抗乙酰胆碱受体单链抗体对致病性抗体结合活性的抑制作用

为探讨单链抗体 (ScFv )对重症肌无力 (MG )的特异性免疫治疗作用 ,从分离自MG患者胸腺的抗乙酰胆碱受体(AchR )抗原结合片段Fab6 37构建单链抗体ScFv6 37。应用放射免疫测定法测定了ScFv6 37与刺激抗原人AchR的特异性结合活性以及与相关抗原大鼠AchR和电鳐AchR的交叉反应 ,应用竞争性ELISA测定抑制致病性抗AchR完整抗体IgG6 37与人AchR结合活性。结果发现ScFv6 37只能与人AchR结合 ,不能与大鼠和电鳐的AchR结合。对IgG6 37与人AchR结合的抑制率为 17 5 %～ 32 9%。表明单链抗体对AchR具有一定的“保护”作用。     

Major histocompatibility complex class I peptide presentation after Salmonella enterica serovar typhimurium infection assessed via stable isotope tagging of the B27-presented peptide repertoire

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.     

Development of a transformation system for Crinipellis perniciosa,the causal agent of witches' broom in cocoa plants

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.     

Heparin-interacting sites of bovine lactoferrin are involved in anti-adenovirus activity

Lactoferrin, a member of the transferrin family of approximately 80 kDa, consists of a single polypeptide chain folded in two symmetric, globular lobes (N- and C-lobes), each able to bind one ferric ion. This glycoprotein, found in physiological fluids of mammals, plays an important role in immune regulation and in defense mechanisms against bacteria, fungi, parasites, and viruses. Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate. We have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin antiviral effect toward adenovirus infection in HEp-2 cells. The results obtained demonstrated that the anti-adenovirus activity of lactoferrin is mediated by the N-terminal half of the protein as the N-lobe was able to inhibit adenovirus infection, even if at lower extent than undigested lactoferrin, whereas C-lobe was ineffective. The results also showed that the anti-adenovirus action of lactoferrin and of its N-terminal peptide lactoferricin took place on virus attachment to cell membrane, mainly through competition for common glycosaminoglycan receptors. The data provide evidence that the anti-adenovirus activity of lactoferrin is mediated mainly by the cluster of positive charges at the N-terminus of whole molecule and that the N-terminal peptide lactoferricin alone is sufficient to prevent infection.     

Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.     

The α1β1 and α2β1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.     

Postnatal changes in membrane properties of mice trigeminal ganglion neurons

Intracellular recordings from neurons in the mouse trigeminal ganglion (TG) in vitro were used to characterize changes in membrane properties that take place from early postnatal stages (P0-P7) to adulthood (>P21). All neonatal TG neurons had uniformly slow conduction velocities, whereas adult neurons could be separated according to their conduction velocity into Adelta and C neurons. Based on the presence or absence of a marked inflection or hump in the repolarization phase of the action potential (AP), neonatal neurons were divided into S- (slow) and F-type (fast) neurons. Their passive and subthreshold properties (resting membrane potential, input resistance, membrane capacitance, and inward rectification) were nearly identical, but they showed marked differences in AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and afterhyperpolarization (AHP) duration. Adult TG neurons also segregated into S- and F-type groups. Differences in their mean AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and AHP duration were also prominent. In addition, axons of 90% of F-type neurons and 60% of S-type neurons became faster conducting in their central and peripheral branch, suggestive of axonal myelination. The proportion of S- and F-type neurons did not vary during postnatal development, suggesting that these phenotypes were established early in development. Membrane properties of both types of TG neurons evolved differently during postnatal development. The nature of many of these changes was linked to the process of myelination. Thus myelination was accompanied by a decrease in AP duration, input resistance (R(in)), and increase in membrane capacitance (C). These properties remained constant in unmyelinated neurons (both F- and S-type). In adult TG, all F-type neurons with inward rectification were also fast-conducting Adelta, suggesting that those F-type neurons showing inward rectification at birth will evolve to F-type Adelta neurons with age. The percentage of F-type neurons showing inward rectification also increased with age. Both F- and S-type neurons displayed changes in the sensitivity of the AP to reductions in extracellular Ca(2+) or substitution with Co(2+) during the process of maturation.     

明胶-聚乳酸载药纳米微球的制备及其体外释药研究

采用复合乳液—溶剂挥发法制得明胶—聚乳酸载五氟脲嘧啶(5—Fu)微球，以混合型乳化剂Tween—80：Span—80＝5：1—作为初乳乳化剂，O—羧甲基壳聚糖作为复乳乳化剂，考察了明胶—聚乳酸载药微球的制备条件对微球的成球性、药物包封率及体外释药的影响。结果表明乳化剂的选择、内部水相药物浓度和PLA分子量等均对载药微球的结构与性能产生影响，经优化条件得到了成球性和体外释放都比较好的载药微球。     

Genetic and phylogeographic structure of populations of<Emphasis Type="Italic"> Pulex simulans</Emphasis> (Siphonaptera) in Peru inferred from two genes (<Emphasis Type="Italic">CytB</Emphasis> and<Emphasis Type="Italic"> CoII</Emphasis>)

In this paper we discuss the potential usefulness of determining the phylogeographic and phylogenetic patterns of a vector for understanding the spread of pathogens or insecticide resistance. We do so using the example of Pulex simulans in Peru. Six populations from six different localities were investigated. Mitochondrial DNA sequences were obtained and branching patterns were inferred using phylogenetic reconstruction methods and nested clade analyses. Ten different haplotypes were discovered. Phylogenetic analysis revealed P. simulans in Peru as a monophyletic group, containing clades that were generally not geographically correlated. The data suggest that P. simulans is not a single genetic entity but rather that this species shows a high degree of intraspecific variation. Restricted gene flow with long distance dispersal coupled with range expansion and long distance colonization are likely to have contributed to the observed patterns of variation.