The ability of peripheral blood mononuclear cells (PBMC) of syphilitic patients to produce IL-2

Abstract The cell-mediated immune response of importance in protection against Treponema pallidum , is distinctly suppressed in some stages of the disease. This may be a result of decreased ability of cells to produce IL-2, or IL-2 absorption by different factors. The experiments were designed to evaluate the ability of peripheral blood mononuclear cells (PBMC) of patients with different stages of syphilis to produce IL-2, and to investigate the causes which could possibly limit its activity. The ability of the PBMC of syphilitic patients to produce IL-2 develops at the beginning of the disease, reaching a maximum in primary seropositive syphilis. In the next stages of the disease this capability is distinctly lowered. The lowest was in malignant syphilis and tabes dorsalis, i.e. during severe disease. Absorption of adherent cells from PBMC increased the ability of lymphocytes to produce IL-2. The highest level of this interleukin was observed at the stages of the disease where suppression was the deepest. Sera of both control and syphilitic patients contained IL-2 inhibitor. Its level was the highest in early and late latent syphilis where no symptoms of disease were present. In all syphilitic sera a distinctly elevated level of soluble IL-2 receptors (sIL-2R) was also found. Its high level was noted in sera of patients in which PBMC had the weakest ability to produce IL-2. These findings suggest that sIL-2R may be bound to IL-2 and in this way would lead to weakening of T cell function and of resistance against Treponema pallidum infection.     

The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively

 The strains BALB/cHeA (BALB/c) and STS/A (STS) differ in production of IL-4 and IL-10, two Th2 cytokines, after stimulation of spleen cells with Concanavalin A, STS being a low and BALB/c a high producer. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains. Each CcS/Dem strain contains a different, random set of approximately 12.5% genes of the "donor" strain STS and approximately 87.5% of the "background" strain BALB/c. We selected for further analysis the RC strain production intermediate between BALB/c and STS. In (CcS-20×BALB/c)F₂ hybrids we found that different loci control expression of IL-4 and IL-10. Cypr1 (cytokine production 1) on chromosome 16 near D16Mit15 controls IL-4 production, whereas the production of IL-10 is influenced by loci Cypr2 near D1Mit14 and D1Mit227 on chromosome 1 and Cypr3 marked by D5Mit20 on chromosome 5. In addition, the relationship between the level of these two cytokines depends on the genotype of the F₂ hybrids at a locus cora1 (correlation 1) on chromosome 5. This differential genetic regulation may be relevant for the understanding of biological effects of T-helper cells in mice of different genotypes. Received: 2 March 1998 / Revised: 8 June 1998     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

The IgM antibody level against ganglioside GM2 correlates to the disease status of HIV-1-infected patients

HIV-1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)-mediated cytolysis of HIV-1-infected cells. Since GM2 is immunogenic in human, we proposed that an anti-GM2 IgM Ab may be produced by some HIV-1-infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV-1-infected patients and 111 seronegative donors. As expected, the anti-GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4+ cell count and the HIV-RNA load in the HIV-1-infected patients. The results showed a positive correlation between the anti-GM2 IgM Ab titer and CD4+ cell count but a negative correlation between the anti-GM2 IgM Ab titer and HIV-RNA load. These suggest that anti-GM2 IgM Ab induced and/or enhanced by HIV-1 infection causes C-mediated cytolysis of HIV-1-infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV-RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV-1 infected patients.     

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1β (IL-1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1β to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein-1. After treatment with IL-1β, glucosamine increased the expression of mRNA encoding the type II IL-1β receptor. These results emphasize the potential role of two regulating proteins of the IL-1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.     

Inhibitory effects of interleukin-6 on release of PGI2 by cultured human pulmonary artery smooth muscle cells

We evaluated the effect of interleukin-6 (IL-6) on the production of prostacyclin (PGI₂) by cultured human pulmonary artery smooth muscle cells (HPASMC). Incubation of these cells for up to 48 h with IL-6 led to a dose- and time-dependent decrease in the concentration of PGI₂ in the culture medium. The incubation of HPASMC with 10 μg/ml of lipopolysaccharide (LPS), 200 U/ml of IL-1β or 500 U/ml of TNFα for 24 hr significantly increased the concentration of PGI₂ in the medium. However, the addition of IL-6 to a medium containing LPS, IL-1β, or TNFα significantly inhibited the stimulatory effect of those substances on PGI₂ production. Such inhibition was closely related to the concentration of IL-6. IL-6 may counteract the roles of LPS and of other cytokines on the regulation of pulmonary vascular tension in endotoxin- and cytokine-mediated disorders such as sepsis and the acute respiratory distress syndrome (ARDS).     

