High-resolution copy number analysis of paraffin-embedded archival tissue using SNP BeadArrays

High-density SNP microarrays provide insight into the genomic events that occur in diseases like cancer through their capability to measure both LOH and genomic copy numbers. Where currently available methods are restricted to the use of fresh frozen tissue, we now describe the design and validation of copy number measurements using the Illumina BeadArray platform and the application of this technique to formalin-fixed, paraffin-embedded (FFPE) tissue. In fresh frozen tissue from a set of colorectal tumors with numerous chromosomal aberrations, our method measures copy number patterns that are comparable to values from established platforms, like Affymetrix GeneChip and BAC array-CGH. Moreover, paired comparisons of fresh frozen and FFPE tissues showed nearly identical patterns of genomic change. We conclude that this method enables the use of paraffin-embedded material for research into both LOH and numerical chromosomal abnormalities. These findings make the large pathological archives available for genomic analysis, which could be especially relevant for hereditary disease where fresh material from affected relatives is rarely available.     

Oestrogen modulates cardiac ischaemic remodelling through oestrogen receptor-specific mechanisms

Aim: Observational and clinical studies suggest different responses upon sex hormone replacement therapy in ischaemic heart disease. Few studies, however, have examined the impact of oestrogen receptor‐dependent mechanisms on the extent of injury after myocardial infarction (MI). Therefore, we set out to evaluate the effect of oestrogen (E2) replacement on infarct size and remodelling, and the respective role of the oestrogen receptors (ER)α and ‐β in this process, using ERα‐ and ERβ‐deficient mice. Methods: Wild type (WT) (ERα^+/+ and ERβ^+/+), ERα‐deficient (ERα^−/−) and ERβ‐deficient (ERβ^−/−) mice were ovariectomized and subsequently supplemented with E2 or placebo using subcutaneous 60‐day release pellets. MI was induced by left coronary artery ligation. Two weeks following MI, haemodynamic function was assessed and infarct size was determined. Results: There was no significant difference in infarct size between E2‐ or placebo‐treated WT (ERα^+/+ and ERβ^+/+) mice. Surprisingly, E2 treatment did result in smaller infarct sizes in ERα^−/− mice, but increased the infarct size in ERβ^−/− mice. Increase of the left ventricular mass post‐MI was significantly larger in the E2‐treated ERα^−/− animals compared with placebo‐treated animals. E2 treatment also significantly increased post‐MI mortality in ERα^+/+, ERβ^+/+ and ERα^−/− animals, but not in ERβ^−/− mice. Conclusions: Although E2 modulates the infarct size in ERα^−/−, it also appears to be responsible for the higher mortality following MI. ERβ appears to be the receptor involved in the modulating effects of E2 in the infarcted heart.     

Lower leg amputation due to critical limb ischaemia: morbidity, mortality and rehabilitation potential

A total of 3 patients, a female aged 66 years, a male aged 67 years and a female aged 82 years, presented with rest pain or gangrene of their lower extremities as a result of critical limb ischemia (CLI). Radiographic studies showed non-reconstructable arterial obstructive disease in the 66-year-old female patient, who underwent an uncomplicated primary amputation and subsequent rehabilitation. Bypass surgery was initially performed in the male patient. However, failed reconstruction and progression of ischaemia necessitated transfemoral amputations to be performed, which was followed by impaired rehabilitation. In the 82-year-old female patient gangrene was the indication for primary amputation. She died due to progressive sepsis following surgery. In about 40% of patients with CLI, major lower extremity amputation is the ultimate solution. The morbidity, mortality and rehabilitation potential in CLI and the factors that influence these outcomes are discussed. The most clinically relevant determinants of morbidity, mortality and rehabilitation following major lower extremity amputation are comorbidity, age, preoperative mobility and amputation level.     

转录因子T-bet与哮喘大鼠气道炎症及川芎嗪的干预效应

目的:转录因子T-bet在支气管哮喘的发病中起重要作用,川芎嗪治疗哮喘有效。实验拟观察川芎嗪对哮喘大鼠气道炎症的影响和转录因子T-bet的调控作用。方法:实验于2005-11/2006-06在南京医科大学完成。①实验材料及分组:72只SPF级SD大鼠随机分为正常对照组、哮喘模型组、川芎嗪小剂量组(20mg/kg)、川芎嗪中剂量组(40mg/kg)、川芎嗪大剂量组(80mg/kg)和地塞米松组,每组12只。实验用磷酸川芎嗪注射液为丽珠集团利民制药厂生产)。②实验过程及评估:以卵蛋白腹腔注射并雾化吸入制备大鼠哮喘模型,末次雾化后24h内麻醉后处死大鼠。观察6组大鼠肺组织形态学变化;测定支气管壁厚度、支气管平滑肌厚度、嗜酸粒细胞和淋巴细胞数。采用免疫组织化学半定量法测定肺组织T-bet蛋白的表达;进行转录因子T-bet蛋白表达量与气道炎症的相关性分析。实验过程中对动物处置符合动物伦理学标准。结果:①哮喘模型组嗜酸粒细胞、淋巴细胞、支气管壁厚度和支气管平滑肌厚度,明显高于正常对照组相应指标(P均<0.01);与哮喘模型组比较,川芎嗪小、中、大剂量组和地塞米松组上述4项指标均减少(P均<0.01)。②哮喘模型组、川芎嗪小、中、大剂量组和地塞米松组的T-bet表达量低于正常对照组(P均<0.01);与哮喘模型组比较,川芎嗪小、中、大剂量组T-bet表达量增加(P均<0.01);随着川芎嗪剂量增加,T-bet表达量亦相应增加,川芎嗪小、中、大剂量组之间两两比较,差异均有统计学意义(P均<0.01)。③相关分析显示哮喘模型组T-bet蛋白表达量与嗜酸性粒细胞和淋巴细胞浸润数呈负相关(r=-0.81,-0.85,P<0.01),与支气管管壁厚度和支气管平滑肌厚度呈负相关(r=-0.77,-0.79,P<0.01)。结论:支气管哮喘大鼠存在T-bet低表达;川芎嗪可抑制气道炎症,增加转录因子T-bet蛋白的表达,纠正Th1/Th2失衡,从而治疗支气管哮喘。     

Adherence of L1210 murine leukemia cells to sephacryl- aminopropylcobalamin beads treated with transcobalamin-II

Sephacryl beads containing an immobilized aminopropylcobalamin- transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.     

Characterization of the human neutrophil C1q receptor and functional effects of free ligand on activated neutrophils

The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.     

Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA,factor H binding protein,Neisseria heparin-binding antigen and Neisseria adhesin A)

BACKGROUND:Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB.OBJECTIVE:To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine.METHODS:Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated.RESULTS:The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD.CONCLUSION:From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada.