TLR4受体抑制剂对脂多糖诱导的小胶质细胞炎症因子的影响

目的探讨TLR4受体抑制剂TAK-242对脂多糖(LPS)诱导的BV2小胶质细胞炎症因子的影响。 方法用脂多糖诱导BV2细胞构建炎症反应模型。(1)利用CCK-8法检测不同浓度的TAK-242预处理BV2小胶质细胞后的细胞存活率,确定最佳TAK-242浓度。(2)BV2小胶质细胞加入0,0.1,0.5,1μg/mL脂多糖(LPS)刺激24 h后,实时荧光定量PCR(qRT-PCR)法检测TLR4炎症因子mRNA表达的变化。(3)将BV2小胶质细胞分为四组：对照组,TAK-242组,LPS组和TAK-242预处理组。实时荧光定量PCR(qRT-PCR)法检测TLR4、MyD88,IL-1β、IL-6炎症因子mRNA表达的变化。 结果(1)CCK-8：低剂量的TAK-242不影响BV2细胞活力;与LPS组相比,TAK-242预处理组细胞活力明显升高(P<0.05)。(2)实时荧光定量PCR法：与正常对照组相比,LPS处理组TLR4的mRNA表达明显增加,差异具有统计学意义(P<0.05)。(3)实时荧光定量PCR法：与正常对照组相比,LPS处理组TLR4、MyD88、IL-1β、IL-6的mRNA表达明显增加,差异具有统计学意义(P<0.05);与LPS组相比,TAK-242预处理组TLR4、MyD88、IL-1β、IL-6的mRNA表达明显下降,差异有统计学意义(P<0.05)。 结论LPS可激活TLR4信号通路;TAK-242可降低疾病因子TLR4、MyD88、IL-1β、IL-6等的mRNA表达,并从而增加细胞存活率。     

CysLT1受体拮抗剂montelukast对全脑缺血海马神经元损伤的保护作用

目的 观察全脑缺血后海马神经元损伤及半胱氨酰白三烯1( cysteinyl leukotriene 1,CysLT1)受体表达变化,评价CysLT1受体选择性拮抗剂montelukast对全脑缺血海马神经元损伤的保护作用.方法 两侧颈总动脉阻断结合降压药诱导大鼠全脑缺血模型,海马切片检查CA1神经元病理改变.免疫组化染色观察CysLT1受体的表达变化,montelukast缺血前后多剂量灌胃给药,评价其对全缺血后CA1神经元损伤的影响.结果 全脑缺血后2～3d,海马CA1区神经元损伤、CysLT1受体表达上调,Montelukast(0.5和1.0 mg/kg)剂量依赖性地减轻全脑缺血后海马神经元损伤(P＜0.05和P＜0.01);montelukast(0.5 mg/kg,连续给药5d)在缺血后30 d对CA1神经元损伤仍有保护作用(P＜0.05).结论 CysLT1受体参与全脑缺血后海马神经元损伤,CysLTt受体拮抗剂montelukast对全脑缺血CA1神经元损伤有剂量依赖性和长期的保护作用.     

锰暴露诱导BV2小胶质细胞环氧合酶2表达变化

目的 观察锰暴露诱导BV2小胶质细胞活化过程中COX2分子表达的变化。方法 实验中BV2细胞分3组,为对照组,50μM锰暴露组和100μM锰暴露组,分别处理12h,24h和48h。免疫荧光化学染色方法观察BV2细胞锰暴露24h后的活化情况,荧光实时定量PCR（qRT-PCR）和Western Blot法分别检测COX2分子mRNA和蛋白表达水平的改变。结果 免疫荧光化学染色结果显示,锰暴露24h后BV2小胶质细胞形态发生改变,细胞胞体变大变圆,突起变短或消失,活化细胞占总细胞数的比例增加（P<0.05）;实时定量PCR结果显示,锰暴露12h和24h后COX2mRNA水平增加（P<0.05）;Western Blot结果显示,锰暴露24h和48h后COX2蛋白表达水平增加（P<0.05）。结论 锰暴露能够诱导BV2细胞COX2分子表达增加,COX2可能参与了锰诱导小胶质细胞活化的过程。     

鱼藤酮对体外培养星型胶质细胞毒性作用

目的观察鱼藤酮对体外培养星型胶质细胞毒性作用的特点。方法体外培养大鼠中脑星型胶质细胞,采用胶质纤维酸性蛋白（GFAP）免疫荧光法鉴定。观察染毒星型胶质细胞形态,检测细胞活力,通过生长曲线分析鱼藤酮对星型胶质细胞增殖的影响以及RT-PCR检测缝隙连接蛋白（connexin43,CX43）mRNA表达。结果0.5μmol/L鱼藤酮染毒24 h即可引起明显的细胞形态改变;0.75,1.0,2.0μmol/L鱼藤酮染毒时星型胶质细胞活力明显降低,乳酸脱氢酶释放量显著增加;0.5μmol/L以上鱼藤酮可明显抑制胶质细胞增殖,CX43 mRNA表达降低。结论鱼藤酮对体外培养星型胶质细胞可以产生明显的毒性损伤作用,但与神经元比较,星型胶质细胞对相同剂量的鱼藤酮染毒具有更强的耐受能力。     

C5aR/C5L2在小胶质细胞炎症中的作用及机制

目的观察棕榈酸(PA)及C5aR拮抗剂(PMX53)干预后小胶质细胞的炎症改变,探讨C5a受体(C5aR)及C5L2在PA诱导的小胶质细胞炎症中的作用。方法取新生1日龄小鼠,分离脑组织,原代培养小胶质细胞并进行分离纯化及鉴定。纯化小胶质细胞随机分成3组:正常对照组、PA处理组、PA+PMX53处理组。分别应用Western blot检测PA及PA+PMX53干预后小胶质细胞C5aR、C5L2、p38MAPK蛋白表达;RT-PCR检测C5L2 mRNA表达及ELISA法检测IL-6表达变化。结果通过免疫组化鉴定,成功培养小胶质细胞。PA干预后,C5aR蛋白表达较对照组明显上升(P0.001),而加入PMX53后,C5aR蛋白表达较PA组显著下降(P0.001);PA干预后,C5L2蛋白及mRNA表达较对照组增加(P0.001),而加入PMX53后,C5L2蛋白表达与PA组差异无统计学意义(P=0.978),而C5L2 mRNA表达较PA组明显上升(P0.001);PA组较对照组p38MAPK蛋白的表达增加(P=0.002),PA+PMX53组p38MAPK蛋白表达较PA组降低(P=0.004);PA干预后,IL-6浓度较对照组显著升高(P0.001),而PMX53干预后,PA+PMX53组较PA组IL-6浓度显著下降(P0.001)。结论 PA可能通过C5a-C5aR激活MAPK信号途径中的p38MAPK通路,从而诱发小胶质细胞炎症反应,而C5L2在此过程中可能发挥抗炎作用。     

从神经科学角度分析脱髓性疾病——免疫细胞，神经胶质细胞，神经细胞三者之间的相互作用

小胶质细胞（Microglia ）对炎症性脱髓鞘性病变-多发性硬化症（MS）的发展起到至关重要的作用。作为抗原呈递细胞,小胶质细胞会对炎性淋巴细胞发挥作用,并产生炎性细胞因子,谷氨酸和活性氧。现在大家普遍认为神经退行性疾病是一种脱髓鞘病变,它会影响到多发性硬化症的预后表现。在神经退行性疾病-多发性硬化症的早期阶段,神经树突和轴突的串珠状肿胀的出现是神经病理学的标志。由小胶质细胞产生的谷氨酸和活性氧能启动串珠形成。最近我们的研究表明,当多发性硬化症发症时,小神经胶质细胞能减少细胞死亡及诱导神经营养因子分泌,而且产生抗炎症细胞因子和抗氧化剂酶来发挥神经保护作用。神经细胞被认为是小胶质细胞的被动目标,同时它也能够通过各种通路来控制小胶质细胞的活性,包括：细胞因子和趋化因子。受损的神经细胞一方面能分泌可溶性不规则趋化因子（sFKN;CX3CR1）并促进小胶质细胞吞噬神经细胞的碎片,同时也诱导小胶质细胞产生抗氧化血红素加氧酶-1（HO-1）。另外,我们还发现神经细胞分泌的 IL-34可以诱导小胶质细胞的神经保护作用,同时也发现星形细胞也具有神经保护作用。由星形细胞产生的IL-33能诱导小胶质细胞的活化,但是,星形细胞的受体-Toll样受体却会诱导神经毒性分子分泌。因此,进一步探讨神经胶质细胞和神经细胞之间的良性互动的平衡关系,将来对于探讨神经退行性疾病的治疗策略来说是至关重要的。     

