中老年男性692例血清前列腺特异性抗原检测分析

目的:探讨中老年男性血清前列腺特异性抗原(PSA)浓度与年龄、生活习惯的关系.方法:以692例中老年男性为研究对象,取样后分离血清,采用双抗体夹心法,根据标准曲线计算出样品的浓度;生活习惯采取问卷调查的方式.结果:血清PSA浓度随年龄的增长逐渐升高,并且与生活习惯有一定关系. 结论:血清PSA浓度与年龄呈正相关,一些不良生活习惯可使血清PSA升高.     

Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies

In the autoimmune disease myasthenia gravis (MG), antibodies against the muscle nicotinic acetylcholine receptor (AChR) cause loss of functional AChR in the neuromuscular junction. To isolate AChR-specific human antibody fragments (Fab), a phage-display library was constructed from an MG patient's thymic B lymphocytes. The first Fab isolated had a low affinity for human AChR, but two sequential antibody chain shufflings using the MG donor heavy and light chain gene repertoires resulted in isolating two new Fab with an approximately 30-fold higher binding ability. The selected Fab contained extensively mutated heavy and light chains and probably represent intraclonal variants of a common progenitor having diverged in vivo by somatic hypermutation. Interestingly, the isolated Fab bound to an extracellular highly immunogenic region located either on an alpha-subunit site affected by the gamma/epsilon-subunits or on the interface between alpha- and gamma/epsilon-subunits. This region is not the previously described "main immunogenic region" (MIR), although it seems to be close to it, as one improved Fab and an anti-MIR mAb competed for AChR binding with distinctly different subpopulations of MG sera. Furthermore, this Fab protected surface AChR in cell cultures against MG autoantibody-induced antigenic modulation, suggesting a potential therapeutic use in MG, especially in combination with a human anti-MIR Fab.     

Development of a cluster of differentiation antibody-based protein microarray

Protein microarrays combine aspects of DNA microarrays and ELISA for the parallel interrogation of a biological sample using a multiplex of protein biomarkers. Here we report the development of a protein microarray consisting of a subset of CD antibodies and CRP. Several preparations (culture supernatant, ascites fluid and purified Ig) of each antibody were used in a forward phase protein microarray. Microarrays were fabricated using a non-contact printer delivering 300 pL (+/-30 pL) to specific locations on polyacrylamide gel-based substrates. Following production, microarrays were blocked for non-specific binding and incubated with sera conjugated directly with Cy3. Using CRP as a control biomarker, 12 clinical samples (inflammatory conditions and controls) were interrogated using the protein microarray format and results compared to CRP measured by conventional immunoassay. The data obtained from the microarray correlated with CRP assessed by immunoassay. Subsequently CRP 'positive' samples were interrogated for CD antigen expression; which revealed CD25 and CD45RO expression in all samples. Whilst this study focussed on a subset of CD antibodies, it is anticipated that this array could be expanded to include a larger number of CD antibodies and allow screening of sera from multiple conditions in order to identify disease markers.     

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.     

树突状细胞体外刺激对HBV特异性细胞毒T细胞影响的研究

目的探讨通过聚肌胞体外作用树突状细胞后改善慢性乙型肝炎患者树突状细胞功能,并在体外活化自身T细胞获得高频数HBV特异性细胞毒性T细胞(CTL)的方法。方法分离慢性乙型肝炎病人外周血单个核细胞,在粒巨噬细胞集落刺激因子(GM CSF)和白细胞介素4(IL4)的诱导下培养树突状细胞。培养的第7天加入聚肌胞刺激获得成熟树突状细胞,经HBVcore1827肽负载后与自身T淋巴细胞共培养,通过酶联斑点计数法(Elispot)及MHC肽四聚体法(Tetramer)比较病人T细胞未经自身树突状细胞刺激组、经HBVcore1827肽负载的自身树突状细胞刺激组、经聚肌胞促成熟的HBVcore1827肽负载的自身树突状细胞刺激组中HBV特异性的CTL的功能和频数。结果慢性乙型肝炎病人外周血单核细胞体外经GM CSF和IL4诱导可转化为树突状细胞,树突状细胞转化过程中聚肌胞的刺激可显著上调树突状细胞表面分子CD80、CD83的表达(P<0.01),促进树突状细胞的成熟。Elispot法检测分泌IFNγ的CTL的频数病人T细胞未经自身树突状细胞刺激组分泌频数为(9～28)/1×105T细胞,均值16;经HBVcore1827肽负载的自身树突状细胞刺激组频数为(30～67)/1×105T细胞,均值为46;经聚肌胞促成熟的HBVcore1827肽负载的自身树突状细胞刺激组频数为(59～130)/1×105T细胞,均值为98。三组数据     

Expression of peroxisome proliferator-activated receptors in the liver of gray mullet (Mugil cephalus)

