency than CXCR5⁻ CD8⁺ T cells, especially after PD-1 blockade. CXCR5⁺ CD8⁺ T cells also demonstrated significantly higher granzyme B synthesis and release, as well as higher level of degranulation. Tumor cells were more readily eliminated by CXCR5⁺ CD8⁺ T cells than by CXCR5⁻ CD8⁺ T cells. Interestingly, we found that B cells were more resistant to CXCR5⁺ CD8⁺ T cell-mediated killing than tumor cells, possibly through IL-10-mediated protection. In addition, the CXCR5⁺ CD8⁺ T cell-mediated cytotoxic effects on tumor cells could be significantly enhanced by PD-L1 blockade. Together, we presented that in patients with in HBV-related hepatocellular carcinoma, CXCR5⁺ CD8⁺ T cells could mediate tumor cell death more potently than the CXCR5⁻ CD8⁺ T cells in vitro while the autologous B cells were protected.     

Gabapentin-induced changes of plasma cortisol level and immune status in hysterectomized women

AimWe have examined the effects of gabapentin (GBP) on stress-related changes of cortisol and catecholamines in patients who underwent hysterectomy because of uterine fibrinoids. Additionally, we have observed the effect of GBP on the immune status in the acute stress response to surgery.MethodsSixty patients scheduled for an abdominal hysterectomy were randomly assigned to the GBP administration 1 h before surgery (n = 30 pts), or to the placebo group (n = 30 pts). Blood samples were collected before and 24 h after the surgery. The intensity of pain was assessed by a visual analogue scale (VAS) every 8 h at rest. Immunomodulatory effects of GBP were determined by flow cytometry. We followed the total proportion of CD3⁺ lymphocytes, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ B lymphocytes, CD16⁺CD56⁺CD3⁻NK cells and CD16⁺CD56⁺CD3⁺ NKT cells before and 24 h after hysterectomy. The plasma cortisol and catecholamines concentration was used to estimate the level of the stress response.ResultsVAS pain score at rest was significantly lower in the GBP group than in the placebo group (P = 0.003). Application of GBP significantly decreased the plasma cortisol level 24 h after the operation in comparison to the placebo group (P < 0,001). We found significant positive correlation between the VAS pain score and concentration of cortisol in all patients (P = 0.025). GBP reduced the concentration of catecholamines (p < 0.05). The proportion of CD3⁺ (P = 0.027) and CD3⁺CD4⁺cells (P = 0.006) was significantly lower in the GBP group 24 h after operation, while the contribution of CD19⁺ (P = 0.033) was significantly higher.ConclusionPreoperative administration of GBP reduced the pain scores at rest in patients at 0, 16 and 24 h after abdominal hysterectomy. Additionally, GBP reduced the stress response and changed immune parameters in the reaction to surgery.     

Evaluation of an ompA-based phage-mediated DNA vaccine against Chlamydia abortus in piglets

Chlamydia abortus (C. abortus) is an obligate intracellular pathogen that causes abortion in pigs and poses a zoonotic risk in pregnant women. Although attenuated and inactivated vaccines are available, they do not provide complete protection in animals underlining the need to develop new vaccines. In this study, we tested the hypothesis that intramuscular immunization with an ompA-based phage-mediated DNA chlamydial vaccine candidate will induce significant antigen-specific cellular and humoral immune responses. Thus, groups of piglets (five per group) were immunized intramuscularly with the phage-MOMP vaccine (λ-MOMP) or a commercial live-attenuated vaccine (1B vaccine) or a GFP-expressing phage (λ-GFP) or phosphate buffered saline (PBS) (control) and antigen-specific cell-mediated and humoral immune responses were evaluated. By day 63 post-immunization, the λ-MOMP vaccine elicited significantly higher (P < 0.05) levels of antigen-specific serum IgG antibody responses than the 1B vaccine or control did. Also, piglets immunized with λ-MOMP vaccine had significantly higher (P < 0.05) MOMP-specific lymphocyte proliferative responses compared to those immunized with the 1B vaccine or control. Furthermore, the total T-cell numbers (CD3 +) and the proportion of CD4 + and CD8 + T-cell subsets as well as the ratio of CD4 +/CD8 + T cells elicited following immunization were comparable between the λ-MOMP- and 1B-vaccinated animals on both days 63 and 70. Interestingly, although the proportion of CD3+CD4−CD8− double negative T cells on day 63 was significantly higher (P < 0.05) in the 1B vaccine group compared to the λ-MOMP-immunized group, there was a significant decrease in the proportion of this T-cell population on day 70 in the 1B compared to the λ-MOMP vaccinated group. These results indicate that the λ-MOMP DNA vaccine is capable of inducing antigen-specific cellular and humoral immune responses that may provide protective immunity against a live challenge infection with C. abortus.     