凝血酶诱导人单核细胞分泌白细胞介素-6的分析

目的凝血酶通过蛋白酶激活受体（PARs）调节细胞的功能而在炎症和免疫反应中起重要作用，对于其诱导单核细胞产生细胞因子的报道则结果不-致。方法用凝血酶及PARs的激动肽分别激发高度纯化后的单核细胞16h后，用ELISA检测单核细胞培养上清液中的1L-6。结果凝血酶呈浓度依赖性地诱导单核细胞产生IL-6，当凝血酶酶活性被抑制后，诱导单核细胞产生IL-6的能力被抑制。PAR-1激动肽SFLI。R—NH2和PAR-4激动肽GYPGQV—NH2呈浓度依赖性地诱导单核细胞产生IL-6，PAR-1、PAR-4不能诱导单细胞产生IL-6，而PAR-3激动肽TFRGAP—NH2和反激动肽PAGRFr—NH2对于单核细胞产生IL-6无明显作用。结论凝血酶可以诱导高度纯化的成人外周血单核细胞产生IL-6，它们的作用依赖于酶的活性，说明它可能是通过PARs激活单核细胞。     

鱼藤酮对大鼠星形胶质细胞CaMKⅡ基因和蛋白表达的影响

目的 研究鱼藤酮对大鼠原代培养星形胶质细胞钙/钙调素依赖性蛋白激酶Ⅱ(CaMK Ⅱ)活性的影响.方法 MTT法检测染毒星形胶质细胞活力,激光共聚焦结合Fluo-4荧光法动态测定细胞内游离钙浓度([Ca2 ]i),采用RT-PCR技术检测CaMK Ⅱα和CaMK Ⅱβ mRNA的表达,Western-blot技术观察磷酸化CaMK Ⅱ蛋白活性的变化.结果 1.0和2.0 μmol/L鱼藤酮染毒时星形胶质细胞活力明显降低,染毒星形胶质细胞[Ca2 ]i呈浓度依赖性升高,CaMK Ⅱα亚基mRNA明显下调(P<0.05),CaMK Ⅱ蛋白活性显著降低(P<0.01).结论 鱼藤酮染毒星形胶质细胞内游离钙浓度的升高,CaMK Ⅱ基因表达和蛋白活性显著降低,可能是其诱导神经细胞变性损伤的重要原因之一.     

鱼藤酮下调对BV-2小胶质细胞中Drosha蛋白水平和白介素-1β表达影响的研究

目的在鱼藤酮处理BV-2小胶质细胞后,观测细胞中Drosha蛋白水平的变化以及相关炎症因子的表达改变。方法在不同浓度及时间的鱼藤酮处理后,用蛋白免疫印迹检测细胞中Drosha的蛋白水平,同时通过荧光定量PCR的方法检测Drosha下游miR-181a及其靶向炎症因子白介素-1β的表达。随后,在使用miRNA mimics补偿miR-181a的水平降低后,进一步观测白介素-1β的表达改变。结果在鱼藤酮处理BV-2小胶质细胞后,Drosha蛋白水平呈现为浓度和时间依赖地降低,同时,其下游miR-181a的表达水平也被下调,而miR-181a的靶向炎症因子白介素-1β的表达增强。在使用miRNA mimics增加miR-181a的水平后,可抑制白介素-1β的表达增强。结论鱼藤酮在BV-2小胶质细胞通过影响Drosha蛋白及其下游miR-181a的水平,调节白介素-1β的表达。     

鱼藤酮下调对ＢＶ ２小胶质细胞中Ｄｒｏｓｈａ 蛋白水平和白介素 １β表达影响的研究

摘要：目的　在鱼藤酮处理ＢＶ ２小胶质细胞后，观测细胞中Ｄｒｏｓｈａ蛋白水平的变化以及相关炎症因子的 表达改变。方法　在不同浓度及时间的鱼藤酮处理后，用蛋白免疫印迹检测细胞中Ｄｒｏｓｈａ的蛋白水平，同 时通过荧光定量ＰＣＲ 的方法检测Ｄｒｏｓｈａ下游ｍｉＲ １８１ａ及其靶向炎症因子白介素 １β的表达。随后，在使 用ｍｉＲＮＡ ｍｉｍｉｃｓ补偿ｍｉＲ １８１ａ的水平降低后，进一步观测白介素 １β的表达改变。结果　在鱼藤酮处理 ＢＶ ２小胶质细胞后，Ｄｒｏｓｈａ蛋白水平呈现为浓度和时间依赖地降低，同时，其下游ｍｉＲ １８１ａ的表达水平 也被下调，而ｍｉＲ １８１ａ的靶向炎症因子白介素 １β的表达增强。在使用ｍｉＲＮＡ ｍｉｍｉｃｓ增加ｍｉＲ １８１ａ的水 平后，可抑制白介素 １β的表达增强。结论　鱼藤酮在ＢＶ ２ 小胶质细胞通过影响Ｄｒｏｓｈａ蛋白及其下游 ｍｉＲ １８１ａ的水平，调节白介素 １β的表达。 关键词：ＢＶ ２小胶质细胞；鱼藤酮；Ｄｒｏｓｈａ蛋白；白介素 １β；ｍｉＲ １８１ａ；帕金森病 中图分类号：Ｒ７４２　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１７）０６ ０４４７ ０５     

Agaricus blazei extract attenuates rotenone-induced apoptosis through its mitochondrial protective and antioxidant properties in SH-SY5Y neuroblastoma cells

The present study was aimed to find out the effect of Agaricus blazei mushroom extract against rotenone-induced cellular model. SH-SY5Y neuroblastoma cells are divided into four experimental groups (control, rotenone (100?nM), A. blazei (5?μg/ml) + rotenone (100?nM), and A. blazei alone treated) based on MTT assay, cells were allowed to measure the ROS, TBARS levels, and antioxidants activities. Finally, mitochondrial transmembrane potential (MMP) and expressions of apoptotic proteins were also analyzed. Pre-treatment with A. blazei significantly enhanced cell viability, attenuated rotenone-induced ROS, MMP, and apoptosis. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone-induced cytotoxicity. Therefore, it may be concluded that A. blazei can be further developed as a promising drug for the treatment of Parkinson’s disease (PD).     

白三烯受体拮抗剂对人鼻黏膜上皮细胞黏蛋白MUC5ACmRNA表达的影响

目的探讨白三烯受体拮抗剂孟鲁司特对鼻黏膜上皮细胞黏蛋白MUC5AC mRNA表达的影响。方法取第二代培养的鼻黏膜上皮细胞分成四组：对照组、IL-1β组、白三烯受体拮抗剂＋IL-1β组、白三烯受体拮抗剂组,培养24小时后通过荧光定量巢式RT-PCR检测鼻黏膜上皮细胞黏蛋白的定量表达。结果 MUC5AC mRNA的定量表达在照组为（4.32±2.53）×106拷贝/μg,白三烯受体拮抗剂组为（2.96±2.17）×106拷贝/μg,IL-1β组为（3.90±1.79）×107拷贝/μg,白三烯受体拮抗剂＋IL-1β组为（5.13±2.47）×106拷贝/μg。四组中MUC5AC mRNA定量表达的差异具有统计学意义（F=21.45,P〈0.01）。白三烯受体拮抗剂＋IL-1β组的MUC5AC mRNA定量表达低于IL-1β组,差异具有统计学意义（t=4.57,P〈0.01）。IL-1β组的MUC5AC mRNA定量表达高于对照组,差异具有统计学意义（t=4.68,P〈0.01）。白三烯受体拮抗剂组和对照组比较,MUC5AC mRNA定量表达差异无统计学意义（t=1.00,P〉0.05）。结论白三烯受体拮抗剂孟鲁司特降低IL-1β诱导的鼻黏膜上皮细胞黏蛋白MUC5AC mRNA的表达,对正常的鼻黏膜上皮细胞黏蛋白的表达无影响。     

Fish Hydrolysate Supplementation Containing n-3 Long Chain Polyunsaturated Fatty Acids and Peptides Prevents LPS-Induced Neuroinflammation

