Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate-putamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay.Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.     

DNA prime followed by protein boost enhances neutralization and Th1 type immunity against FMDV

Prime-boost strategy has been exhibited its potency to enhance immune responses, which would be important to the success to develop a vaccine against the foot-and-mouth disease virus (FMDV). An eukaryotic expression construct encoding the FMDV capsid VP1 protein with a recombinant VP1 protein or a commercial FMDV vaccine were tested in the prime-boost strategy in mice and cattle trials. The levels of induced specific antibodies, T cell proliferations, and DTH activities were significantly higher in the prime-boost groups than in those vaccinated with DNA, protein or FMDV vaccine alone. More importantly, the levels of neutralizing antibodies in the former groups were significantly higher than others and could last for at least four months in cattle trials. This study suggests that the prime-boost strategy significantly improves the effective immunity and may provide a longer protection against FMDV infection.     

双表达外源性4-1BBL/IL-15的K562细胞活化外周血淋巴细胞的研究

为肿瘤过继免疫治疗开发体外激活T细胞、NK细胞的高效途径,研究双表达外源性4-1BBL和IL-15的K562细胞刺激外周血淋巴细胞活化的能力。采用分子克隆技术,分别将4-1BBL和IL-15基因插入双表达载体pVITRO-2,命名为pV4-1BBL-IL-15。经测序鉴定后,利用脂质体介导的转染及潮霉素筛选,获得稳定双表达4-1BBL、IL-15分子的K562细胞(K562/4-1BBL/IL-15)。经流式细胞仪(FACS)分选后,K562/4-1BBL/IL15细胞和K562细胞分别用丝裂霉素C处理,与外周血淋巴细胞孵育24 h,FACS检测淋巴细胞表面活化性受体CD69的表达。对NK细胞不仅同时检测活化性受体NKG2D的表达,还用乳酸脱氢酶释放法观察NK细胞受不同刺激细胞作用后,细胞毒活性的变化。结果显示受K562/4-1BBL/IL15细胞刺激后,T细胞CD69的表达无明显变化。γδT细胞CD69表达增长5倍。NK细胞CD69表达增长6倍,而NKG2D的表达增加1.5倍;NK细胞受K562/4-1BBL/IL15细胞作用72 h后,细胞毒活性明显提高。提示双表达4-1BBL/IL-15的K562细胞能够高效激活γδT细胞及NK细胞,有望用于肿瘤的过继免疫治疗。     

(His)6 -eIF3s4融合蛋白在人乳腺癌细胞Bcap37中的表达

目的：构建真核细胞翻译起始因子3第4亚单位（eIF3s4）真核表达载体用于进一步功能研究。方法：设计引物,以K562细胞的总RNA为模板,采用RT-PCR方法扩增eIF3s4的全长编码区cDNA,克隆于pGEM-TEasy载体,经DNA测序并与GenBank数据库序列进行比对后,将该cDNA序列亚克隆于真核表达载体pcDNA4/HisMaxB载体,构建成（His）6-eIF3s4融合蛋白的表达载体,用LipofectamineTM2000瞬时转染人乳腺癌细胞株Bcap37,利用Westernblot方法检测（His）6-eIF3s4融合蛋白的表达。结果：DNA序列测定表明,利用RT-PCR方法克隆的eIF3s4全长编码区cDNA序列与GenBank数据库序列一致,Westernblot结果证实（His）6-eIF3s4融合蛋白在Bcap37细胞中获得预期表达。结论：成功地构建了eIF3s4的真核表达载体并在人乳腺癌细胞Bcap37中表达,为建立稳定的eIF3s4高表达细胞系,进而研究eIF3s4与肿瘤细胞多药耐药相关性打下了基础。     

Shigella infection in a SCID mouse-human intestinal xenograft model: role for neutrophils in containing bacterial dissemination in human intestine

Shigellae infect human intestine and cause intense inflammation and destruction of colonic and rectal mucosa. To model the interactions of shigella with human intestine in vivo, we have studied shigella infection in human intestinal xenografts in severe combined immunodeficient mice (SCID-HU-INT mice). Inoculation of shigella into human intestinal xenografts caused severe inflammation and mucosal damage, which was apparent as soon as 4 h following infection. Shigella infection was associated with human intestinal production of interleukin-1B (IL-1B) and IL-8 and a marked neutrophil influx into the graft. Depletion of neutrophils from SCID-HU-INT mice reduced inflammation in the human intestinal xenograft in response to shigella infection but failed to significantly alter tissue damage. However, the number of intracellular bacteria was more than 20-fold higher in the human intestinal xenografts from neutrophil-depleted SCID-HU-INT mice. Infection of human intestinal xenografts with an attenuated vaccine strain of shigella (CVD1203) induced lower levels of IL-1B and IL-8 than wild-type shigella and caused only moderate damage to the intestinal permeability barrier. Our studies establish the SCID-HU-INT mouse as a viable model for studying the interactions between shigella and human intestine and indicate that neutrophils are important for controlling the invasion of human intestine by shigella.     

Intrapulmonary cystic lymphangioma in a 2-month-old infant.

