Reduction of Nitric Oxide with L–/Vc–Monomethyl Arginine in lnterleukin–2 and Anti–CD3 Monoclonal Antibody Combination Therapy

We evaluated nitric oxide induction in antitumor therapy consisting of anti–CD3 monoclonal antibody (anti–CD3) and interleukin–2 (IL–2), then determined the effect of nitric oxide reduction with L–N^G–monomethyl arginine (LNMA) on the therapeutic methods. Female C57BL/6 mice, MCA102 (a non immunogenic, NK–resistant murine fibrosarcoma cell line), and 145–2C11 (hamster anti–murine–CD3 mAb) were utilized in an experimental hepatic metastasis model developed by injecting a tumor cell suspension into the spleen of mice. A marked increase in serum NO₂^–+ NO₁ was observed at 19 hours after anti–CD3 (10 μ, IV) and additional IL–2 administrations (40times10¹ U, twice, If) induced a further increase. The NO₂, + NO_3- elevation in spot urine in the combination therapy was not suppressed with LNMA at a dose of 100 μg/h but was significantly lowered at 300 μg/h. The efficacy of the anti–CD3 + IL–2 therapy was not diminished by LNMA administration either at 100 μg/h or at 300 μg/h.     

慢性髓性白血病细胞来源的树突状细胞大容量诱导及其功能的实验研究

目的　探索大容量诱导慢性髓性白血病 (CML)细胞来源的树突状细胞 (DCs)的适宜方法 ;研究CML DCs刺激自体T淋巴细胞增殖并分泌γ 干扰素 (IFN γ)的能力。方法　用CS 30 0 0 plus血细胞分离机采集初诊CML病人的外周血单个核细胞 (PBMNCs) ;单采的CML PBMNCS转入组织培养袋 ,加入重组人粒 巨细胞集落刺激因子 (rhGM CSF)和重组人白介素 4 (rhIL 4 ) ,培养诱导 7d ;在诱导前后 ,用流式细胞仪分别检测细胞表面HLA DR、CD1a、CD80和CD86的表达水平 ;用3 H TdR掺入法检测CML DCs和CML PBMNCs刺激自体和异体T细胞增殖的能力 ;用ELISA法检测在自体混合淋巴细胞培养 (MLR)时T细胞分泌的IFN γ浓度。结果　用血细胞分离机收集的CML PBMNCs ,在组织培养袋内经细胞因子培养诱导 ,HLA DR、CD1a、CD80、CD86的表达均有明显上调 ,细胞形态也表现典型的DC特征 ;CML DCs能显著刺激自体和异体T细胞增殖 ,而CML PBM NCs仅能刺激异体T细胞的增殖 ,刺激自体T细胞增殖的能力很弱 ;刺激自体T细胞增殖时分泌的IFN γ浓度 ,CML DCs组为 (877± 2 14 )pg/mL ;CML BPMNCs组仅为 (14± 1.7) pg/mL。 结论　单采的CML PBMNCs转入组织培养袋 ,加入rhGM CSF和rhIL 4 ,可收获大容量的CML DCs;CML DCs在体外具有显著刺激自体T细胞增殖     

Comparison of the use of bulk to micro culture of cell preparations for lymphokine-activated cytotoxicity assays.

Different assay systems have been used to quantitate lymphokine-induced natural cytotoxic activity as a measure of immune status. This study compares the effects of inducing cytotoxicity in a bulk culture system, where effector cells are transferred to a micro culture well for assay, to a micro culture system where the effector cells are not transferred. The effector/target ratio for both the bulk and micro culture systems was calculated using the number of viable effector cells present at the time of target cell addition. After overnight incubation with interleukin-2 (IL-2), the lytic activity of murine spleen cells to targets using a micro culture system was increased two-fold over the bulk culture method. This increase was amplified further after 5 days of activation with IL-2, in that the micro culture system resulted in a four-fold increase in cytotoxic activity. The loss of some adherent cells in the bulk culture system did not explain the overall decrease in recovered cytotoxicity. The difference appeared to be related to cell loss during centrifugation. Therefore, the E/T ratios are different in the two systems if not corrected for the number of viable cells.     

甲基强的松龙治疗实验性变态反应性脑脊髓炎的作用机制

目的:研究细胞因子、T细胞凋亡和淋巴细胞增殖在实验性变态反应性脑脊髓炎(EAE)形成中的作用及甲基强的松龙(MP)治疗EAE的作用机制。方法:采用人脑纯化的髓鞘碱性蛋白(MBP)与完全福氏佐剂免疫Lewis大鼠,建立EAE动物模型。用双抗体夹心ELISA法检测各组大鼠血清中IL-10、TNF-α、IFN-γ的含量:流式细胞仪检测外周血T细胞凋亡;3H-TdR释放法检测外周血淋巴细胞转化率。结果:与对照组比较,EAE组的外周血IFN-γ、TNF-α水平明显增高,IL-10水平明显降低,MP治疗后IFN-γ和TNF-α水平下降,IL-10浓度上调。MP还诱导外周血T细胞凋亡和抑制MBP致敏淋巴细胞增殖并呈剂量依赖性。结论:应用人MBP成功建立EAE大鼠模型,MP可能通过调节Th细胞因子格局、促进Th2细胞因子分泌、抑制MBP致敏淋巴细胞增殖及外周血T细胞凋亡而发挥治疗多发性硬化的作用。     

Maculopapular eruption with enlarged macrophages in eight patients receiving G-CSF or GM-CSF

In the last years, granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF) are being increasingly used and several cutaneous eruptions have been reported in relation to these treatments. In 1991 Horn et al. described three patients with maculopapular eruption that paralleled the time of infusion of GM-CSF. Two of the cases showed an increase in the number and size of macrophages in the biopsy specimen. Since then, several cases have been reported showing this histopathological alteration that has been considered characteristic of reaction to G-CSF or GM-CSF. Although maculopapular eruption with enlarged macrophages can appear after chemotherapy treatment, we have found that the presentation of this eruption after the beginning of cytokine treatment is suggestive of the involvement of G-CSF and GM-CSF in the eruption. We described eight cases of patients treated with G-CSF or GM-CSF that developed maculopapular eruptions with enlarged macrophages.     