Neuroinflammation constitutes a normal part of the brain immune response orchestrated by microglial cells. However, a sustained and uncontrolled production of proinflammatory factors together with microglial activation contribute to the onset of a chronic low-grade inflammation, leading to neuronal damage and cognitive as well as behavioral impairments. Hence, limiting brain inflammatory response and improving the resolution of inflammation could be particularly of interest to prevent these alterations. Dietary n-3 long chain polyunsaturated fatty acids (LC-PUFAs) and low molecular weight peptides are good candidates because of their immunomodulatory and proresolutive properties. These compounds are present in a fish hydrolysate derived from marine-derived byproducts. In this study, we compared the effect of an 18-day supplementation with this fish hydrolysate to a supplementation with docosahexaenoic acid (DHA) on lipopolysaccharide (LPS)-induced inflammation in mice. In response to peripherally injected LPS, the fish hydrolysate supplementation decreased the hippocampal mRNA expression of the proinflammatory cytokines IL-6 (p < 0.001), IL-1β (p = 0.0008) and TNF-α (p < 0.0001), whereas the DHA supplementation reduced only the expression of IL-6 (p = 0.004). This decline in proinflammatory cytokine expressions was associated with an increase in the protein expression of IκB (p = 0.014 and p = 0.0054 as compared to the DHA supplementation and control groups, respectively) and to a modulation of microglial activation markers in the hippocampus. The beneficial effects of the fish hydrolysate could be due in part to the switch of the hippocampal oxylipin profile towards a more anti-inflammatory profile as compared to the DHA supplementation. Thus, the valorization of fish byproducts seems very attractive to prevent and counteract neuroinflammation.     

慢性鱼藤酮中毒诱导的细胞凋亡机制初步探讨

目的观察小剂量鱼藤酮长期暴露对大鼠纹状体一氧化氮（nitricoxide,NO）、细胞色素C（cytochromeC,Cyt-C）含量及Caspase-3蛋白表达的影响,探讨鱼藤酮诱导的中脑多巴胺神经元凋亡机制。方法采用Wistar大鼠72只,随机分为对照组、鱼藤酮中毒0.335mg/kg、0.670mg/kg、1.005mg/kg组,连续皮下注射鱼藤酮30d、60d和90d,以生化法和Western-blotting法检测大鼠纹状体Cyt-C含量及Caspase-3蛋白的表达。结果小剂量鱼藤酮长期中毒可导致大鼠纹状体内NO和Cyt-C含量增加（P〈0.05）,且呈时间—剂量效应关系。Westernblotting结果证实小剂量鱼藤酮重复注射可导致纹状体Caspase-3蛋白表达增强。结论低剂量鱼藤酮重复注射可使动物脑组织NO、Cyt-C含量增高及Caspase-3蛋白表达增强,NO介导的细胞凋亡机制可能参与了鱼藤酮中毒所致的多巴胺神经元死亡。     

Die Rolle von Zytokinen in der Pathogenese und Therapie chronisch-entzündlicher Darmerkrankungen

Crohn's disease and ulcerative colitis are chronic disorders of the gastrointestinal tract. Crohn`s disease is characterised by a transmural inflammation that can affect any part of the human gastrointestinal tract, whereas ulcerative colitis is restricted to the colon.Both diseases are disorders mediated by a pathogenic proinflammatory cytokine pattern produced by T-cells and macrophages. Inflammatory bowel diseases arise in individuals who are genetically susceptible as a result of a pathological immune response to mucosal bacterial antigens. In Crohn's disease, the interaction of macrophages and T-cells induces the production of proinflammatory cytokines, such as TNF-α, IL-12, IL-6 and IFN-γ. In the last decade these molecular mediators, their receptors and their mechanisms of action were characterised.Since then treatment has changed and new approaches based on the knowledge about cytokine interactions has been developed.One possibility to inhibit a proinflammatory cytokine response is to inhibit cell-cell interactions by blocking adhesion molecules (α4β1, α4β7) or costimulatory molecules (CD40L). The pathologic cytokine action in the inflamed gut can be counteracted in different ways. On the one hand clinical trials showed, that the function of proinflammatory cytokines can be neutralized by cytokine-antibodies (TNF-α, IL-12),by cytokine receptorantibodies (TNF-α, IL-6R) or by inhibiting the synthesis of proinflammatory cytokines by the application of antisense-oligonucleotides (TNF-α converting enzyme).On the other hand the application of protective antiinflammatory recombinant cytokines (IL-10, IL-11 and IFN-α) might protect individuals from an overwhelming proinflammatory immune response.The most effective cytokine antibody in the treatment of Crohn's disease so far is the anti-TNF-antibody infliximab,which may also cause serious side effects (e.g. tuberculosis reactivation).     

