Differential effect of sodium arsenite during the activation of human CD4+ and CD8+ T lymphocytes

Contamination of water with arsenic is a problem affecting several regions of the world. Peripheral blood mononuclear cells (PBMC) from chronically exposed individuals show a lower replicating activity than non-exposed individuals when stimulated with phytohemagglutinin (PHA). We have previously reported that PBMC from healthy donors treated in vitro with 1 muM sodium arsenite (NaAsO2) and stimulated with PHA showed a reduction in proliferation by a delay in cell cycle entry and a decrease in the rounds of cell division. In this paper we tested the effect of 1-5 muM NaAsO2 on the proliferation, viability, blast transformation, expression of the CD4 and CD8 molecules, and during the activation and proliferation of both CD4+ and CD8+ T lymphocytes. We found a reduction in cell proliferation and an increase in non-dividing cells with higher concentrations of NaAsO2 (2-5 microM) when proliferation was studied by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. The use of 7-aminoactinomycin D (7-AAD) in CFSE-labeled cells allowed us to detect an increase in percentage of non-dividing cells, and an increase in apoptotic/dead cells mainly in non-proliferating cells. Analysis of the expression of CD4 and CD8 molecules on these cells showed that concentrations > or = 2 microM NaAsO2 reduced the expression of the CD8 molecule and induced apoptosis/death in CD4+ cells. Analysis of blast transformation by flow cytometry showed an accumulation of CD8+ resting cells in the presence of NaAsO2. Analysis of CD25 and CD69 expression in kinetics experiments in both subtypes showed a delay in the expression of CD25 and a delay in the downregulation of the CD69 molecule, in both CD4+ and CD8+ cells. However, in the case of CD8+ cells, we detected an accumulation of a CD25- CD69- population in the presence of increasing concentrations of NaAsO2. Altogether, our results show that NaAsO2 alters the expression kinetics of the early activation molecules CD25 and CD69 similarly in both subtypes. In addition, activated and non-activated CD4+ cells die by apoptotic mechanisms and although a percentage of CD8+ cells also die by apoptosis, a subpopulation of these cells is unable to activate and thus accumulates as resting cells.     

Azithromycin modulates immune response of human monocyte-derived dendritic cells and CD4+ T cells

Azithromycin (AZM) is a macrolide antibiotic that exhibits anti-inflammatory activity aside from its antimicrobial effect, a feature that may ameliorate certain inflammatory disorders and prevent graft-versus-host disease in patients receiving stem cell transplantation. In the present study, we investigated the ability of AZM to influence the function of human monocyte-derived dendritic cells (DCs) and CD4⁺ T cells. We found that AZM down-regulated CD80, CD86, and HLA-DR expression in lipopolysaccharide (LPS)-stimulated DCs and suppressed interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor-alpha production in these cells. In addition, AZM increased endocytosis and/or expression of Toll-like receptor (TLR)2, TLR4, and TLR9 in DCs and suppressed anti-CD3/CD28–induced CD4⁺ T cell proliferation and interferon-gamma production, an effect that was synergistic with dexamethasone. Finally, AZM suppressed DC-induced allogeneic T cell proliferation and cytokine production. Our study demonstrates that AZM modulates DC and CD4⁺ T cell function and may be of therapeutic benefit in various inflammatory disorders.     

香菇多糖对原发性肝癌TACE前后外周血CD4+CD25+调节性T细胞的影响

目的 观察香菇多糖在原发性肝癌(TACE)前后对外周血CD4+CD25+调节性T细胞的变化,探讨香菇多糖对原发性肝癌患者的免疫调节机制.方法 原发性肝癌46例,随机分为治疗组24例,对照组22例,治疗组在TACE术后第1天给予香菇多糖1 mg,3次/周,两组化疗方案相同,在TACE前及TACE后4周分别抽外周血,应用流式细胞术检测CD4+CD25+调节性T细胞的变化.结果 外周血CD4+CD25+Treg细胞占CD4+T细胞的百分率:治疗组在TACE前为(10.59±4.34)%、TACE后4周(6.72±2.60)%;对照组在TACE术前为(10.21±4.42)%、TACE后4周为(8.56±3.12)%.在TACE前两组间差异无统计学意义(P>0.05);TACE后4周时治疗组较对照组低,两组间差异有统计学意义(P<0.05).结论 香菇多糖可提高恶性肿瘤患者的免疫功能,在TACE后立即给药效果更佳.     

In-vitro generation and characterisation of murine CD4+CD25+ regulatory T cells with indirect allospecificity

Naturally arising CD4(+)CD25(+) regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4(+)CD25(+) T cells with antigen-specificity for an K(d) peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells pulsed with the K(d) peptide, we generated T cell lines from purified CD4(+)CD25(+) T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4(+)CD25(+) T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-gamma. Importantly, they were more potent than freshly isolated CD4(+)CD25(+) T cells in suppressing proliferation and cytokine secretion by effector CD4(+) T cells. Furthermore, the CD4(+)CD25(+) T cell lines could be expanded to large cell numbers and maintained in culture up to 1 year. The K(d)-specific CD4(+)CD25(+) T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying a full mismatch BALB/c heart.     

Changes of phenotype and function of human CD4+CD25-T cells induced by transfection of Foxp3

The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4⁺ T cells. The pMSCV-Foxp3 retroviral vector encoding Foxp3 gene was transduced into the PT67 packaging cell line. Virus-containing supernatant was applied to differentiate CD4⁺CD25⁻ T cells. The resulting cells were sorted with flow cytometry. The expressions of CD25, CD127, CTLA-4 and the proliferation of transfected T cells were examined. The effect of transfected CD4⁺ T cells on the proliferation and cytokine production of CD4⁺CD25⁻ T cells was examined. Foxp3-gene transfected CD4⁺ T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4, but down-regulated CD127 expression. After transfection, the proliferation of CD4⁺ T cells was eliminated. Transfected T cells inhibited the proliferation of CD4⁺CD25⁻ T cells. CD4⁺CD25⁻ T cells acquired a regulatory phenotype and function after it was transduced with the Foxp3 gene. This suggested a key role of Foxp3 in the generation of CD4⁺CD25⁺ regulatory T cells. __________ Translated from Acta Academiae Medicinae Militaris Tertiae, 2008, 30(3): 186–188 [译自: 第三军医大学学报]     

哮喘患者外周血单个核细胞CD4~+CD25~+调节性T细胞及IL-4分泌水平的检测

目的:探讨CD4+CD25+调节性T细胞在哮喘发病机制中的作用,为哮喘的临床治疗提供新的思路和方法。方法:分离哮喘患者和正常人外周血单个核细胞(PBMC),用流式细胞仪分析CD4+CD25+调节性T细胞在外周血单个核细胞中的比例;酶联免疫吸附法(ELASA)检测外周血PBMC培养上清IL-4的分泌状况。结果:哮喘组外周血CD4+CD25+调节性T细胞的百分率为(3.2±1.5)%,健康对照组为(6.5±2.5)%,哮喘组明显低于对照组(P<0.05);哮喘组外周血PBMC培养上清IL-4分泌水平为(57.3±13.5)ng/L,健康对照组为(28.6±7.2)ng/L,哮喘组明显高于对照组(P<0.05)。结论:CD4+CD25+调节性T细胞可能参与了哮喘的发生与发展。     

Activation of the aryl hydrocarbon receptor reduces the number of precursor and effector T cells,but preserves thymic CD4CD25Foxp3 regulatory T cells

Aryl hydrocarbon receptor (AhR) activation suppresses immune responses, including allergic sensitization, by increasing the percentage of regulatory (Treg) cells. Furthermore, AhR activation is known to affect thymic precursor T cells. However, the effect of AhR activation on intrathymic CD4⁺CD25⁺Foxp3⁺ Treg cells is unknown. Therefore, we investigated the effect of AhR activation on the percentage and number of CD4⁺CD25⁺Foxp3⁺ Treg cells during allergic sensitization in relevant immunological organs.     

