小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.     

Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.     

CD3+ leukemic large granular lymphocytes utilize diverse T-cell receptor V beta genes

CD3+ large granular lymphocyte (LGL) leukemia is a disease of unknown etiology characterized by clonal proliferation of T cells that usually express T-cell receptor (TCR) alpha beta heterodimers. The purpose of this study was to identify the variable (V), joining (J), and diversity (D) region TCR beta-chain genes expressed by CD3+ LGL leukemic cells in an attempt to gain insights into the etiology of this disorder. Twelve patients with LGL leukemia were studied, including seven with both LGL leukemia and rheumatoid arthritis (RA). RA is also a disease of unknown etiology that occurs frequently in patients with LGL leukemia. Clonally expanded T cells that express specific TCR V beta genes have been identified in fluid and tissue specimens from the joints of patients with RA. In this study, V beta expression was determined by PCR using a panel of 22 unique V beta primers to amplify cDNA prepared from peripheral blood mononuclear cells (PBMC). A dominant V beta gene product was readily apparent in all patients. To confirm that the dominant V beta gene originated from a clonal expansion, DNA fragments corresponding to the dominant V beta genes were subcloned into plasmids and independently isolated recombinants were sequenced. V-D-J region sequences that occurred repeatedly indicated clonality. The V beta and J beta genes expressed by the leukemic cells showed a pattern of distribution that followed the frequency with which these genes are represented in the peripheral blood. The residues corresponding to the third complementarity-determining region of the TCR beta chain were different in all cases. A specific pattern of VDJ usage was not identified for those patients with both LGL leukemia and RA; however, utilization of V beta-6 by LGL clones (N = 3) was observed only in the setting of RA. These data suggest that leukemic CD3+ LGL cells have been clonally transformed in a random fashion with respect to the TCR beta chain.     

Determining resectability for hepatocellular carcinoma: the role of laparoscopy and laparoscopic ultrasonography

We reviewed our experience with preoperative determination of resectability in patients with hepatocel‐lular carcinoma (HCC) over the last 10 years, and evaluated the role of laparoscopy with laparoscopic ultrasonography (USG) since we instituted this technique in June 1994. From January 1989 to December 1998, 500 of 1741 patients with HCC (28.7%) were considered suitable for hepatic resection after preoperative assessment. Significantly more contrast‐enhanced computed tomography (CT) scans and fewer percutaneous USGs or hepatic arteriograms were performed in the 299 patients managed since June 1994 (group 2) than in the 201 patients managed before then (group 1). One hundred and ninety‐eight patients in group 2 (66%) underwent laparoscopy with laparoscopic USG. Unresectable disease was found in 41 patients in group 1 (20.4%) (all at laparotomy), and in 68 patients in group 2 (22.7%) (16 at laparotomy without laparoscopic examination, 31 at laparoscopic examination alone, and 21 at laparotomy after an inconclusive laparoscopic examination) (P = 0.5). The most common features of unresectable disease were the presence of bilobar intrahepatic metastases and an inadequate liver remnant with cirrhosis. The adoption of the laparoscopic examination after June 1994 improved the overall resection rate at laparotomy in group 2 from 77.3% to 86.2%, which was better than that in group 1 (79.6%,P = 0.057). For patients with unresectable disease, the operation time and hospital stay were significantly shorter in group 2. The postoperative morbidity and mortality rates were 9.8% and 4.9%, respectively, in group 1, and 5.9% and 2.9% in group 2. There was no operative morbidity in the 31 patients who had unresectable disease detected by the laparoscopic examination alone. Laparoscopy with laparoscopic USG avoids unnecessary laparotomy, and has a definite role in determining resectability in patients with HCC.     

ISOLA器械治疗小儿脊柱侧凸

目的定期随访评价ISOLA器械治疗小儿脊柱侧凸.方法41例小儿脊柱侧凸患儿,特发性脊柱侧凸32例,神经肌肉性脊柱侧凸5例,其他类型的脊柱侧凸4例,术前、术中、术后均摄X线片,记录Cobb角,顶椎的移位及旋转,躯干偏离中线程度.结果Cobb角术前平均为66°,术后1年为36°,术后2年为36°.1年后,所有患儿的脊柱均完全融合.顶椎的偏离改善了14mm.躯干偏离改善了5mm.特发性脊柱侧凸患儿1年后有基本的正常活动.术后4例出现并发症,1例深部感染,1例骶尾部褥疮,1例横钩脱落,1例L3椎弓根螺丝钉脱落.结论目前有多种后路器械治疗小儿脊柱侧凸.严格掌握手术指征及ISOLA的原理,该术式能获得较为满意的疗效.     

Spermatic vein embolization with hot contrast material: fertility results

Spermatic venography with hot contrast material embolization was undertaken in 81 patients with varicoceles and infertility. Long-term follow-up information was available in 91% of the patients, and there was an overall conception rate of 40.5%. Embolization with hot contrast material was easily performed without special embolization devices and proved to be a safe and effective technique.