骨形态发生蛋白信号去除后Sonic hedgehog对小脑颗粒细胞发育的影响

目的:骨形态发生蛋白信号在神经细胞的生长、分化、增殖、凋亡等方面发挥作用,而Sonic hedgehog能调节小脑颗粒细胞的增殖,故实验设计了去除骨形态发生蛋白信号后Sonic hedgehog信号在小脑颗粒细胞发育中作用的观察。方法:实验于2004-11/2005-10在美国费城儿童医院Crenshaw实验室和北京大学医学部解剖学神经研究实验室完成。①实验材料:C57Bl/6J小鼠40只;Bmpr1b敲除鼠系来源于Karen Lyons博士。②实验过程:通过敲除骨形态发生蛋白Ⅰ型受体的Bmpr1a和Bmpr1b两个亚型基因,去除骨形态发生蛋白信号在小脑内的传导。③实验评估:使用原位杂交和免疫组织化学方法检测了新生幼鼠小脑中Sonic hedgehog信号的表达情况。结果:①在胚胎11.5d,磷酸化SMAD免疫阳性的细胞被观察到在正常鼠神经管背侧的菱形唇附近,受体基因双敲除的小鼠背侧神经管中未发现免疫阳性细胞,此结果提示通过骨形态发生蛋白Ⅰ型受体传导的骨形态发生蛋白信号已消失。当小鼠出生后,磷酸化SMAD免疫阳性细胞主要位于正常小鼠的内颗粒层,在基因敲除鼠只有一小部分区域有磷酸化SMAD蛋白的表达。②Sonic hedgehog表达在正常新生鼠小脑的颗粒细胞层和蒲肯野氏细胞层;基因敲除鼠Sonic hedgehog信号只表达在小脑一小部分区域的颗粒细胞层和蒲肯野氏细胞层,而大部分区域则缺少它的表达。PTCH和N-myc在小脑中的表达与Sonic hedgehog表达部位类似。这提示Sonic hedgehog信号及其PTCH和N-myc都参与了小脑颗粒细胞的发育。结论:实验阐明了骨形态发生蛋白信号去除后,Sonic hedgehog信号对小脑颗粒细胞发育所起到的重要作用。     

手性毛细管气相色谱法测定人尿中美芬妥英光学异构体含量的方法学研究

A gas chromatographic method equipped with nitrogen-phosphorus detector was developed for the determination of the S-and R-enantiomers of the anticonvulsant, mephenytoin (MP) in human urine. Dichioromethane (4 ml) was added to 1 ml urine, the mixture was shaken and centrifuged. The organic phase was transferred to another tube and blown to dryness under nitrogen on water bath (37℃). The residue was dissolved in 10 μl ethylacetate and 1～2 μl was injected into the GC. Our results showed that direct enantiomeric separation of mephenytoin was obtained by using a chiral capillary column, the retention times for S-and R-mephenytoin were 25. 5 and 26. 2 min respectively, with a detection limit less than 50 ng/ml of mephenytoin. Similar linear and reproducible standard curves were obtained over the concentration range of 53.2 to 2128. 0 ng/ml (for S-MP, r=0. 9914±0. 0070, n=6; and for R-MP, r=0. 9939±0. 0070, n=6), and the mean recoveries of S-and R-MP were 95. 4% and 95. 8% respectively. The within--day relative standard deviations were less than 8. 8% for both S-and R-MP, and that of between--days were less than 14.3%. There was a good reproducibility of the urine S/R mephenytoin determined in China and in Sweden by using similar method in 107 Chinese volunteers after a single oral dose of 100 mg racemic mephenytoin (r=0. 9091, P<0. 001).     

Immunomodulation by macrolide antibiotics

Macrolide antibiotics are strongly concentrated within host cells, a property that sustains their activity against intracellular pathogens and is likely responsible for the modulation of cell metabolism and function. There is extensive literature on the subject of macrolide-induced modulation of immune responses. Erythromycin A derivatives seem to display anti-inflammatory activity in vitro, in some animal models and in various clinical settings such as diffuse panbronchiolitis (DPB). The underlying mechanisms are not yet fully understood: inflammatory cytokine and oxidant production by phagocytes is down-regulated by these drugs, but other possible targets include bacterial virulence factors, bronchial and epithelial cells, etc. Also, a link has been suggested between the macrolide transmembrane carrier system and the P-glycoprotein family, which comprises MDR (multiple drug resistance) and CFTR (cystic fibrosis transmembrane conductance regulator), which are respectively involved in the chemotherapeutic resistance of cancer cells and in the genesis of cystic fibrosis.     