Effects of a soluble CD4 and CD4-Pseudomonas exotoxin A chimeric protein on human peripheral blood lymphocytes: lymphocyte activation and anti-HIV activity in vitro.

Recombinant sCD4-based proteins were evaluated for their effects on antigen-stimulated proliferation of human peripheral blood mononuclear cells (PBMC) and for antiviral activity against PBMC infected with human immunodeficiency virus (HIVD34). Two sCD4-based proteins were solubilized, refolded, and purified to homogeneity from recombinant E. coli and consisted of the 178 amino-terminal residues of CD4 fused with the translocating and catalytic domains of Pseudomonas exotoxin A (sCD4-PE40) or 183 amino-terminal residues of CD4 (sCD4-183); a third sCD4 consisting of 369 amino acids of CD4 was purified from recombinant mammalian cells for comparative purposes (sCD4-369). Increasing molar concentrations of these sCD4s were evaluated for inhibition of PBMC proliferation induced by alloantigen (MLR), by tetanus toxoid (TTOX), or in response to crosslinking with antibody to CD3 (OKT3). In addition, the concentrations of each protein required to inhibit replication of the HIVD34 isolate in primary PBMC was determined by quantitation of HIV p24 antigen released into supernatant fluids by infected cells. By comparing antiviral activity with anti-proliferative activity a relative estimate of the selectivity index for each recombinant sCD4 was determined. Proliferation of PBMC in response to alloantigen or OKT3 was less sensitive to inhibition than proliferation induced by TTOX, and the selectivity indices estimated for sCD4-PE40 were 170, 170 and 17, respectively. The selectivity index for sCD4-183 was greater than 350 under all assay conditions. Comparative evaluation of alloantigen-stimulated proliferation with antiviral activity of sCD4-183 versus sCD4-369 suggested that the E. coli-derived sCD4-183 may have a higher selectivity index under these conditions than its mammalian cell-derived counterpart.     

TF3-H4对CD4~+CD25~+调节性T细胞和CD4~+CD25~-效应性T细胞早期活化的影响

目的观察1',2',3',7'-四氢茶黄素-3,3'-双没食子酸酯(TF3-H4)对CD4+CD25+调节性T细胞和CD4+CD25-效应性T细胞早期活化的影响,分析TF3-H4对两群功能相反的CD4+T细胞的作用。方法免疫磁珠分离BALB/c小鼠脾脏CD4+CD25+调节性T细胞和CD4+CD25-效应性T细胞,加入ConA或PDB,和TF3-H4共同孵育12 h后收集细胞,流式细胞仪分析细胞表面早期活化标志CD69的表达。结果在ConA和PDB的作用下,两群细胞早期活化标志CD69的表达均升高。TF3-H4(20 mg.L-1)不能抑制PKC激动剂PDB激活的CD4+CD25-效应性T细胞CD69的表达,却能抑制ConA激活的CD4+CD25-效应性T细胞CD69表达;同时,TF3-H4也能明显抑制ConA激活的CD4+CD25+调节性T细胞的CD69表达。结论茶黄素衍生物TF3-H4可能经由TCR活化途径的上游抑制CD4+CD25-效应性T细胞活化;TF3-H4对CD4+CD25+调节性T细胞和CD4+CD25-效应性T细胞早期活化的抑制作用,可能是该类化合物发挥抗炎、抗肿瘤作用的机制之一。     

