Identification of the blood component responsible for increased susceptibility to gut-derived infection

It has previously been reported that the transfusion of allogeneic whole blood increases sepsis-related mortality and decreases the ability of the host to kill bacteria that have translocated from the intestinal tract. To determine which blood component contributes to this adverse effect, the impact of the transfusion of white cells (WBCs), red cells (RBCs), and plasma on microbial translocation, bacteria killing, and mortality rate was studied. Blood from C3H/HeJ mice was separated into WBCs, RBCs, and plasma, and these fractions were transfused to Balb/c mice. Controls received sterile saline. Five days after transfusion, all Balb/c mice underwent a 20-percent burn and gavage with 1 × 10(10) Escherichia coli labeled with 14C-glucose. Mortality was observed for 10 days. Four additional groups, receiving the same treatment as above, were sacrificed 4 hours after the burn, and mesenteric lymph nodes, liver, kidney, and blood were harvested aseptically. For each tissue, quantitative colony counts, radionuclide counts, and percentage of translocated bacteria that remained alive were calculated. By radionuclide counts, no difference was observed in the degree of 14C E. coli translocation among the groups. In contrast, the percentage of viable bacteria and the mortality rate were significantly higher in the group receiving allogeneic WBCs than in all other groups (p < 0.05). It is concluded that WBCs are the component in transfused blood that has an adverse effect on host resistance to gut- derived infection.     

Transfusion and cytomegalovirus-associated AIDS-defining disease in hemophiliacs. Hemophilia Malignancy Study

BACKGROUND: Whether transfusion increases the risk of AIDS-defining cytomegalovirus (CMV) infection (CMV AIDS) in immunosuppressed patients is not known. Because of concerns about the risk of transfusion transmission of CMV and potential exposure to multiple strains of CMV through transfusion, the National Hemophilia Foundation recently recommended that CMV-negative blood be used in human immunodeficiency virus-positive hemophiliacs, regardless of their CMV serologic status. Although the multiple strains of CMV cause different CMV disease manifestations in transplant recipients, there are no data on CMV disease in human immunodeficiency virus-positive hemophiliacs. STUDY DESIGN AND METHODS: It was hypothesized that if the transmission of CMV through transfusion causes CMV disease in human immunodeficiency virus- positive hemophiliacs, then hemophiliacs with CMV AIDS would be more likely to have received transfusions than those with AIDS-defining disease not caused by CMV (non-CMV AIDS). The number and type of transfusions were evaluated in 334 hemophiliacs with AIDS (35 with CMV AIDS and 299 with non-CMV AIDS) enrolled in the multicenter Hemophilia Malignancy Study. RESULTS: There were no differences between hemophiliacs with CMV AIDS and those with non-CMV AIDS in age, type, and severity of hemophilia; the proportion receiving transfusions; or the mean number of units transfused. These findings persisted after correction for transfusion practice, (i.e., CMV-unscreened blood vs. CMV-negative and/or white cell-reduced blood). There was no difference between the groups in CMV lgG titers or in the proportion who were CMV seropositive, and there was no difference between these parameters in those who had received transfusion(s) and those who had not. CONCLUSION: Transfusion appears to have little, if any, effect on the development of CMV AIDS or CMV lgG seroprevalence in patients with hemophilia.     

腺病毒介导人骨形态发生蛋白2基因在兔脂肪干细胞的表达与成骨分化效应

目的:采用腺病毒介导人骨形态发生蛋白2基因转染兔脂肪干细胞,观察目的基因在脂肪干细胞中的表达效率及向成骨细胞定向分化的能力。方法:实验于2005-12/2006-09在武汉大学医学院中心实验室和三峡大学医学院病理实验室进行。①实验材料:4月龄健康新西兰大耳白兔2只。质粒pAd-BMP-2由美国哈佛医学院分子骨科中心Oliver博士惠赠;携带β-半乳糖酐酶基因的腺病毒对照载体由李康博士惠赠;大肠杆菌DH5a以及293细胞由本实验室保存。②实验方法:新西兰兔肌注麻醉后,完整取出双侧腹股沟脂肪垫,清除外包膜、明显的结缔组织和小血管,剪碎,离心,体外分离培养脂肪干细胞。表达载体pAd-hBMP-2经293细胞包装重组腺病毒后,取第3代生长良好的脂肪干细胞,按5×105接种于60mm培养皿,将Ad-hBMP-2病毒以感染复数10～20感染细胞。将成功转染骨形态发生蛋白2的脂肪干细胞作为转染组,以转染携带β-半乳糖酐酶基因腺病毒载体的脂肪干细胞作为对照组。两组均置入不含骨形态发生蛋白2的无血清成骨诱导培养基中进行诱导分化。③实验评估:定期观察细胞的形态学变化,MTT法绘制生长曲线,计算增殖时间;成骨诱导培养后测定碱性磷酸酶活性,光倒置显微镜下观察钙化结节形成情况;RT-PCR、免疫组织化学法、Westernblot法检测目的基因人骨形态发生蛋白2和成骨细胞标志性蛋白Ⅰ型胶原、骨钙素的表达。结果:①脂肪干细胞转染前后形态学变化、生长曲线及倍增时间:Ad-hBMP-2基因修饰的脂肪干细胞经诱导培养后形态规则,多角型细胞增多,转染组的细胞生长曲线倍增时间明显短于对照组。②碱性磷酸酶活性及矿化结节形成:转染组脂肪干细胞碱性磷酸酶活性呈增加趋势,成骨诱导培养7,10,14d均显著高于未转染组(P<0.01)。转染组成骨细胞四环素标记显示圆形矿化结节呈金黄色,数量多,结节大。③脂肪干细胞人骨形态发生蛋白2与Ⅰ型胶原、骨钙素的表达:成骨诱导培养7,14d,RT-PCR、免疫组织化学法、Western blot法检测结果显示转染组目的基因人骨形态发生蛋白2、成骨标志性蛋白Ⅰ型胶原、骨钙素的分泌表达均明显强于对照组。结论:经Ad-hBMP-2基因修饰的兔脂肪干细胞在体外能定向分化为成骨细胞,且分化增殖能力强;目的基因骨形态发生蛋白2表达高、持续时间长。     

