RESUMO
BACKGROUND: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown. METHODS: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65-/-) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65-/- and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting. RESULTS: BTB09089 significantly promoted motor outcomes in WT but not in GPR65-/- mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65-/- mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65-/- mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65-/- mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice. CONCLUSIONS: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.
Assuntos
AVC Isquêmico , Camundongos Knockout , Neurônios , Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/agonistas , Camundongos , AVC Isquêmico/metabolismo , Masculino , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Reabilitação do Acidente Vascular Cerebral , Fármacos Neuroprotetores/farmacologia , Camundongos Endogâmicos C57BLRESUMO
ABSTRACT: Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors nitric oxide and prostaglandins. The voltage-dependent K + channel (K V ) blocker (4-aminopyridine) and selective K Ca blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that K V and K Ca channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K + channel (K ir ) blocker (barium chloride) and the K ATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by K ir channels. The vasodilation effect and expression of K ir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca 2+ levels in the isolated thoracic aorta. These results suggest that K ir channels play a more important role than K ATP channels in regulating vascular tone in larger arteries and that the activity of the K ir channel is enhanced by the PKC pathway.
Assuntos
Aorta Torácica , Músculo Liso Vascular , Proteína Quinase C , Simendana , Vasodilatação , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Simendana/farmacologia , Masculino , Vasodilatação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína Quinase C/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Vasodilatadores/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
AIMS: Few treatments are available in the subacute phase of traumatic brain injury (TBI) except rehabilitation training. We previously reported that transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects against cerebral ischemia/reperfusion injury. In this study, it was hypothesized that delayed CO2 postconditioning (DCPC) starting at the subacute phase may promote neurological recovery of TBI. METHODS: Using a cryogenic TBI (cTBI) model, mice received DCPC daily by inhaling 5%/10%/20% CO2 for various time-courses (one/two/three cycles of 10-min inhalation/10-min break) at Days 3-7, 3-14 or 7-18 after cTBI. Beam walking and gait tests were used to assess the effect of DCPC. Lesion size, expression of GAP-43 and synaptophysin, amoeboid microglia number and glia scar area were detected. Transcriptome and recombinant interferon regulatory factor 7 (Irf7) adeno-associated virus were applied to investigate the molecular mechanisms. RESULTS: DCPC significantly promoted recovery of motor function in a concentration and time-course dependent manner with a wide therapeutic time window of at least 7 days after cTBI. The beneficial effects of DCPC were blocked by intracerebroventricular injection of NaHCO3 . DCPC also increased puncta density of GAP-43 and synaptophysin, and reduced amoeboid microglia number and glial scar formation in the cortex surrounding the lesion. Transcriptome analysis showed many inflammation-related genes and pathways were altered by DCPC, and Irf7 was a hub gene, while overexpression of IRF7 blocked the motor function improvement of DCPC. CONCLUSIONS: We first showed that DCPC promoted functional recovery and brain tissue repair, which opens a new therapeutic time window of postconditioning for TBI. Inhibition of IRF7 is a key molecular mechanism for the beneficial effects of DCPC, and IRF7 may be a potential therapeutic target for rehabilitation after TBI.
Assuntos
Lesões Encefálicas Traumáticas , Dióxido de Carbono , Fator Regulador 7 de Interferon , Animais , Camundongos , Lesões Encefálicas Traumáticas/metabolismo , Dióxido de Carbono/metabolismo , Dióxido de Carbono/uso terapêutico , Modelos Animais de Doenças , Proteína GAP-43/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Fator Regulador 7 de Interferon/uso terapêutico , Sinaptofisina/metabolismo , Sinaptofisina/uso terapêuticoRESUMO
Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.
Assuntos
Adenilato Quinase/metabolismo , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Destreza Motora/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Masculino , Metformina/farmacologia , Camundongos , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacosRESUMO
Most chemotherapeutic drugs commonly suffer from low aqueous solubility that can potentially limit drugs absorption. Drug nanomerization is an advanced approach to overcoming their poor water-solubility. In this study, class I hydrophobin recombinant HGFI (rHGFI)-based curcumin (Cur) nanoparticles (rHGFI-Cur) were prepared by freeze-drying method. The rHGFI-Cur nanocomposites were characterized by contact angle, transmission electron microscopy, fluorescence microscopy and dynamic light scattering. The results showed that rHGFI could lead to the wettability conversion and stability improved of Cur in water. X-ray photoelectron spectroscopy and Fourier transform infrared suggested that rHGFI could non-covalently bind to Cur to render them hydrophilic through hydrophobic forces. Additionally, drug release and cytotoxicity assays illustrated that rHGFI-Cur nanoparticles could facilitate Cur release and exhibited higher cytotoxicity than free Cur for human esophageal cancer cells TE-1. Thus, it suggested that rHGFI has a great potential application for hydrophobic drug delivery without toxicity.[Formula: see text].
Assuntos
Curcumina , Nanopartículas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Solubilidade , ÁguaRESUMO
We hypothesized that activation of the central histaminergic system is required for neuroprotection induced by hypoxic preconditioning. Wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice were preconditioned by 3 hours of hypoxia (8% O(2)) and, 48 hours later, subjected to 30 minutes of middle cerebral artery (MCA) occlusion, followed by 24 hours of reperfusion. Hypoxic preconditioning improved neurologic function and decreased infarct volume in WT or HDC-KO mice treated with histamine, but not in HDC-KO or WT mice treated with α-fluoromethylhistidine (α-FMH, an inhibitor of HDC). Laser-Doppler flowmetry analysis showed that hypoxic preconditioning ameliorated cerebral blood flow (CBF) in the periphery of the MCA territory during ischemia in WT mice but not in HDC-KO mice. Histamine decreased in the cortex of WT mice after 2, 3, and 4 hours of hypoxia, and HDC activity increased after 3 hours of hypoxia. Vascular endothelial growth factor (VEGF) mRNA and protein expressions showed a greater increase after hypoxia than those in HDC-KO or α-FMH-treated WT mice. In addition, the VEGF receptor-2 antagonist SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume and reversed increased peripheral CBF in WT mice. Therefore, endogenous histamine is an essential mediator of hypoxic preconditioning. It may function by enhancing hypoxia-induced VEGF expression.
Assuntos
Histamina/fisiologia , Hipóxia Encefálica/complicações , Acidente Vascular Cerebral/etiologia , Animais , Western Blotting , Química Encefálica/fisiologia , Circulação Cerebrovascular/fisiologia , Cromatografia Líquida de Alta Pressão , Cinamatos/farmacologia , Histamina/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Histidina Descarboxilase/fisiologia , Hipóxia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/patologia , Ataque Isquêmico Transitório/genética , Ataque Isquêmico Transitório/fisiopatologia , Precondicionamento Isquêmico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of Delta Psi(m) and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen-glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Carnosina/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Carnosina/administração & dosagem , Ácido Glutâmico/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , N-Metilaspartato/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologiaRESUMO
Vascular endothelial growth factor (VEGF or VEGF-A) is a hypoxia induced angiogenic growth factor that is potent in neurotrophy,neuroprotection, anti-apoptosis and cell proliferation. Recent reports suggest that VEGF is related to many central nervous system diseases, such as cerebral ischemic disease, Alzheimer's disease and Parkinson's disease. Further study of the relationship between VEGF and central nervous system diseases,and investigation of VEGF related drugs will shed light on a new way for treatment of central nervous system diseases.
Assuntos
Doença de Alzheimer/metabolismo , Isquemia Encefálica/metabolismo , Doença de Parkinson/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Humanos , Fármacos Neuroprotetores/farmacologia , Fator A de Crescimento do Endotélio Vascular/classificação , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIM: To study the effect and mechanism of BCG on human nature killer cells. METHODS: PBMC or purified NK cells were isolated from normal human peripheral blood with negative anti-Mycobacterium tuberculosis antibody and cultured with BCG, IL-12, BCG plus IL-12 and BCG plus anti-IL-12R beta 1 mAb (2B10), respectively. The levels of IFN-gamma and IL-12p40 in the culture supernatants were measured by ELISA. The frequency of IFN-gamma and granzyme B producing cells were analyzed by ELISpot. The cytolytic activity was detected by MTT reduction assay. The surface expression of IL-12R beta 1 on NK cells was detected by flow cytometry. RESULTS: BCG significantly induced IFN-gamma production by PBMC in a dose-dependent manner. When PBMC was stimulated with BCG, the frequency of granzyme B producing cells was higher than that in unstimulated PBMC (P<0.05). BCG enhanced the cytotoxic activity of PBMC. BCG alone didn't induce IFN-gamma production by purified NK cells, but it can augment IL-12-induced IFN-gamma production by purified NK cells. The cytotoxic activities of BCG-stimulated and unstimulated purified NK cells were not significantly different (P>0.05). BCG induced IL-12 production by PBMC in a dose-dependent manner and enhanced IL-12R beta 1 expression on different subsets of NK cells. Blocking the effect of IL-12 by anti-IL-12R beta 1 mAb (2B10) inhibited BCG-induced IFN-gamma production and granzyme B releasing by PBMC. CONCLUSION: BCG can indirectly promote biologic activity of NK cells and the production of endogenous IL-12 combined with up-regulation IL-12R beta 1 expression on the surface of NK cells is a part of the mechanisms of IL-12 on human NK cells.
Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-12/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Mycobacterium bovis/imunologia , Receptores de Interleucina-12/metabolismo , Linhagem Celular Tumoral , Humanos , Interferon gama/biossíntese , Interleucina-12/metabolismo , Células Matadoras Naturais/microbiologiaRESUMO
SARS-CoV infection of human results in antigen-specific cellular and humoral immune responses. However, it is critical to determine whether SARS-CoV-specific memory T cells can persist for long periods of time. In this study, we analyzed the cellular immune response from 21 SARS-recovered individuals who had been diagnosed with SARS in 2003 by using ELISA, CBA, ELISpot and multiparameter flow cytometry assays. Our results demonstrated that low levels of specific memory T cell responses to SARS-CoV S, M, E and N peptides were detected in a proportion of SARS-recovered patients, and IFN-gamma was the predominant cytokine produced by T cells after stimulation with peptides. Cytometry analysis indicated that the majority of memory CD8(+) T cells produced IFN-gamma, whereas memory CD4(+) T cells produced IFN-gamma, IL-2 or TNF-alpha. These results might provide valuable information on the cellular immune response in recovered SARS-CoV patients for the rational design of vaccines against SARS-CoV infection.
Assuntos
Memória Imunológica , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Linfócitos T/imunologia , Adulto , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Desenho de Fármacos , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interleucina-2/biossíntese , Masculino , Pessoa de Meia-Idade , Valores de Referência , Linfócitos T/virologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossíntese , Adulto JovemRESUMO
Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.
Assuntos
Sangue Fetal/citologia , Imunofenotipagem , Células Matadoras Naturais/classificação , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/citologia , Moléculas de Adesão Celular/metabolismo , Feminino , Humanos , Memória Imunológica , Recém-Nascido , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , GravidezRESUMO
Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.
Assuntos
Células Matadoras Naturais , Leucócitos Mononucleares , Subpopulações de Linfócitos , Fenótipo , Adulto , Biomarcadores/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Proliferação de Células , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/classificação , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Subpopulações de Linfócitos/classificação , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMO
BACKGROUND & OBJECTIVE: Currently, 5-fluorouracil (5-FU) is still one of the widely applied chemotherapeutic agents for tumors. Interleukin-12 (IL-12) can promote the differentiation of Th1 cells and induce the production of interferon-gamma (IFN-gamma) by CD8+ T cells. This study was to investigate the suppression mechanism of 5-FU on the immune response mediated by T cells from normal human peripheral blood, and to determine the effect of IL-12 on the immune suppression induced by 5-FU. METHODS: The effects of 5-FU on the proliferation of peripheral blood mononuclear cells (PBMCs) and liver cancer cell line HepG2 were examined. PBMCs were stimulated with either anti-CD3 alone or anti-CD3 plus anti-CD28 in the presence or absence of 5-FU at different concentrations (0.20-50.00 microg/ml). The level of IFN-gamma in the culture supernatant was determined by ELISA. PBMCs were pretreated with 5-FU and stimulated with anti-CD3 and anti-CD28 for 2 days. The proportions of CD4+IFN-gamma+, CD8+IFN-gamma+, CD4+IL-2+ and CD8+IL-2+ T cells, and the expression of CD25 on CD4+ and CD8+ T cells were examined by flow cytometry (FCM). PBMCs were cultured in different combinations with anti-CD3 plus anti-CD28, IL-12 and/or 5-FU for 48 h. IFN-gamma level in the supernatant was detected by ELISA. The expression of IFN-gamma on CD4+ and CD8+ T cells were examined by FCM. RESULTS: 5-FU inhibited the proliferation of HepG2 cells and PBMCs, and suppressed INF-gamma production in PBMCs in a dose-dependent manner. The proportions of immune T cells were lower in 5-FU-pretreated PBMCs than in control PBMCs (0.7% vs. 2.1% for CD4+IFN-gamma+ T cells, 2.2% vs. 3.9% for CD8+IFN-gamma+ T cells, 0.7% vs. 2.5% for CD4+IL-2+ T cells, 0.2% vs. 0.4% for CD8+IL-2+ T cells). Both the positive rate and mean fluorescence intensity (MFI) of CD25 on CD4+ and CD8+ T cells were decreased after pretreatment of 5-FU. When stimulated by anti-CD3 and anti-CD28, the proportions of CD4+IFN-gamma+ and CD8+IFN-gamma+ T cells were 1.1% and 3.2% before adding IL-12, and 1.6% and 4.1% after treatment of IL-12. When stimulated by anti-CD3, anti-CD28, and 5-FU, the proportions of CD4+IFN-gamma+ and CD8+IFN-gamma+ T cells were 0.5% and 1.1% before adding IL-12, and 1.0% and 2.5% after treatment of IL-12. CONCLUSIONS: 5-FU could inhibit the proliferation of HepG2 cells and the immune function of PBMCs. IL-12 could restore the T-cell immune function inhibited by 5-FU.