Lymphangioma is an abnormal collection of lymphatics that are developmentally isolated from the normal lymphatic system. Lymphangioma rarely presents as a solitary pulmonary lesion. We report a rare case of intrapulmonary cystic lymphangioma involving the upper lobe of the right lung, which presented with dyspnea in a 2-month-old infant. High-resolution computed tomography (HRCT) of the chest demonstrated a well-circumscribed, multiseptate, cystic lesion in the upper lobe of the right lung, mimicking the feature of type I congenital cystic adenomatoid mal-formation. The tumor was removed by bilobectomy of the upper and middle lobes of the right lung, and its pathologic examination confirmed the diagnosis of an intra-pulmonary cystic lymphangioma.     

抗Ig融合蛋白Fc段单克隆抗体的制备、鉴定与应用研究

目的：制备并鉴定抗Ig融合蛋白Fc段的单克隆抗体(mAb)，建立用于检测Ig融合蛋白的夹心ELISA法和纯化Fc融合蛋白的亲和层析法。方法：以hBCMA—Ig融合蛋白为抗原免疫BALB／c小鼠，通过细胞融合制备抗Fc段mAb，用ELISA等方法鉴定mAb的Ig亚类、表位以及种属特异性，建立用于检测Ig融合蛋白的夹心ELISA法;Western blot检测mAb对变性Ig融合蛋白的反应性。将mAb与Sepharose4B交联，制备亲和层析柱，对LAIR1-Ig融合蛋白进行纯化。结果：获得7株稳定分泌抗Fc段mAb的杂交瘤(FMUFcl-FMUFc7)。利用FMUFc4作为包被mAb，FMUFc5作为酶标记mAb，成功地建立了检测Ig融合蛋白的ELISA法，敏感度达到2μg／L；在7株mAb中，FMUFc6可用于Ig融合蛋白的western blot检测。用FMUFc 6mAb制备的亲和层析柱，可有效地纯化LAIRl—Ig融合蛋白。结论：成功地制备了抗Ig融合蛋白Fc段的mAb，建立了可用于Ig融合蛋白检测和纯化的方法，为Ig融合蛋白的应用提供了有力手段。     

The involvement of calcium mobilization in the calcium-activated potassium currents activated by hyposmotic swelling in gastric antral circular myocytes of the guinea-pig

In our study of the effects of hyposmotic swelling on the Ca(2+)-activated potassium currents [I(K(Ca))] and its mechanism, we employed the whole-cell patch clamp technique using the gastric antral circular myocytes of the guinea-pig. Hyposmotic swelling efficiently increased I(K(Ca)), and the extent of changes in I(K(Ca)) was sharply dependent on the osmolarity of the perfusion solutions. When the calcium-free solution (EGTA 10 microM added in calcium-free solution) was superfused, I(K(Ca)) was not increased by the hyposmotic swelling. Gadolinium (Gd(3+)) 100 nM, a blocker of the stretch-activated nonselective cation channel, blocked the activation of I(K(Ca)) induced by hyposmotic swelling, but nicardipine 5 microM (the L-type calcium channel blocker) did not. Heparin 3 mg/ml, a potent inhibitor of inositol triphosphate receptor (InsP(3)R), did not inhibit the response, and caffeine 1 mM (the agonist for calcium-induced calcium release [CICR]) imitated the effect of hyposmotic swelling. Ryanodine (15 microM), markedly inhibited the effect. These results suggest that hyposmotic swelling activates I(K(Ca)), and the activation is associated with CICR, which is triggered by extracellular calcium influx through the stretch-activated channel (SA channel).     

Immunogenicity and Safety of Two Different Haemophilus influenzae Type b Conjugate Vaccines in Korean Infants

The incidence of invasive diseases, including meningitis caused by Haemophilus influenzae type b (Hib) was markedly decreased after routine immunization of Hib vaccine through diverse schedules in many countries. The purpose of this study was to evaluate the immunogenicity and safety of Hib conjugate vaccines in Korean children before the implementation of a national immunization program against Hib in Korea. A multicenter controlled trial was performed on two different Hib vaccines in Korean children. A total of 319 infants were enrolled: 199 infants were immunized with the Hib polysaccharide conjugated to the tetanus toxoid (PRP-T) and 120 infants with the Hib polysaccharide conjugated to the outer-membrane protein of Neisseria meningitides (PRP-OMP). Immunogenicity was evaluated by enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay. Both vaccines showed good immunologic responses after primary immunization. After 2 doses of PRP-T or PRP-OMP, 78.9% and 91.7% of infants achieved an antibody level of ≥1.0 µg/mL, respectively. Both vaccines were safe and well-tolerated. No serious adverse events were observed. Thus, Hib conjugate vaccines appear to be safe and show good immunogenicity in Korean infants. These results will be important reference data for the implementation of Hib vaccine in the national immunization program of Korea.     

Prokaryotic expression, purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli

AIM: To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein. METHODS: The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.coli expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E.coli BL21 (DE3) pLysS and induced to express fusion protein VEGFR2 D3.4/GST with IPTG. The expressed protein was purified by washing in urea and detected by SDS-PAGE and Western blot. RESULTS: SDS-PAGE analysis showed that a novel protein with the expected molecular mass (M(r);) about 46 000 was expressed with the inducement of IPTG. And it existed mostly in the form of inclusion body. Grayscale scanning showed that the expressed VEGFR2 D3.4/GST fusion protein accounted for 38.6% of the total bacterium protein. After the purified product was washed by urea, its purity reached 87.1%. Western blot confirmed the recombinant protein was VEGFR2 D3.4/GST fusion protein. CONCLUSION: High purification VEGFR2 D3.4/GST fusion protein is obtained through the E.coli expression system.