Resistance to activated protein C and factor V Leiden

Over the last four years, there has been an explosion of knowledge about APCr and factor V Leiden. However, there remain a considerable number of difficult clinical areas in which there are no clear answers. Undoubtedly, factor V Leiden is commonly found in association with venous thromboembolic disease in whatever manifestation, but equally it has an unusually high frequency in the general population. Only a small proportion of those that carry the mutation develop a thrombosis. It is estimated that only 6% of those that carry the mutation will develop a thrombosis over a 30-year period, whilst for antithrombin, Protein C or Protein S deficiency, this figure is nearer 60%. Particular areas of difficulty remain in relation to the use of the combined OCP and in the management of the asymptomatic carrier of the mutation in pregnancy. Although the scientific basis of APCr and factor V Leiden is well established, its natural history remains relatively poorly understood, probably as a consequence of its relative novelty. Despite the plethora of new data that have appeared, there remains much to be learnt about factor V Leiden and the APCr phenotype.      

可吸收生物材料聚羟基乙酸与滑膜细胞共同构建组织工程化腱鞘

目的:目前对于腱鞘的修复主要运用网腱膜、自体静脉和生物膜等腱鞘的替代品,其缺点是无生物学功能,不能达到真正意义上的结构与功能的重建。实验采用可吸收生物材料聚羟基乙酸与滑膜细胞共同构建组织工程化腱鞘,验证其可行性。方法:实验于2005-09/2007-03在上海市第九人民医院组织工程实验室完成。取Leghorn鸡滑膜腱鞘,体外消化培养获得滑膜细胞并大量扩增后,取第2代滑膜细胞与聚羟基乙酸生物材料复合,形成大小1.5cm×1.0cm,厚1.0mm左右的细胞生物学支架,体外培养6d后,将支架包绕在硅胶管上回植到裸鼠皮下及将支架直接回植于鸡爪原位腱鞘缺损处,以未接种细胞的单纯聚羟基乙酸包绕硅胶管为对照组,体内培养3,6周后取材观察。整个实验过程中对滑膜细胞及两种情况下构建形成的组织工程化腱鞘进行形态学和组织学观察。结果:①所获得的滑膜细胞分为A、B型滑膜细胞,A型滑膜细胞形态呈巨噬细胞样,B型滑膜细胞形态呈成纤维样,且随着滑膜细胞的扩增,A型滑膜细胞比例逐渐减少,到第2代时,基本全部为B型细胞。②在裸鼠皮下构建的组织工程化腱鞘第3周、第6周时有新生组织产生,且具有弹性,失去支撑后仍可保持形状,而单纯聚羟基乙酸对照组在第3周时只有少量新生组织,到第6周时新生组织消失。③在Leghorn鸡原位处构建的组织工程化腱鞘第3周时内壁光滑,与肌腱无粘连,第6周时形成与正常腱鞘相似鞘膜。结论:应用组织工程技术可以构建与正常组织相似的组织工程腱鞘。     

Monocytes and macrophages synthesize and secrete thrombospondin

Thrombospondin, one of the major glycoproteins released from alpha- granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate- polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.     