凉血解毒方药对原发免疫性血小板减少症患者外周血CD4＋CD2＋5调节性 T 细胞水平的影响

目的：观察原发免疫性血小板减少症（ ITP）患者治疗前后外周血CD4＋CD2＋5调节性T细胞水平变化,探讨凉血解毒方药对ITP患者CD4＋CD2＋5调节性T细胞的作用。方法选取56例ITP患者,男12例,女44例;年龄18～62岁,中位年龄29岁。给以凉血解毒方药治疗（地黄止血胶囊2‘．0 g,3次／d,口服,凉血解毒中草药煎剂升板汤,1剂／d）。患者于治疗前、治疗后90 d分别采取外周静脉血,采用流式细胞术检测CD4＋CD2＋5调节性T细胞水平。20例健康体检者为正常对照组。结果治疗后第90天,总有效率为73．2％。56例ITP患者治疗前外周血 CD4＋CD2＋5调节性T细胞表达水平低于正常对照组[（1．01±0．67）％,（2．81±0．52）％],差异有统计学意义（ P ＜0．05）;重症ITP患者CD4＋CD2＋5调节性 T细胞水平低于非重症患者[（0．71±0．23）％,（1．48±0．64）％],差异有统计学意义（ P ＜0．01）;患者治疗后90 d CD＋4 CD2＋5调节性T细胞水平高于治疗前[（1．93±0．53）％,（10．1±0．67）％],与治疗前比较差异有统计学意义（ P ＜0．05）;治疗后有效组CD4＋CD2＋5调节性T细胞水平高于无效组,差异有统计学意义（P ＜0．05）。结论 ITP 患者外周血CD 4＋CD 2＋5调节性 T 细胞水平低于正常对照组（ P ＜0．05）;凉血解毒方药提升CD4＋CD2＋5调节性T 细胞水平可能是治疗ITP的疗效机制。     

鹰嘴豆芽素A抗HIV-1活性及抑制CD4~+淋巴细胞早期活化作用

目的研究鹰嘴豆芽素A(biochanin A,BioA)对HIV-1活性及CD4+淋巴细胞早期活化作用的影响。方法①以MT-2和H9/HIV-1ⅢB细胞或HIV-1病毒共培养建立HIV-1感染模型,以不同浓度的BioA干预该过程,观察细胞融合情况和测定培养上清中的p24抗原含量变化,以评价BioA的抗HIV-1活性。②以PHA刺激剂诱导人外周血CD4+淋巴细胞活化,利用流式细胞术检测不同浓度BioA对早期活化标志CD69分子的表达影响。结果①BioA在本实验所设浓度下均能减少由HIV-1介导的细胞融合数(EC50=5.1μmol.L-1,SI=39)及p24抗原的表达量(EC50=38μmol.L-1,SI=5.2)。②终浓度5、10、25、50μmol.L-1的BioA对CD4+淋巴细胞早期活化抗原CD69表达具有明显抑制作用(P<0.01),其中50μmol.L-1达到最大抑制率,能使活化率从(60.42±0.52)%降低到(19.70±0.38)%。结论BioA具有抗HIV-1介导的细胞融合和HIV-1复制的活性及抑制CD4+淋巴细胞早期活化作用。     

CD4+CD25+调节性T细胞及Th17细胞与Graves病机制研究

目的：探讨CD4+CD25+调节性T细胞以及Th17细胞与Graves病的关系。方法：检测GD患者和对照组外周血单个核细胞CD4+CD25+Tregs、Th17细胞的数量,PBMC中TGF-β、IL-10和FoxP3 mRNA的表达水平以及血清TGF-β、IL-10和IL-17水平。结果：GD组外周血Th17数量高于对照组,血清中IL-17水平也高于对照组（P＜0.05）;而CD4+CD25+Tregs的数量低于对照组,而且TGF-β、FoxP3表达水平低于对照组（P＜0.05）;GD组血清中TGF-β、IL-10水平高于对照组（P＜0.05）。结论：CD4+CD25+Tregs数量的减少或功能障碍以及Th17细胞数量的升高,可能参与GD的发病过程。     

HIV-1-induced depletion of CD4+ T cells in the gut: mechanism and therapeutic implications

The mechanism of CD4+ T cell depletion remains a central unresolved issue in AIDS research. Recent studies have examined the massive depletion of CD4+ T cells observed in macaque models of acute HIV infection. A key question is whether the observed CD4+ T cell death is due to direct consequences of viral infection and to indirect mechanisms including increased expression of mediators of T-cell apoptosis. The therapeutic implications of the early CD4+ T cell loss are discussed.     

Effects of immunoglobulin D on expression of IgD receptor and protein tyrosine kinase signaling in human CD4~+ T cells

OBJECTIVE To observe whether human CD4~+T cells could be activated by immuno-globulin D(IgD) via IgD receptor(IgDR)-Lck.METHODS Human CD4~+T cells were purified from peripheral blood mononuclear cells(PBMCs) with microbeads.The viability of T cells were detected by CCK-8.The binding affinity and expression of IgDR on T cells were detected by flow cytometry.The protein expression of IgDR,Lck and P-Lck were analyzed by western blot.RESULTS IgD could concentration-dependent bind to IgDR on CD4~+T cells.The expression of IgDR was increased in response to treatment with IgD in a time-dependent and concentration-dependent manner.Stimulating by IgD resulted in enhanced phosphorylation of Lck compared with that in the medium control sample.The expression of Lck was not changed.As inhibitor of PTK,Herbimycin A or A770041,which combined with IgD could significantly inhibit phosphorylation of Lck(Tyr~(394)).The proliferation promoting effect of IgD was blocked by Herbimycin A or A770041.IgD could stimulate CD4~+T cell activation and proliferation through upregulating activating tyrosine residue of Lck(Tyr~(394)) phosphorylation.CONCLUSION These results demonstrate that IgD exaggerates CD4~+T cell activities,which may be through promoting Lck phosphorylation.     

Influence of anticancer agents on cell survival,proliferation, and CD4+CD25+Foxp3+ regulatory T cell-frequency in human peripheral-blood mononuclear cells activated by T cell-mitogen

Many anticancer agents currently used are considered to be cytotoxic not only to cancer cells but also to functional immune cells. To learn more about the immunosuppressive adverse influence of chemotherapeutic drugs in cancer chemotherapy, we examined the effects of arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate on the survival, proliferation, cytokine production, and CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Arsenic trioxide, dacarbazine, and 5-fluorouracil increased trypan-blue stained (dead) cell rates and suppressed the mitogen-activated proliferation of PBMCs significantly at 1–100 μM (p < 0.05). Methotrexate also significantly increased the percentages of dead cells and suppressed the mitogen-activated PBMC-proliferation at concentrations of more than 0.05 μM (p < 0.01). Arsenic trioxide significantly inhibited the production of interferon γ, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 5 μM (p < 0.05). In contrast, the anticancer agents significantly increased Treg cell-frequency in the activated PBMCs at concentrations of more than 0.1 μM for methotrexate, 5 μM for arsenic trioxide and 5-fluorouracil, and 50 μM for dacarbazine, respectively (p < 0.05). These agents did not significantly influence the production of transforming growth factor (TGF) β from the activated PBMCs at a concentration range of 0.05–50 μM. Our data suggest that the anticancer agents: arsenic trioxide, dacarbazine, 5-fluorouracil, and methotrexate attenuate T cell mediated immunity by not only inhibiting the proliferative response of T cells but by also increasing the frequency of Treg cells, which may result in the suppression of the effector T cell function.     

特异性免疫治疗对哮喘大鼠白细胞介素-10和CD_4~＋CD_（25）~＋调节性T细胞的影响

目的：探讨特异性免疫治疗（specific immunotherapy,SIT）对哮喘大鼠白细胞介素-10（IL-10）和CD4＋CD25＋调节性T细胞（CD4＋CD25＋Tr）的影响。方法：40只健康雄性清洁级Wistar大鼠随机均分成正常对照组、哮喘组、SIT对照组和SIT治疗组,每组10只。通过卵蛋白（OVA）雾化吸入的方法对致敏大鼠进行SIT干预,观察各组支气管肺泡灌洗液（BALF）中细胞分类及计数结果、血清和BALF中IL-10水平及外周血CD4＋CD25＋Tr百分率变化。结果：正常对照组BALF和血清中IL-10浓度分别高于哮喘组和SIT对照组（均为P〈0.01）,哮喘组和SIT对照组BALF和血清中IL-10浓度低于SIT治疗组（P〈0.01,P〈0.05）;正常对照组外周血CD4＋CD25＋Tr百分率显著高于哮喘组、SIT对照组和SIT治疗组（P〈0.01）,而SIT治疗组CD4＋CD25＋Tr百分率分别高于哮喘组和SIT对照组（P〈0.05）。结论：通过上调体内IL-10和CD4＋CD25＋Tr趋于正常状态可能是SIT治疗哮喘有效的重要机制。     

Induction of chromosomal aberrations and micronuclei by 2-hydroxy-4-methoxybenzophenone (oxybenzone) in human lymphocytes

Oxybenzone or benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is a filter used in a variety of personal care products for protection of human skin and hair from damage by ultraviolet radiation. BP-3 is suspected to exhibit endocrine disruptive properties. Indeed, it was found to be able to interact with the endocrine system causing alteration of its homeostasis, with consequent adverse health effects. Moreover, it is ubiquitously present in the environment, mostly in aquatic ecosystems, with consequent risks to the health of aquatic organisms and humans. In the present study, we analyzed the cytogenetic effects of BP-3 on human lymphocytes using in vitro chromosomal aberrations and micronuclei assays. Blood samples were obtained from five healthy Italian subjects. Lymphocyte cultures were exposed to five concentrations of BP-3 (0.20, 0.10, 0.05, 0.025, and 0.0125?μg/mL) for 24 and 48?h (for chromosomal aberrations and micronuclei tests, respectively). The concentration of 0.10?µg/mL represents the acceptable/tolerable daily intake reference dose established by European Union, whereas 0.20, 0.05, 0.025, and 0.0125?µg/mL represent multiple and sub-multiple of this concentration value. Our results reported cytogenetic effects of BP-3 on cultured human lymphocytes in terms of increased micronuclei and chromosomal aberrations’ frequencies at all tested concentrations, including concentrations lower than those established by European Union. Vice versa, after 48-h exposure, a significant reduction of the cytokinesis-block proliferation index value in cultures treated with BP-3 was not observed, indicating that BP-3 does not seem to produce effects on the proliferation/mitotic index when its concentration is equal to or less than 0.20?μg/mL.     

Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir

Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate. We compared the membrane permeation characteristics of these two compounds at 20 degrees C in human erythrocytes and in human T-lymphoblastoid CD4+ CEM cells, using a "papaverine-stop" assay. In erythrocytes, abacavir influx was rapid, nonsaturable (rate constant=200 pmol/s/mM/microl cell water), and unaffected by inhibitors of nucleoside or nucleobase transport. CBV influx was slow, saturable, strongly inhibited by adenine or hypoxanthine, and occurred via both the nucleobase carrier (Vmax=0.67 pmol/s/microl cell water; Km=50 microM) and the nucleoside carrier (Vmax=0.47 pmol/s/microl cell water; Km=440 microM). Similar qualitative results were obtained with CD4+ CEM cells, although CBV influx rates were somewhat higher and abacavir influx rates lower, compared to the corresponding rates in erythrocytes. Equilibrium studies further revealed that both compounds are concentrated intracellularly, but nonmetabolically, in both cell types, apparently due to cytosolic protein binding (absent in erythrocyte ghosts). We conclude that, in both cell types, while CBV influx is slow and carrier-dependent, abacavir influx occurs rapidly by nonfacilitated diffusion. The membrane permeation characteristics of abacavir are consistent with its superior oral bioavailability and its impressive ability to penetrate the central nervous system.