Pilot study of an HLA-A2 peptide vaccine using flt3 ligand as a systemic vaccine adjuvant

A pilot vaccine study was conducted to test the safety and immunological efficacy of four monthly immunizations of an MHC class I peptide vaccine, the E75 HLA-A2 epitope from HER-2/neu, using flt3 ligand as a systemic vaccine adjuvant. Twenty HLA-A2-expressing subjects with advanced stage prostate cancer were randomly assigned to one of four immunization or treatment schedules: (a) Flt3 ligand (20 g/kg per day) administered subcutaneously daily for 14 days on a 28-day cycle, monthly for four months; (b) flt3 ligand course as above with the E75 peptide vaccine administered on day 7 of each flt3 ligand cycle; (c) flt3 ligand course as above with the E75 peptide vaccine administered on day 14 of each flt3 ligand cycle; or (d) E75 peptide admixed with granulocyte–macrophage colony-stimulating factor and administered intradermally once every 28 days, as has previously been reported. The primary endpoints of the study were the determination of safety and immunological efficacy in generating E75-specific T cells as determined by peptide-specific interferon-gamma ELIspot. Adverse events included one grade 3 skin reaction and the development of grade 2 autoimmune hypothyroidism in two subjects with preexisting subclinical autoimmune hypothyroidism. Dendritic cells were markedly increased in the peripheral blood of subjects receiving flt3 ligand with each repetitive cycle, but augmentation of antigen-presenting cells within the dermis was not observed. Apart from a single subject, no significant peptide-specific T-cell responses were detected by ELIspot, whereas delayed-type hypersensitivity responses were detectable in control subjects and in subjects receiving peptide vaccine early in the course of flt3 ligand administration. The absence of robust peripheral immune responses in the current study may be attributable to the small numbers of subjects or differences in the subject population. In addition, the inability of flt3 ligand to augment the number of peripheral skin antigen-presenting cells may have contributed to the absence of robust peptide-specific immunity detectable in the peripheral blood of immunized subjects treated with flt3 ligand.     

Deletions in the ligand for CD40 in X-linked immunoglobulin deficiency with normal or elevated IgM (HIGMX-1)

Patients with X-linked Ig deficiency with normal or elevatedIgM (HIGMX-1) fail to switch from IgM/IgD to other Ig isotypes.Interaction between the B cell antigen CD40 and the CD40 ligandexpressed on activated T cells is critical for T cell drivenisotype switching. We have reported that T lymphocytes fromthree unrelated male patients with HIGMX-1 failed to expressCD40 ligand on their surface, but the mRNA for CD40 ligand wasof an apparently normal size and level. Analysis of CD40 ligandcDNA from two of the patients revealed deletions that alterthe reading frame. Patient 1 displayed two mutations: a C Atransversion at nucleotide 590 and the deletion of an adjacentC nucleotide. The second patient had a 58 bp deletion from nucleotides289–346. Furthermore, neither patient expressed a proteinproduct detectable by the CD40L mAb, 5c8.     

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins in the cyst fluid of a patient with astrocytoma

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins (IGFBPs) were demonstrated in the cyst fluid of a patient with a hypothalamic astrocytoma. The astrocytoma cyst fluid was subjected to gel chromatography at low pH and the IGF-I and IGF-II levels were measured by specific radioimmunoassays. Immunoreactive IGF-I and IGF-II levels were 19 ng/ml and 78 ng/ml respectively. Several-fold higher IGF-II values were obtained when cyst fluid was not extracted or was extracted with acid ethanol before radioimmunoassay analysis. The immunoreactive IGFBP-1 concentration was 26 ng/ml. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western ligand blotting with [¹²⁵I]IGF-II revealed bands at 200, 34.5, 29.5, 24 and 21 kD as visualized by autoradiography. Binding studies demonstrated that these binding proteins bind specifically [¹²⁵I]IGF-I and [¹²⁵I]IGF-II. These observations suggest that IGFs as well as IGF-binding proteins are produced by astrocytoma cells and may act in a paracrine or autocrine fashion capable of modulating the growth of astrocytoma tumours.     

Role of phloretin as a sensitizer to TRAIL-induced apoptosis in colon cancer

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, −8 and −9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.     

缺氧诱导因子HIF-1α、3α在血管瘤不同时期的表达和意义

目的 探讨缺氧在血管瘤不同时期的表达和作用.方法 采用免疫组化SP法测定24例增生期血管瘤和18例消退期血管瘤中缺氧染色阳性率,HIF-1α、HIF-3α、VEGF、Ki-67和细胞凋亡表达情况.结果 24例增生期血管瘤中缺氧染色阳性率为80%(19/24),HIF-1α阳性指数为(23.40±4.73)、HIF-3α为(7.90±2.15)、VEGF为(16.90±3.34)、Ki-67为(57.60±11.33)、细胞凋亡指数为(4.50±1.51);而消退期血管瘤中缺氧染色阳性率为90%(16/18),HIF-1α为(9.50±2.67)、HIF-3α为(19.80±2.43)、VEGF为(2.70±0.32)、Ki-67为(11.20±2.65)、细胞凋亡指数为(11.40±2.67).不同时期血管瘤表达的HIF-1α、HIF-3α、VEGF、Ki-67、细胞凋亡指数均有显著性差别(P＜0.05).结论 缺氧是血管瘤不同时期的普遍现象,但对增殖期血管瘤的作用是通过HIF-1α促进内皮细胞繁殖,而对消退期血管瘤是通过HIF-3α促进其凋亡.     

低氧诱导因子-1α在成纤维细胞中的表达及对毛囊的影响

目的：探讨外源性人低氧诱导因子（HIF）-1α基因在成纤维细胞中的表达及对体外培养人毛囊的影响。方法：通过脂质体将含有HIF—1α cDNA的真核表达载体pcDNA3．0瞬时转染成纤维细胞,应用反转录（RT）-PCR、免疫组化等方法检测HIF-1α在成纤维细胞中的表达。同时通过ELISA检测转染细胞上清液中血管内皮生长因子（VEGF）的表达情况。进一步将该上清加至体外培养的人毛囊和细胞中,显微镜下测量毛囊的平均生长长度,并观察毛囊的形态学变化。并通过四甲基偶氮唑蓝（MTT）法检测加入转染上清的细胞活性。结果：成功地将HIF—1α真核表达载体HIF—1α pcDNA3．0瞬时转染了成纤维细胞,用RT-PCR、免疫组化染色可检测出细胞中HIF—1α的表达,MTT检测转染后细胞活性增强,并且该上清液可以检测出VEGF的表达。该上清可以明显促进体外培养的人毛囊生长．延缓其进入退行期。结论：应用脂质体能够成功地将外源性人HIF—1α基因转染成纤维细胞,并进行有效表达,其表达的HIF—1α可增强细胞活性,且可诱导转染细胞上清液中VEGF的表达,在体外具有促进人毛囊生长的生物学活性。     

人牙髓组织缺氧耐受机制的初步研究

目的：研究缺氧诱导因子-1α（Hypoxia inducible factor-1α,HIF-1α）和环氧化酶-2（Cyclooxygenase-2,COX-2）在人正常牙髓和炎症牙髓中的免疫定位,探讨缺氧耐受机制在牙髓炎病程中和牙髓自身修复过程中的作用和意义。方法：通过SP法对第1组10例健康牙髓、第2组10例深龋（有过敏症状但无牙髓炎症状）患牙牙髓、第3组15例急性牙髓炎牙髓和第4组15例慢性牙髓炎牙髓进行免疫组化染色,分别对HIF-1α和COX-2进行免疫定位和半定量分析。结果：①HlF-1α在第1组健康牙髓和第2组深龋牙髓标本中,只有个别标本呈弱阳性表达,二组问无显著性差异（P〉0．05）,但对比其它二组标本则有显著性差异（P〈0．001）：第3组急性牙髓炎牙髓呈强阳性表达,第四组慢性牙髓炎牙髓亦呈阳性染色与第3组比较总体偏弱,有显著性差异（P〈0．05）。②COX-2在第1组牙髓中个别标本呈弱阳性表达;第2组标本绝大多数染色呈弱阳性,与第1组比较有显著性差异（P〈0.05）;第3组标本染色均呈强阳性,与前两组比较有显著差异性伊值分别为P〈0.001,P〈0．05）;第4组标本亦呈阳性染色,总体上阳性程度明显高于第l组（P〈0．001）,高于第2组（P〈0．05）,弱于第3组（P〈0．05）。HIF-1α与COX-2的表达关系主要在第3组和第4组牙髓标本中体现明显,在第3组二者的变化大致呈平行关系;在第4组基本呈反向变化关系。结论：HIF-1α和COX-2在牙髓炎病程中可能发挥着重要的生理和病理作用,在牙髓炎进程中和牙髓自身修复过程中可能存在着缺氧环境和缺氧耐受机制。     

Effect of Clopidogrel on Platelet Membrane CD40 Ligand in Coronary Artery Disease Patients Undertaking Percutaneous Coronary Intervention

Objectives To investigate the change and clinical significance of clopidogrel on platelet membrane CD40L in coronary artery disease patients before and after percutaneous coronary intervention(PCI). Methods 30 cases who were diagnosis coronary artery diseases(CAD) by coronary angiography, mean age 56±9 years old. All the patients who had no antiplatelet aggregation contraindication, were treated with standard anti angina pectoris drugs. Before PCI, all the patients took clopidogrel 75 mg per day. Activated platelet membrane CD40L express rate was measured by flow cytometry before and after PCI 6 hours. Results Activated platelet membrane CD40L express rate were 3.73±2.15 and 2.46±0.90, respectively in 30 patients before and after PCI 6 hours. Activated platelet membrane CD40L express rate was significantly decrease after PCI 6 hours than that before PCI(P<0.01). Conclusions Clopidogrel has significance effect on platelet membrane CD40L in coronary artery disease patients undergoing PCI. Clopidogrel can suppression platelet activation and prevent thromboembolism event occurrence.