Effective treatment of high-grade lymphoproliferative disorder after renal transplantation using autologous lymphocyte activated killer cell therapy

Posttransplantation lymphoproliferative disorders (PTLD) is not uncommon and can occur in 2% to 5% of solid organ recipients on immunosuppression. Epstein-Barr virus (EBV) infection or reactivation and intensive anti-T lymphocyte treatment are important pathogenetic factors for a large proportion of these disorders. Nonclonal lesions with polymorphous histology have a potential for regressing when the immunosuppressants are reduced or stopped. Clonal tumors with a monomorphous histology carry a poor prognosis, and the mortality rate for monoclonal lymphoma has been reported as high as 80%. We report a renal transplant recipient who developed high-grade monoclonal lymphoma only 4 months after a live-donor transplantation. The tumor was EBV positive. Reduction of immunosuppressants resulted in minimal regression of the tumor. The patient was treated with adoptive immunotherapy using ex vivo generation of autologous lymphocyte activated killer (LAK) cells. She had leukapheresis, and autologous peripheral blood mononuclear cells were obtained and cultured in interleukin-2 (IL-2)-rich medium for 9 to 10 days. The IL-2-activated LAK cells were reinfused into the patient without any systemic administration of IL-2. The patient experienced no side effects during the infusion. There was no rejection episode, and the renal function of the patient remained stable after treatment. Computed tomography scan performed 2 months after the infusion showed marked regression of the lesions in the liver and spleen. Five months later, magnetic resonance imaging showed complete resolution of the tumor lesions. Ultrasonography 13 months after the LAK cell infusion showed no lesion. The allograft function was not affected after treatment. Adoptive immunotherapy using IL-2-activated autologous LAK cells was effective in treating this renal transplant patient with EBV-positive high-grade lymphoma. The patient's kidney allograft functioned well without any rejection.     

Moxifloxacin modifies corneal fibroblast-to-myofibroblast differentiation

Background and Purpose

Fibroblast-to-myofibroblast differentiation is associated with scarring, an important issue in corneal surgery. Moxifloxacin (MOX), commonly applied to prevent post-operative infection, would benefit more if it modifies fibroblast-to-myofibroblast differentiation other than antimicrobial activity. Our purpose was to explore whether MOX has anti-fibrotic effect in human corneal fibroblasts (HCFs).

Experimental Approach

HCFs were incubated in MOX-containing medium concurrently with TGF-β1 (co-treatment), before (pretreatment) or after (post-treatment) adding TGF-β1. HCF contractility was evaluated with a type I collagen gel contraction assay. Expression of α-smooth muscle actin (α-SMA), Smad2, phospho-Smad2-Ser467, Smad4 and Smad7 was determined by immunoblotting. Formation of α-SMA-positive filaments and distribution of active Smad2 were observed under confocal microscopy. Expression of TGF-β receptor types I (TGFBR1) and II (TGFBR2) was assessed with flow cytometry.

Key Results

MOX did not affect gel contractility or α-SMA filament formation in HCFs without TGF-β1 stimulation. MOX did, however, retard HCF-containing gel contractility and α-SMA filament formation following TGF-β1 stimulation in the pretreatment and co-treatment groups but not in the post-treatment group. MOX blocked the expression of Smad2, phospho-Smad2-Ser467 and TGFBR1 under TGF-β1 incubation. Additionally, MOX enhanced Smad7 expression in TGF-β1-incubated HCFs, but did not interfere with TGF-β-triggered Smad2 nuclear translocation or Smad4 expression.

Conclusions and Implications

MOX inhibited TGF-β-induced fibroblast-to-myofibroblast differentiation via blocking TGFBR1 and enhancing Smad7 expression. MOX should be used before or during surgery to achieve these effects. These results suggest a de novo mechanism by which MOX participates in corneal wound healing.     

Heparin therapy in the Chinese--lower doses are required

Warfarin requirements are lower in the Chinese, but it is not known if this applies to heparin. We investigated the optimal dose for heparin therapy in Chinese patients, and to assess relationship between i.v. heparin dosage and anticoagulation efficacy. One hundred Chinese patients requiring intravenous heparin therapy were given an initial bolus followed by continuous intravenous infusion. The main outcome measures were: (i) Efficacy of anticoagulation assessed by blood coagulation studies (APTT) compared to heparin dosage, (ii) Determinants of dosage variation-age, gender, body weight, height, indication for heparin therapy and number of medications, other disease, and serum albumin level. It was found that the mean therapeutic infusion dose requirement of heparin was 848.7 +/- 274.7 units/h, 79% required a dose of 1000 units/h or less. Heparin dose correlated negatively with age (r = -0.40; p < 0.001) and positively with weight (r = 0.44 p < 0.001) and height (r = 0.49; p < 0.001). Chinese subjects require lower heparin doses (about 800 units/h) than usually recommended for Caucasians (usual dose 1000-1500 units/h). This can be partly explained by the lower body weight in Chinese patients.