Changes in the plasma levels of type 1 and type 2 plasminogen activator inhibitors in normal pregnancy and in patients with severe preeclampsia

This report defines the nature of the molecules responsible for the increased plasma plasminogen activator inhibitor (PAI) activity in preeclamptic patients and the relationship of these inhibitors to the severity of placental damage in preeclampsia. Clinical groups consisting of pregnant women with either severe preeclampsia or chronic hypertension with superimposed severe preeclampsia, as well as normal pregnant and nonpregnant women, were analyzed in a panel of functional and immunologic assays for PAI-1 and PAI-2. Pure severe preeclamptic patients in their third trimester showed a significant increase in both antigenic (136 ng/mL) and functional (5.76 U/mL) type 1 PAI (PAI-1) as compared with normal third-trimester pregnant women (34.8 ng/mL and 2.57 U/mL, respectively). In contrast, antigenic (186 ng/mL) and functional (5.76 U/mL) levels of type 2 PAI (PAI-2) were significantly lower in the pure severe preeclampsia group as compared with the values of the normal pregnant group (269 ng/mL and 9.58 U/mL, respectively). The patients with chronic hypertension and superimposed severe preeclampsia exhibited PAI-2 levels comparable to those of the pure preeclamptic group, whereas their antigenic and functional PAI-1 levels were intermediate (94 ng/mL and 3.25 U/mL, respectively) between the normal pregnant and the pure preeclamptic groups. During early puerperium of both normal pregnant women and patients, plasma PAI-1 antigen and activity decreased within one day to approximately the levels detected in normal nonpregnant women, while PAI-2 levels remained elevated for over 11 days. Similar results were obtained in plasma samples obtained from citrated blood and blood collected with an anticoagulant/antiplatelet mixture, suggesting that increased PAI-1 levels in preeclamptic patients were not due to platelet activation in vitro. In preeclamptic patients, a positive correlation between birth weight and PAI-2 values was observed (r = .64, P less than .05), whereas birth weight was inversely correlated with both PAI-1 levels and total PAI activity (r = -.6, P less than .005 and r = -.76, P less than .005 respectively). Preeclamptic patients with extensive placental infarction exhibited higher plasma PAI activity (24.1 U/mL v 11.6 U/mL) and PAI-1 values (305 ng/mL v 80.9 ng/mL) than preeclamptic patients without extensive placental infarction. In contrast, PAI-2 levels were reduced in preeclamptic patients with infarction in comparison with those of patients without infarction (141 ng/mL v 212.9 ng/mL). Our data indicate that increases in the level of PAI-1 accounts for the high plasma PAI activity in severe preeclampsia as measured using single-chain t-PA.     

Dyspepsia and Helicobacter pylori in Japanese employees with and without ulcer history

In a Dutch working population, the apparent association between dyspeptic symptoms and Helicobacter pylori infection was found to be entirely due to subjects with an ulcer history. In general populations with a much higher prevalence of H. pylori infection and peptic ulcer disease, such as in Japan, the relationship between dyspepsia and H. pylori has yet to be clarified. A questionnaire on ulcer history and dyspeptic symptoms during the preceding 3 month period was obtained from apparently healthy Japanese employees who underwent a periodic medical examination. In addition, serum samples were analysed for anti-H. pylori IgG antibodies. A total of 196 men and 35 women, aged 23–71 years, participated in the study. Seven women (20%) and 49 men (25%) had a diagnosis of peptic ulcer disease. Among 41 subjects with verified duodenal (26) and/or gastric (17) ulcer, 95% were H. pylori positive while 32% had had frequent dyspeptic symptoms in the 3 months prior to the study (29% of the 35 men and 50% of the 6 women). Among the 147 men and 28 women without an ulcer history, the 3 month period prevalence of frequent dyspepsia was 14 and 32%, respectively. The rate of H. pylori positivity was 80% in non-ulcer dyspeptics and 68% in all other non-ulcer subjects (95% confidence intervals: 61–92 and 61–76%, respectively). Significant differences in symptoms between H. pylori positive and negative subjects could not be detected, neither in the whole population nor in the non-ulcer group. In conclusion, in this Japanese working population, no association was found between dyspeptic symptoms and H. pylori infection, irrespective of the inclusion of subjects with a peptic ulcer history.     

Adequacy of diagnostic tests and surgical management of symptomatic invasive lobular carcinoma of the breast

Introduction

Invasive lobular carcinoma (ILC) presents diagnostic and therapeutic challenges as it produces subtle radiological changes. It has been suggested that it is not suitable for breast conserving surgery (BCS). The aim of this study was to ascertain the diagnostic adequacy of modern mammography and ultrasonography in the context of a fast track symptomatic diagnostic clinic in the UK. It also sought to compare the mastectomy, re-excision and BCS rates for ILC with those for invasive ductal carcinoma (IDC).

Methods

A retrospective analysis of prospectively collected data was carried out on all new symptomatic cancers presenting to the one-stop diagnostic clinic of a single breast unit between 1998 and 2007.

Results

Compared with IDC, ILC was significantly larger at presentation (46mm vs 25mm), needed re-excision after BCS more often (38.8% vs 22.3%) and required mastectomy more frequently (58.8% vs 40.8%). Although mammography performs poorly in diagnosing ILC compared with IDC, when combined with ultrasonography, sensitivity of the combined imaging was not significantly different between these two histological types.

Conclusions

Provided ultrasonography is performed, standard radiological imaging is adequate for initial diagnosis of symptomatically presenting ILC but some additional preoperative workup should clearly be employed to reduce the higher number of reoperations for this histological type.     

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.