During the last decade, peroxisome proliferation has emerged as a novel biomarker of exposure to certain organic chemical pollutants in aquatic organisms. Peroxisome proliferation is mediated by nuclear receptors, peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described in mammals: PPAR alpha, PPAR beta and PPAR gamma. PPARs have also been discovered in several fish species. The aim of the present study was to investigate the expression of PPAR subtypes and their cellular distribution patterns in the liver of gray mullet Mugil cephalus, a fish species widely distributed in estuaries and coastal areas in Europe and used as sentinel of environmental pollution. For this purpose, antibodies were generated against the three subtypes of mouse PPARs and different protocols of antigen retrieval were used. In western blots, main bands were detected of approximately 44 kDa for PPAR alpha, two bands of 44 and 58 kDa for PPAR beta and a single band of 56 kDa for PPAR gamma. Similar results were obtained in mouse liver and may indicate antibody recognition of two forms of the protein in certain cases. PPAR alpha was the subtype most markedly expressed in gray mullet liver, being expressed mainly in melanomacrophages, nuclei of hepatocytes and sinusoidal cells and connective tissue surrounding bile ducts. PPAR beta was expressed in the same cell types but immunolabeling was generally weaker than for PPAR alpha. PPAR gamma showed very weak expression; positivity was mainly found in melanomacrophages and connective tissue surrounding bile ducts. Our results demonstrate that all the three PPAR subtypes are expressed in gray mullet liver but in different intensities. The cellular distribution patterns of PPAR subtypes in gray mullet liver resembled partly those found in mouse liver with PPAR alpha as the main subtype expressed in hepatocytes. The fact that melanomacrophages, cells of the immune system in fish, show strong expression of both PPAR alpha and PPAR beta whereas PPAR gamma expression is almost restricted to this cell type suggest a significant role of PPAR-mediated regulation of cell function in melanomacrophages.     

Increased islet antigen presentation leads to type-1 diabetes in mice with autoimmune susceptibility

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is frequently used in preclinical and clinical protocols to modulate autoimmune responses, bone marrow transplants, and recovery from immune ablative therapies. The immunological outcome of such therapies is not fully understood. We tested the hypothesis that GM-CSF would enhance the maturation of antigen-presenting cells, facilitating presentation of beta-cell autoantigens to autoreactive T cells. We found that islet expression of GM-CSF greatly enhanced disease in male mice. Islet-derived APC but not splenic APC showed markedly enhanced capacity to stimulate in vitro proliferative responses of islet-antigen-specific autoreactive T cells. In vivo transfer of CD8(+) and CD4(+) T cells demonstrate that autoreactive T cells undergo extensive division in pancreatic lymph nodes of GM-CSF-transgenic mice compared with wild-type NOD male mice. Together, the results presented here demonstrate that expression of GM-CSF in the pancreas can enhance autoimmunity in disease-susceptible mice.     

Malignant gastrointestinal stromal tumor of the gallbladder

Gastrointestinal stromal tumors (GISTs) of the gallbladder are representative of an extremely rare group of tumors. We have encountered a patient with a malignant GIST of the gallbladder and presented it with a review of some articles. A 72-yr-old woman initially presented with right upper quadrant abdominal pain, fever and chills. Emergency cholecystectomy was performed under the impression of gallbladder empyema. Liver metastasis was found at 7 months postoperatively and the patient expired 9 months after the surgery. At the time of cholecystectomy, the gallbladder showed a necrotic serosal surface with an irregular thickened wall. A mass, 6 cm in length and 3 cm in width, encircled the whole wall of the neck and upper body of the gallbladder. Microscopic findings revealed frequent mitotic figures (more than 20/50 HPF) and tumor necrosis. Hyperchromatic, pleomorphic and spindle shaped neoplastic cells that were arranged in a pattern of short fascicles infiltrated the entire layer of the gallbladder. The tumor cells were immunoreactive for CD117 antigen (c-kit protein) and vimentin. They were negative for desmin, smooth muscle actin and S-100 protein. Mutations of the c-kit proto-oncogene were not found in this case. These findings were sufficient to provide enough clinical, histopathological and immunohistochemical evidence in diagnosing our case as a malignant GIST.     

细胞因子E-cadherin、CD34在肝细胞肝癌患者预后评价中的价值

目的研究肝细胞肝癌（hepatocellular carcinoma,HCC）组织中上皮钙黏蛋白（E-cadherin）和血管内皮因子CD34的表达情况,探讨二者之间及与临床病理之间的关系及其对HCC患者预后的评价.方法应用免疫组织化学PV-6000两步法检测41例HCC组织标本中E-cadherin和CD34的表达,并与临床病理学指标和术后生存期进行比较分析.结果41例HCC组织中E-cadherin和CD34表达阳性率分别为48.8%和100%.E-cadherin蛋白的表达在大肝癌、侵袭性高危组和癌组织Edmondson分级Ⅲ、Ⅳ级的HCC患者中明显降低（x^2=4.1881、4.8118、6.2695;P均＜0.05）.E-cadherin表达阴性的患者肝切除术后2年复发率较阳性表达组明显增加,5年生存率明显降低.CD34的表达与HCC患者年龄、侵袭性呈显著负相关（t=1.9371、1.9010;P＜0.05）,CD34阳性表达[微血管密度（MVD）≤40]组的2年复发率和5年生存率与MVD＞40组相比,差异无统计学意义.E-cadherin与CD34的表达不相关.结论HCC组织中E-cadherin表达阴性的患者预示着较差的预后,CD34的阳性表达与肿瘤复发和患者生存率无相关关系,HCC组织中E-cadherin和CD34的共同表达无相关性.     

TAP and TAP-like — Brothers in arms?

The transporter associated with antigen processing like (TAPL, ABCB9) is a member of the ATP-binding cassette (ABC) transporter family. Moreover, TAPL belongs to the TAP family due to its high sequence homology to TAP1 and TAP2. TAPL forms a homodimer which is localized in lysosomes with a minor fraction in the ER. It functions as an ATP-dependent peptide transporter which shows a broad peptide specificity ranging from 6-mer up to 59-mer peptides. In contrast to TAP, TAPL transports peptides with low affinity but high efficiency. This review will briefly summarize current knowledge about the structural organization and possible physiological function of TAPL in antigen processing and presentation.