Characterization of T cell epitopes in αs1-casein in cow''s milk allergic, atopic and non-atopic children

BACKGROUND: One to two percent of infants suffer from IgE-mediated allergic reactions against cow's milk proteins. Most children develop clinical tolerance, but approximately 15% are still allergic by the age of 10 years. Little is known about the T cell epitopes in individual cow's milk protein in relation to allergy and tolerance. OBJECTIVE: To identify T cell epitopes in alphas1-casein, the most abundant milk protein, and to investigate T cell responses toward these epitopes in allergic, atopic and non-atopic children. METHODS: Allergen-specific T cell lines (TCLs) were derived from peripheral blood mononuclear cells of 11 cow's milk allergic, nine atopic and nine non-atopic children. T cell responses were measured to alphas1-casein and to overlapping peptides (18-mers), spanning the alphas1-casein molecule. Proliferation was determined by incorporation of (3)H-thymidine, and cytokine production (IL-10, IL-13 and IFN-gamma) was measured by ELISA. RESULTS: Four main regions (amino acid (AA) residues 43-66, 73-96, 91-114 and 127-180) in the alphas1-casein molecule were immunogenic to T cells, among which the AA residues 133-156 spanned the immunodominant part. Only subtle differences were found in peptide recognition between the subject groups. Some of the peptides induced slightly Th1- or Th2-skewed cytokine responses. The increased levels of IL-10 in response to alphas1-casein observed in TCLs from atopic children appeared not to be linked to recognition of specific IL-10-inducing epitopes. CONCLUSIONS: The immunodominant sequence in alphas1-casein is spanned by AA residues 133-156. Tolerance towards alphas1-casein in atopic children may be mediated by an overall induction of IL-10 and not by recognition of certain T cell epitopes. The identified T cell epitopes in children with cow's milk allergy may be useful targets in developing peptide immunotherapy.     

Role of intercellular adhesion molecule-1 in a murine model of toluene diisocyanate-induced asthma

BACKGROUND: Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) are thought to contribute to the airway inflammation and airway hyper-responsiveness (AHR) of allergic asthma. Some differences from allergic asthma have been noted, including airway neutrophilia, and the involvement of ICAM-1 in toluene diisocyanate (TDI) asthma is currently unclear. OBJECTIVE: We utilized mice lacking ICAM-1 expression (ICAM-1(-/-)) to investigate the role of ICAM-1 in airway inflammation and AHR in TDI-induced asthma. METHODS: Male C57BL/6J mice (ICAM-1(+/+)) and ICAM-1(-/-) mice were intranasally sensitized to TDI solution or solvent alone. Airway inflammation, AHR and cytokine secretion were assessed 24 h after challenge by TDI or solvent. The production of antigen-specific IgG and IgE by TDI sensitized and non-sensitized mice was determined. RESULTS: TDI challenge to ICAM-1(+/+) mice induced an increase in airway inflammatory cell numbers, AHR and cytokine secretion of TNF-alpha, macrophage inflammatory protein-2 (MIP-2), IL-4, IL-5 and IFN-gamma into the bronchoalveolar lavage fluid. All these pathophysiological changes were reduced in ICAM-1(-/-) mice. Serum levels of TDI-specific IgG and IgE of ICAM-1(-/-) and ICAM-1(+/+) mice were comparable. CONCLUSION: These results suggest that ICAM-1 plays an essential role in airway inflammation and AHR in TDI-induced asthma.     

Influence of age and HLA type on interferon-gamma (IFN-gamma) responses to a naturally occurring polymorphic epitope of Plasmodium falciparum liver stage antigen-1 (LSA-1)

Antigenic polymorphism and HLA restriction may limit the immunogenicity of a subunit vaccine against liver-stage Plasmodium falciparum. We examined 59 clinical isolates and five laboratory clones of P. falciparum for polymorphism in the N- and C-terminal regions of LSA-1, evaluated binding of the corresponding peptides to selected HLA class I alleles, and measured IFN-gamma responses in residents of a malaria-endemic area of Papua New Guinea where HLA-A*1101, -24, -B13, and -B40 are the most common class I alleles. LSA-1 polymorphism was limited to a single non-synonymous mutation encoding serine (S), proline (P), or threonine (T) at amino acid 85. Nine-mer 84-92 peptides with S, T, or P at the primary anchor position bound differentially to HLA-A11, -A2, and -B7. IFN-gamma ELISPOT responses increased with age in malaria-exposed subjects: 14-16% and 30-36% of 2-5- and 6-54-year-olds, respectively, had > or =10 IFN-gamma-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant (P<0.05). IFN-gamma responses to all three peptides were also greater for older than younger individuals. No children < 3 years old had lymphocytes that responded to all three 84-92 peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-gamma responses. These data support the notion that age-related cumulative exposure to P. falciparum increases the frequency of IFN-gamma responses to polymorphic epitopes of liver-stage antigens such as LSA-1.