Gut–brain and brain–gut axis in Parkinson's disease models: Effects of a uridine and fish oil diet

Recent investigations have focused on the potential role of gastrointestinal (GI) abnormalities in the pathogenesis of Parkinson's disease (PD). The ‘dual-hit’ hypothesis of PD speculates that a putative pathogen enters the brain via two routes: the olfactory system and the GI system. Here, we investigated (1) whether local exposures of the neurotoxin rotenone in the gut or the brain of mice could induce PD-like neurological and GI phenotypes as well as a characteristic neuropathology in accordance with this ‘dual-hit hypothesis’ and (2) the effects of a diet containing uridine and fish oil providing docosahexaenoic acid (DHA), in both models. Mice were given rotenone either orally or by an injection in the striatum. Dietary interventions were started 1?week before rotenone exposures. We found that (1) both oral and intrastriatal administration of rotenone induced similar PD-like motor deficits, dopaminergic cell loss, delayed intestinal transit, inflammation, and alpha-synuclein accumulation in the colon; (2) the uridine and DHA containing diet prevented rotenone-induced motor and GI dysfunctions in both models. The models suggest possible bidirectional communication between the gut and the brain for the genesis of PD-like phenotype and pathology. The dietary intervention may provide benefits in the prevention of motor and non-motor symptoms in PD.     

Effects of low-dose irradiation on enhancement of immunity by dendritic cells

Low-doses of irradiation have been reported to have beneficial effects, particularly anti-tumor effects. In this paper, we show the effects of the low-dose irradiation on T cell activation induced by dendritic cells (DCs). DCs, which had been pre-irradiated at 0.02-1.0 Gy from a (137)Cs source, were cultured with allogeneic T cells, and the proliferation of T cells was then examined. The 0.05Gy-pre-irradiated DCs showed the highest proliferation capacity of T cells. The 0.05Gy-irradiation does not augment the expression of major histocompatibility complexes (MHCs) or costimulatory molecules on DCs, as with non-irradiated DCs or 1Gy-irradiated DCs, but does augment the production of IL-2, IL-12 and IFN-gamma DCs. These results suggest that the low-dose irradiation augments T cell-activation capacity through cytokine production by DCs, which might shift na?ve helper T cells to Th1 cells.     

The effect of ozone exposure on the ability of human surfactant protein a variants to stimulate cytokine production.

Ozone exposure can cause inflammation and impaired lung function. Human surfactant protein A (SP-A) may play a role in inflammation by modulating cytokine production by macrophages. SP-A is encoded by two genes, SP-A1 and SP-A2, and several allelic variants have been characterized for each gene. These allelic variants differ among themselves in amino acids that may exhibit differential sensitivity to ozone-induced oxidation and this may produce functional differences. We studied the effects of SP-A variants before and after ozone exposure on the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8. These are important proinflammatory cytokines and are expressed by the macrophage-like THP-1 cells. Eight variants were expressed in vitro, characterized by gel electrophoresis, and studied. These included six single-gene SP-A alleles and two SP-A variants derived from both genes. Variants were exposed to ozone at 1 ppm for 4 hr at 37 degrees C, and we compared their ability to stimulate cytokine (TNF-alpha and IL-8) production by THP-1 cells to air-exposed and unexposed SP-A variants. We found that a) SP-A2 variants (1A, 1A(0), 1A(1) stimulate significantly more TNF-alpha and IL-8 production than SP-A1 variants (6A, 6A(2), 6A(4); b) coexpressed SP-A variants (1A(0)/6A(2), 1A(1)/6A(4) have significantly higher activity than single gene products; c) after ozone exposure, all SP-A variants showed a decreased ability to stimulate TNF-alpha and IL-8 production, and the level of the decrease varied among SP-A variants (26-48%); and d) human SP-A from patients with alveolar proteinosis exhibited a minimal decrease (18% and 12%, respectively) in its ability to stimulate TNF-alpha and IL-8 after in vitro ozone exposure. We conclude that biochemical and functional differences exist among SP-A variants, that ozone exposure modulates the ability of SP-A variants to stimulate cytokines by THP-1 cells, and that SP-As from bronchoalveolar lavage (BAL) fluid of certain alveolar proteinosis patients may be oxidized in vivo.