H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.     

脑出血大鼠脑组织膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达及益气活血中药的干预效应

目的:观察益气活血中药对脑出血大鼠脑组织中膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达量的影响,从脑出血损伤区微血管系统重建的角度,探讨益气活血中药治疗脑出血的作用机制。方法:实验于2006-03/10在中南大学湘雅医院中西医结合研究所实验室完成。实验材料:补阳还五汤全方(黄芪,当归,赤芍,红花,川芎,地龙,桃仁按20∶3∶3∶3∶2∶3∶3的比例);补阳还五汤益气成分(黄芪按上述比例);补阳还五汤活血成分(当归,赤芍,地龙,川芎,桃仁,红花按3∶3∶3∶2∶3∶3的比例)。用蒸馏水两次水煎,分别浓缩为1.54,0.81和0.73g/mL。实验分组:155只SD大鼠随机分为正常对照组、假手术组、模型组、益气活血组、益气组、活血组。正常对照组5只大鼠,其余每组30只,再随机分为术后灌胃2,4,7,14,21,28d6个观察时间点,各个时间点5只大鼠。实验干预:造模:采用立体定位技术将胶原酶Ⅶ注入大鼠大脑右苍白球制成脑出血大鼠模型。假手术组大鼠仅注入2μL生理盐水,其余手术过程相同。给药:正常对照组:普通饲养,自由饮水;假手术组和模型组术后予蒸馏水灌胃2次/d,2mL/次;益气活血组、益气组、活血组分别给予补阳还五汤全方、补阳还五汤益气成分、补阳还五汤活血成分30.80,16.20,14.60g/(kg·d)(按体表面积计算为临床70kg成人剂量的3倍)灌胃,2次/d,2mL/次。各组大鼠分别于灌胃2,4,7,14,21,28d麻醉下取脑,制备切片;正常组动物于28d处死。实验评估:免疫组织化学染色方法检测各组灌胃不同时间脑组织基质金属蛋白酶2、基质金属蛋白酶9和膜型基质金属蛋白酶的阳性微血管数。结果:155只大鼠均进入结果分析。①正常组、假手术组皮质偶见膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达。②模型组膜型基质金属蛋白酶、基质金属蛋白酶2呈双峰表达,4d为最高峰,至14～21d再有小高峰出现。③益气活血组给药4d时,膜型基质金属蛋白酶、基质金属蛋白酶2为表达低谷,低于模型组(P<0.01)、益气组和活血组(P<0.05);在中后期,益气活血组膜型基质金属蛋白酶表达高峰为7～14d,较模型组提前出现,21d后与模型组比,各治疗组膜型基质金属蛋白酶已表达极少(P<0.01);益气活血组基质金属蛋白酶2在7d后一直呈高水平维持,高于其他各组(P<0.05),28d后开始逐渐下降。④模型组基质金属蛋白酶9在造模后4d达最高峰(P<0.01),两周后几乎无表达。益气活血组基质金属蛋白酶9高峰推迟至7d出现(P<0.05),之后逐渐下降。结论:益气活血中药可通过调节脑出血后膜型基质金属蛋白酶、基质金属蛋白酶2和基质金属蛋白酶9表达,改善出血后脑组织的微环境,有利于微血管系统重建,促进组织修复。     

骨纤维结构不良的诊断和治疗进展

目的:对长期以来关于骨纤维结构不良大量相关研究及文献进行回顾,综述骨纤维结构不良的诊断和治疗的最新进展。资料来源:通过计算机互联网检索OVID数据库1966-01/2006-10关于骨纤维结构不良的文献,检索词:Osteofibrous dysplasia,限定语言种类为English。同时检索1994-01/2006-10中国全文期刊数据库有关骨纤维结构不良的文献,检索词为:骨纤维结构不良,限定语言种类为中文。资料选择:选择与骨纤维结构不良相关的观察对比研究、经验总结、个案报道、最新研究进展等文献,力求资料全面,排除重复研究。资料提炼:共收集相关国内外文献41篇,排除重复性研究11篇,采用30篇,包括关于骨纤维结构不良定义、发病机制、病理、诊断及治疗等。资料综合:①骨纤维结构不良是一种起源于纤维组织的良性骨肿瘤。发病率低、误诊率高。目前具体发病机制不明,现认为与常染色体显性遗传有关。②骨纤维结构不良好发于胫骨,症状为局部肿块。特征性影像学表现为胫骨中段前侧皮质膨胀性密度减低。确诊方法为病理检查。重点与骨纤维异常增殖症、造釉细胞瘤相鉴别,现有大量研究证明该病与造釉细胞瘤有联系。③治疗上过去认为10岁以前应保守治疗,10岁后选择手术治疗,目前倾向于早期骨膜外切除手术治疗。结论:骨纤维结构不良发病率低,对该病认识较少,误诊率较高,重点需与骨纤维异常增殖症、造釉细胞瘤相鉴别,应提高对该病的认识与重视程度,对可疑者行病理检查,确诊者行骨膜外切除,切除范围较大的病例行重建手术。     