Suppressive effect of β,β-dimethylacryloyl alkannin on activated dendritic cells in psoriasis by the TLR7/8 pathway

β,β-dimethylacryloyl alkannin (DMA) is a key component of Lithospermum and possesses good efficacy for treating psoriasis. DMA inhibits activated dendritic cells (DCs), but the mechanism is unknown. Therefore, this study aimed to explore the modulation of the TLR7/8 pathway by DMA in psoriasis-activated DCs. Models of psoriasis-like skin lesions were established using BALB/c mice; 8 mice were treated with DMA (2.5 mg/kg). Bone marrow cells were isolated and induced into DCs using R848, a TLR7/8 agonist. Splenic CD11c + cells were detected by flow cytometry. Skin CD11c + cells were detected by immunofluorescence. TLR7, TLR8, MYD88, and IRAKM proteins were detected by Western blot. The effects of DMA on surface molecules of DCs were observed by flow cytometry. mRNA expression of inflammatory factors was detected by qRT-PCR. Secreted cytokines were detected by cytometric bead array. Compared with the model group, psoriasis-like skin lesions were alleviated by DMA, the splenic CD11c + cells were significantly decreased (P < 0.01), and CD11c + cell numbers in skin lesions were decreased (P < 0.01). Expression levels of TLR7, MYD88, and IRAKM were significantly decreased (P < 0.05). R848-stimulated DCs showed increased expression of I-A/I-E, CD80, and CD86 (P < 0.01), increased IL-23 and IL-1β mRNA and secretion (P < 0.05), and increased TLR7, TLR8, MYD88, and IRAKM expression (P < 0.01); DMA inhibited all of these effects of the TLR7/8 pathway activation by R848 (P < 0.05). In conclusion, DMA could inhibit psoriasis-activated DCs via the TLR7/8 pathway.     

Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2 + activity in the vascular smooth muscle cell line A10

Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca^2 + indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca^2 + activity [Ca^2 +]_i in A10 cells. This effect was absent in a Ca^2 +-free medium, indicating that OlyA/PlyB-induced [Ca^2 +]_i increase was dependent on Ca^2 + influx into cells. The increase in [Ca^2 +]_i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La^3 +, ii) the inhibitor of the sodium–calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca^2 +-ATPase inhibitor thapsigargin reduced the [Ca^2 +]_i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K⁺ in the medium did not prevent OlyA/PlyB-induced [Ca^2 +]_i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca^2 +]_i is due to an influx of Ca^2 + through a variety of co-existing plasma membrane Ca^2 +-permeable channels, Ca^2 + entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca^2 + release, altogether leading to disturbed Ca^2 + homeostasis in A10 cells.     

5E- and 5Z-farnesylacetones from Sargassum siliquastrum as novel selective L-type calcium channel blockers

A specific blocker of L-type Ca^2 + channels may be useful in decreasing arterial tone by reducing the open-state probability of L-type Ca^2 + channels. The aim of the present study was to evaluate the farnesylacetones, which are major active constituents of Sargassum siliquastrum, regarding their vasodilatation efficacies, selectivities toward L-type Ca^2 + channels, and in vivo antihypertensive activities. The application of 5E-(farnesylacetone 311) or 5Z-farnesylacetone (farnesylacetone 312) induced concentration-dependent vasodilatation effects on the basilar artery that was pre-contracted with depolarization and showed an ignorable potential role of endothelial-derived nitric oxide. We also tested farnesylacetone 311 or 312 to determine their pharmacological profiles for the blockade of native L-type Ca^2 + channels in basilar arterial smooth muscle cells (BASMCs) and ventricular myocytes (VMCs), cloned L- (α1C/β2a/α2δ), N- (α1B/β1b/α2δ), and T-type Ca^2 + channels (α1G, α1H, and α1I). Farnesylacetone 311 or 312 showed greater selectivity toward the L-type Ca^2 + channels among the tested voltage-gated Ca^2 + channels. The ranked order of the potency for farnesylacetone 311 was cloned α1C ≒ L-type (BASMC) ≒ L-type (VMCs) > α1B > α1H > α1I > α1G and that for farnesylacetone 312 was cloned α1C ≒ L-type (BASMCs) ≒ L-type (VMCs) > α1H > α1G > α1B > α1I. The oral administration of the farnesylacetone 311 (80 mg/kg) conferred potent, long-lasting antihypertensive activity in spontaneous hypertensive rats, but it did not alter the heart rate.     

Effects of taurocholic acid on immunoregulation in mice

ContextCurrently, there is a dramatically growing interest in Chinese traditional medicines, especially in the therapy of inflammatory diseases. Taurocholic acid (TCA), as a kind of natural bioactive substance of animal bile acid, has medicinal applications to treat a wide range of inflammatory diseases.ObjectiveThe study was designed to evaluate the effects of TCA on cytokine secretion, such as TNF-α and IL-1β and on the ratio of CD4⁺/CD8⁺, which is beneficial for understanding the mechanism of TCA on immunoregulation preliminarily, and also will benefit our further research.Materials and methodsThe gene and protein expressions of TNF-α and IL-1β were measured by real time RT-PCR and ELISA in serum, spleen and lymphocytes respectively. The ratio of CD4⁺/CD8⁺ in peripheral blood and lymphocytes was measured by flow cytometry.ResultsOur present study has shown that lipopolysaccharide (LPS) and cyclosporin A (CsA) could increase or decrease the gene and protein expressions of TNF-α and IL-1β respectively. TCA (0.25 g/kg, 0.125 g/kg) could recover the suppressed expressions of TNF-α and IL-1β and increase the ratio of CD4⁺/CD8⁺. In vitro, TCA (15 μg/mL) could inhibit the increased production of TNF-α and IL-1β; TCA (0.15 μg/mL–15 μg/mL) could inhibit the increased gene expressions of IL-1β and TNF-α. TCA (0.15 μg/mL) could recover the suppressed expressions of TNF-α and IL-1β.ConclusionThe function of immunoregulation of TCA may be accomplished through modulating the gene and protein expressions of TNF-α and IL-1β and elevating CD4⁺/CD8⁺ T-cell ratio.