长链核酸扩增技术评价病毒灭活效果的研究

目的:探讨用长链核酸扩增技术评价病毒灭活效果的可行性.方法:针对伪狂犬病毒(pseudorabies virus,PRV)糖蛋白gD基因前后的保守区设计预计产物长短不一的5对引物,用半巢式PCR技术扩增经低pH法(4.0±0.1)、巴氏消毒法[(60±1.0)℃]和s/D法(有机溶剂/洗涤荆)处理后的PRV核酸,同时以细胞感染法做平行对照.结果:低pH对PRV核酸有破坏作用,处理时间越长,核酸损伤程度越明显,处理60 min时,6.62 lgTCID50的PRV完全被灭活.5条不同长度PCR扩增产物中,只有3.9 kb的长片段检出与细胞培养结果一致.7.25 lgTCID50的PRV经(60±1.0)℃处理20 min后即被完全灭活,7.13 lgTCID50的PRV经s/D法处理1 h后被完全灭活,但各长度核酸片段扩增均为阳性,与细胞感染试验结果不符.结论:低pH对PRV核酸的损伤程度随处理时间的延长而增加;用长链PCR(3.9 kb)技术来评价经低pH法灭活病毒的效果是可行的,而该法不适合评价巴氏消毒法和S/D法灭活病毒的效果.     

Clinical Impact of Community-Acquired Respiratory Viruses on Bronchiolitis Obliterans After Lung Transplant

Community-acquired viral respiratory tract infections (RTI) in lung transplant recipients may have a high rate of progression to pneumonia and can be a trigger for immunologically mediated detrimental effects on lung function. A cohort of 100 patients was enrolled from 2001 to 2003 in which 50 patients had clinically diagnosed viral RTI and 50 were asymptomatic. All patients had nasopharyngeal and throat swabs taken for respiratory virus antigen detection, culture and RT-PCR. All patients had pulmonary function tests at regular intervals for 12 months. Rates of rejection, decline in forced expiratory volume (L) in 1 s (FEV-1) and bacterial and fungal superinfection were compared at the 3-month primary endpoint. In the 50 patients with RTI, a microbial etiology was identified in 33 of 50 (66%) and included rhinovirus (9), coronavirus (8), RSV (6), influenza A (5), parainfluenza (4) and human metapneumovirus (1). During the 3-month primary endpoint, 8 of 50 (16%) RTI patients had acute rejection versus 0 of 50 non-RTI patients (p=0.006). The number of patients experiencing a 20% or more decline in FEV-1 by 3 months was 9 of 50 (18%) RTI versus 0 of 50 non-RTI (0%) (p=0.003). In six of these nine patients, the decline in FEV-1 was sustained over a 1-year period consistent with bronchiolitis obliterans syndrome (BOS). Community-acquired respiratory viruses may be associated with the development of acute rejection and BOS.     

Workshop on Late Renal Allograft Dysfunction

Despite continued improvement in incidence of acute immune injury and short-term graft survival, late allograft dysfunction remains a significant problem in the renal transplant population. Recent reports suggest that rates of renal function decline are quite varied in the overall recipient population, and that individual rates for many recipients may not change substantially over time. Moreover, analyses also reveal distinct predictive factors for both early and late functional decline. Long-term outcome studies for renal transplantation, however, might be significantly limited by incomplete data sets for assessing clinical endpoints. In view of the heterogeneous factors that may cause progressive allograft injury, more routine biopsy sampling would allow a more complete characterization of induced injuries. Elucidating mechanisms of renal fibrosis in response to injury, in experimental systems and humans, is also an important goal in better understanding chronic allograft damage. Regulation of cell senescence genes and epithelial to mesenchymal transition, studied in other models of renal fibrosis, are likely relevant to studies of renal allograft dysfunction. Recent technical advances in analyzing biological samples may play a pivotal role in identifying and validating surrogate markers of allograft function for future interventional trials in transplantation.     

Mapping the rat somatosensory pathway from the anterior cruciate ligament nerve endings to the cerebrum

The anterior cruciate ligament (ACL) serves as the primary restraint to anterior tibial translation. In addition to this biomechanical function, the ACL appears to have a function in neuromuscular control. This hypothesis was formulated after the discovery of mechanoreceptors within the ACL. The full somatosensory pathway from the ACL to the cerebrum has yet to be elucidated. In order to map this sensory pathway, we conducted a viral trans-synaptic tracing experiment using the neurotropic pseudorabies virus (PRV). The pseudorabies virus was injected into the ACL of rats and allowed to replicate and spread trans-synaptically for 6-7 days. The brain and spinal cord of each sacrificed rat was then removed and processed immunohistochemically to detect the presence of PRV. PRV-immunoreactive neurons were found to be localized in several different regions from the spinal cord to the cerebrum. Four nuclei in the reticular formation of the brain stem demonstrated strong positive labeling: the mesencephalic reticular nucleus, magnocellular reticular nucleus, paragigantocellular reticular nucleus, and gigantocellular reticular nucleus. This finding suggests that the nerve endings of the rat ACL project into the cerebrum and that the reticular formation may play an important role in the afferent pathway of those nerve endings.     