白藜芦醇对动物离体心房收缩力和心率作用的种属差异性比较

目的:观察白藜芦醇对豚鼠、小鼠和家兔离体心肌收缩力和心率的影响。方法:实验于2005-08/2006-12在河北医科大学西校区实验中心完成。①实验分组:离体豚鼠、小鼠和家兔心肌各分为9组:空白对照组、溶剂对照组、递增累积浓度白藜芦醇组(浓度为10-6,3×10-6,10-5,3×10-5,10-4,3×10-4mol/L),白藜芦醇对照组(5×10-5mol/L),ATP敏感性钾通道阻断剂格列苯脲(5×10-5mol/L)预孵育 白藜芦醇组,钙激活钾通道阻断剂四乙胺(10-3mol/L)预孵育 白藜芦醇组,电压依赖性钾通道阻断剂4-氨基吡啶(10-3mol/L)预孵育 白藜芦醇组,内向整流钾通道阻断剂氯化钡(10-4mol/L)预孵育 白藜芦醇组,乙酰胆碱调节钾通道阻断剂阿托品(10-5mol/L)预孵育 白藜芦醇组。②实验方法:不同类型的钾通道阻断剂均预孵育15min后,分别加入白藜芦醇(终浓度为5×10-5mol/L),连续记录30min,与相应动物白藜芦醇对照组相比较。③评估指标:分析不同阻断剂与白藜芦醇联用对心房收缩力下降率及心率抑制率的影响。结果:①白藜芦醇可降低豚鼠和小鼠离体心肌收缩力和心率(P<0.05),并被ATP敏感性钾通道阻断剂格列苯脲和钙激活钾通道阻断剂四乙胺部分阻断。②白藜芦醇可降低家兔离体心肌心率,格列苯脲可阻断白藜芦醇的负性变时作用。③电压依赖性钾通道阻断剂4-氨基吡啶、内向整流钾通道阻断剂氯化钡、乙酰胆碱调节钾通道阻断剂阿托品均不能阻断白藜芦醇对3种不同动物离体心房收缩力和心率的作用(P>0.05)。结论:白藜芦醇可呈剂量依赖性减慢豚鼠、小鼠和家兔的心率,白藜芦醇可减弱豚鼠心肌收缩力,其作用是与开放ATP敏感性钾通道有关,而与电压依赖性钾通道、内向整流钾通道和乙酰胆碱调节钾通道无关。同时,钙激活钾通道也参与了白藜芦醇对豚鼠和小鼠离体心房收缩力和/或心率的抑制作用。白藜芦醇对离体心肌收缩力和心率的作用有种属差异性。     

Randomised clinical trial: otilonium bromide improves frequency of abdominal pain, severity of distention and time to relapse in patients with irritable bowel syndrome

Aliment Pharmacol Ther 2011; 34: 432–442

Summary

Background Otilonium bromide (OB) is a spasmolytic agent that blocks L‐Type Calcium channels in human colonic smooth muscle. Aim To study the efficacy of OB in symptom control in irritable bowel syndrome (IBS). Methods A total of 356 patients (46.16 ± 19 years, 71% female) with IBS participated in a double‐blind, randomised, parallel placebo‐controlled phase IV study. OB (40 mg t.d.s.) or placebo was administered for 15 weeks, and follow‐up was extended 10 additional weeks. Results Otilonium bromide (n = 179) and placebo (n = 177) groups had comparable demographics, symptom severity and IBS subtype. Both OB and placebo reduced abdominal pain and IBS symptoms. The effect of OB was significantly greater than placebo in the reduction of weekly frequency of episodes of abdominal pain at the end of treatment period (primary endpoint, ?0.90 ± 0.88 vs. ?0.65 ± 0.91, P = 0.03), reduction of abdominal bloating (?1.2 ± 1.2 vs. ?0.9 ± 1.1, P = 0.02) and global efficacy by patient assessment (1.3 ± 1.1 vs. 1.0 ± 1.1, P = 0.047). Intensity of abdominal pain, proportion of patient responders, safety and quality of life scores were similarly affected by OB and placebo. During follow‐up, the therapeutic effect of OB remained greater than placebo in terms of withdrawal rate due to symptom relapse (10% vs. 27%, P = 0.009), global efficacy of treatment and relapse‐free probability (P = 0.038). Conclusions This placebo‐controlled double‐blind study shows that otilonium bromide is safe, well tolerated and superior to placebo in reducing the frequency of abdominal pain, severity of abdominal bloating and protecting from symptom relapse in IBS. These results further confirm that patients with IBS can improve during and following treatment with otilonium bromide.

     

Chios mastic treatment of patients with active Crohn＇s disease

AIM： To evaluate the effectiveness of mastic administration on the clinical course and plasma inflammatory mediators of patients with active Crohn＇s disease （CD）. METHODS： This pilot study was conducted in patients with established mild to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Ten patients and 8 controls were recruited for a 4-wk treatment with mastic caps （6 caps/d, 0.37 g/cap）. All patients successfully completed the protocol. CD Activity Index （CDAI）, Nutritional Risk Index （NRI）, C-reactive protein （CRP）, interleukin-6 （IL-6）, tumor necrosis factor-alpha （TNF-α）, monocyte chemotactic protein-1 （MCP-1）, and total antioxidant potential （TAP） were evaluated in the plasma at baseline and at the end of the treatment period. Results were expressed as mean values ± SE and P 〈 0.05 was considered to indicate statistical significance. RESULTS： Patients exhibited significant reduction of CDAI （222.9 ± 18.7 vs 136.3 ± 12.3, P = 0.05） as compared to pretreament values. Plasma IL-6 was significantly decreased （21.2 ± 9.3 pg/mL vs 7.2 ± 2.8 pg/ mL, P = 0.027）, and so did CRP （40.3 ± 13.1 mg/mL vs 19.7 ± 5.5, P = 0.028）. TAP was significantly increased （0.15 ± 0.09 vs 0.57 ± 0.15 mmol/L uric acid, P = 0.036）. No patient or control exhibited any kind of side effects. CONCLUSION： The results suggest that mastic significantly decreased the activity index and the plasma levels of IL-6 and CRP in patients with mildly to moderately active CD. Further double-blind, placebo-controlled studies in a larger number of patients are required to clarify the role of this natural PrOduct in the treatment of patients with CD.     

Alterations in the function of circulating mononuclear cells derived from patients with Crohn＇s disease treated with mastic

AIM： To assess the effects of mastic administration on cytokine production of circulating mononuclear cells of patients with active Crohn＇s disease （CD）. METHODS： The study was conducted in patients with established mildly to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Recruited to a 4 wk treatment with mastic caps （6 caps/d, 0.37 g/cap） were 10 patients and 8 controls, all of who successfully completed the protocol. Interleukin-6 （IL-6）, tumor necrosis factor-alpha （TNF-α）, monocyte chemotactic protein-1 （MCP-1）, macrophage migration inhibitory factor （MIF） and intracellular antioxidant glutathione （GSH） were evaluated in peripheral blood mononuclear cells （PBMC） before and after treatment. RESULTS： Treating CD patients with mastic resulted in the reduction of TNF-α secretion （2.1 ± 0.9 ng/mL vs 0.5 ± 0.4 ng/mL, P = 0.028）. MIF release was significantly increased （1.2±0.4 ng/mL vs 2.5 ± 0.7 ng/mL, P = 0.026） meaning that random migration and chemotaxis of monocytes/macrophages was inhibited. No significant changes were observed in IL-6, MCP-1 and GSH concentrations. CONCLUSION： This study shows that mastic acts as an immunomodulator on PBIC, acting as a TNF-α inhibitor and a MIF stimulator. Although further double-blind, placebo-controlled studies in a large number of patients is required to clarify the role of this natural product, this finding provides strong evidence that mastic might be an important regulator of immunity in CD.