敲低低密度脂蛋白受体相关蛋白1抑制C3H10T1/2多潜能干细胞定向成脂

C3H10T1/2多潜能干细胞成脂过程分为定向和分化两个阶段,骨形成蛋白4(BMP4)可以诱导其定向成前脂肪细胞．已有的研究表明,脂肪组织特异性敲除低密度脂蛋白受体相关蛋白1(Lrp1)的小鼠体重减轻,脂肪组织含量减少,揭示此基因对成脂具有重要作用．然而,目前尚不清楚Lrp1是否在成脂定向过程中发挥作用．采用小干扰RNA技术(RNAi),在体外水平研究低密度脂蛋白Lrp1对C3H10T1/2多潜能干细胞成脂定向的作用．分别在C3H10T1/2成脂的定向期和脂滴成熟期敲低Lrp1,通过显微镜下观察、油红O染色、Western blotting等实验证实,定向期而非脂滴成熟期敲低Lrp1显著抑制C3H10T1/2多潜能干细胞成脂．BMP4通过激活下游Smad1/5/8信号通路发挥作用,而敲低Lrp1显著抑制BMP4诱导的Smad1/5/8磷酸化．这些结果说明：敲低Lrp1通过下调Smad信号通路,抑制BMP4诱导的C3H10T1/2多潜能干细胞成脂定向．     

Pro-inflammatory cytokines IL-6 and CCL2 suppress expression of circadian gene Period2 in mammary epithelial cells

Chronic inflammation is known to contribute to tumor initiation and cancer progression. In breast tissue, the core circadian gene Period (PER)2 plays a critical role in mammary gland development and possesses tumor suppressor function. Interleukin (IL)-6 and C-C motif chemokine ligand (CCL) 2 are among the most abundant cytokines in the inflammatory microenvironment. We found that acute stimulation by IL-6/CCL2 reduced PER2 expression in non-tumorigenic breast epithelial cells. Longer term exposure to IL-6/CCL2 suppressed PER2 to an even lower level. IL-6 activated STAT3/NFκB p50 signaling to recruit HDAC1 to the PER2 promoter. CCL2 activated the PI3K/AKT pathway to promote ELK-1 cytoplasm-to-nucleus translocation, recruit HDAC1 to the proximal PER2 promoter and facilitate DNMT3-EZH2-PER2 promoter association. Ectopic expression of PER2 inhibited IL-6 or CCL2 induced mammosphere forming ability and reduced sphere size indicating that PER2 repression in breast epithelial cells can be crucial to activate tumorigenesis in an inflammatory microenvironment. The diminished expression of PER2 can be observed over a time scale of hours to weeks following IL-6/CCL2 stimulation suggesting that PER2 suppression occurs in the early stage of the interaction between an inflammatory microenvironment and normal breast epithelial cells. These data show new mechanisms by which mammary cells interact with a cancerous microenvironment and provide additional evidence that PER2 expression contributes to breast tumorigenesis.     

hnRNP A2/B1在人永生化表皮HaCaT细胞凋亡过程中的表达及其调控作用研究

该文以姜黄素诱导人永生化表皮HaCaT细胞凋亡为基础,对hnRNP A2/B1在核基质中的存在、分布及其与细胞凋亡相关基因产物的共定位及相互作用关系进行了研究。蛋白质印迹结果显示,hnRNP A2/B1存在于HaCaT细胞核基质蛋白组分中,在经过姜黄素处理后,表达下调;激光共聚焦显微镜观察显示,hnRNP A2/B1在HaCaT细胞中分别与Fas、p53和Bax等基因产物具有共定位关系,姜黄素处理后其共定位区域出现由核膜或核仁向胞质转移的趋势。GST pull-down实验证实,hnRNPA2/B1分别与Fas、p53和Bax有直接相互作用关系。结果表明,hnRNPA2/B1作为一种核基质蛋白,通过与细胞凋亡相关基因产物的相互作用参与HaCaT细胞的凋亡诱导调控过程,这对深入认识核基质蛋白在细胞凋亡过程中的调控机制具有重要意义。     

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.     

The distinct effects of a butanol fraction of <Emphasis Type="Italic">Bidens pilosa</Emphasis> plant extract on the development of Th1-mediated diabetes and Th2-mediated airway inflammation in mice

Bidens pilosa is claimed to be useful for immune or anti-inflammatory disorders; however, little scientific evidence has been published concerning its function. In this paper, immune disease mouse models were used to study the function of a butanol fraction of B.pilosa. We demonstrated treatment with the butanol fraction of B.pilosa ameliorated Th1 cell-mediated autoimmune diabetes in nonobese diabetic (NOD) mice but caused deterioration of Th2 cell-mediated airway inflammation induced by ovalbumin (OVA) in BALB/c mice. We next showed that Th2 cytokines (IL-4 and/or IL-5) increased but Th1 cytokine (IFN-) decreased following injections with the butanol fraction of B.pilosa in both mouse strains. Accordingly, Th2 cytokine-regulated IgE production in mouse serum increased following treatment with this fraction. Finally, we found that the butanol fraction of B.pilosa inhibited Th1 cell differentiation but promoted Th2 cell differentiation. Taken together, the butanol fraction of B.pilosa has a dichotomous effect on helper T cell-mediated immune disorders, plausibly via modulation of T cell differentiation.     

Determination of the serum metallothionein (MT)1/2 concentration in patients with Wilson's disease and Menkes disease

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH₂ terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the spiked MT-1was fully recovered from the plasma.We investigated the normal range of MT1/2 (25–75%tile) in 200 healthy human serum and found it to be 27–48 ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long–Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.     

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca²⁺-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.