Human Herpesvirus 6 in Bronchalveolar Lavage Fluid after Lung Transplantation: A Risk Factor for Bronchiolitis Obliterans Syndrome?

Bronchiolitis obliterans syndrome (BOS) is the limiting factor to long-term survival after lung transplantation. Previous studies suggested respiratory viral tract infections are associated with the development of BOS. To identify the impact of virus detection in bronchoalveolar lavage (BAL) fluid, we analyzed BAL samples from 87 consecutive lung transplant recipients for human herpesvirus (HHV)-6, Epstein-Barr virus, Herpes simplex virus 1/2, Cytomegalovirus, respiratory syncytical virus and adenovirus by PCR. Acute rejection, BOS and death were recorded for a mean follow-up time of 3.27 +/- 0.47 years. Results of PCR analysis and other potential risk factors were entered into a Cox regression analysis of BOS predictors and death. Only acute rejection was a distinct risk factor for BOS of all stages, death and death from BOS. HHV-6 was detected in 20 patients. Univariate and multivariate analysis revealed that HHV-6 was associated with an increased risk to develop BOS > orb = stage 1 and death, separate from the risk attributable to acute rejection. Identification of HHV-6 DNA in BAL fluid is a potential risk factor for BOS. Our results warrant further studies to elucidate a possible causal link between HHV-6 and BOS.     

弥漫性大B细胞淋巴瘤中CD40L的表达及意义

目的探讨弥漫性大B细胞淋巴瘤（DLBCL）组织中CD40L表达与DLBCL预后间的关系及意义。方法免疫组织化学法检测27例弥漫性大B细胞淋巴瘤、20例淋巴结反应性增生组织中CD40L的表达。结果（1）DLBCL中CD40L过度阳性率（25．93％）显著低于淋巴结反应性增生（63．64％），P〈0．05。（2）CD40L在Ⅲ、Ⅳ期DLBCL过度阳性率（14．29％）低于Ⅰ、Ⅱ期（38．46％），P〈0．05。CD40L过度阳性率在有结外浸润DLBCL（11．76％）低于无有结外浸润DLBCL（40％），P〈0．05。（3）DLBCL患者CD40L的过度阳性率与远处转移、临床分期均显著相关，P〈0．05。结论（1）CD40L过度阳性率与结外器官浸润及临床分期密切相关，其可能作为判断DLBCL侵袭性及预后的指标。（2）DLBCL中CD40L表达的减少可能是影响其发病的因素之一。     

Biological properties of interleukin-12 and its therapeutic use in persistent hepatitis B virus and hepatitis C virus infection

Summary. Interleukin (IL)-12 is a pleiotropic cytokine produced by antigen-presenting cells in response to diverse stimuli. IL-12 is a key molecule in the regulation of host's immune responses. In particular, IL-12 influences the balance between the T-helper cells type 1 (TH₁) and type 2 (TH₂); it modulates macrophage responses through the control of interferon-gamma synthesis by TH₁ cells; and, suppresses IgE class antibody production (has a suppressive effect on allergic reactions) and promotes a shift in the IgG subclasses. IL-12 enhances resistance to several infectious diseases, is a powerful antitumor agent in vivo , and acts as a vaccine adjuvant. The biological properties of IL-12 point to the potential therapeutic use in persistent hepatitis B virus and hepatitis C virus infection.     

医院用血液制品HCV污染情况的调查

目的调查医院治疗用血液制品ＨＣＶ污染情况．方法临床治疗所用的血液制品，在输完后留容器之残液备检．采用酶标免疫法（ＥＬＩＳＡ）检测．对ＥＬＩＳＡ检测之阳性标本及在阴性标本中随机抽取９份，采用逆转录双ＰＣＲ方法检测ＨＣＶ＿ＲＮＡ．结果血浆白蛋白抗＿ＨＣＶ阳性率４５８％，新鲜血浆阳性率２６％．两种方法检测同一标本，ＥＬＩＳＡ检测阳性的血液制品中，用ＰＣＲ证实，并非都有病毒复制；而在ＥＬＩＳＡ检测的阴性标本中，仍旧有较高的ＰＣＲ阳性检出．结论血液制品存在着ＨＣＶ污染，抗＿ＨＣＶ对于ＨＣＶ感染的诊断价值很高．检测血液制品中的抗＿ＨＣＶ，合理使用血液成份，可防止ＨＣＶ在医院内传播．     

Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.