Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1- dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post- labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.      

乙稀雌酚肌注致心房纤颤1例

1　病例报告　男 ,71岁 ,因排尿困难于 1999- 0 9- 0 4入院 .B超确诊前列腺增生 5 a,既往无高血压、心脏病及心率失常史 .查体 :Bp17/ 10 k Pa,心界不大 ,心率 6 2次· min- 1 ,心律齐 .下腹部可触及涨大的膀胱 ,约耻骨上 4指 .入院时 ECG正常 .肝肾功能无异常 ,确诊前列腺增生伴急性尿潴留 .首先予导尿 ,导出尿液约 90 0 m L,之后 im乙稀雌酚 1mg,2 h后患者突发心慌、胸闷、气短、心前区疼痛 .发现患者大汗BP12 /6 k Pa,P110次· min- 1 ,R2 8次· min- 1 ,心率绝对不齐 ,脉搏明显短促 ,两肺底可闻及湿性罗音及哮鸣音 ,ECG示房颤 ,心…     

Multilevel likelihood ratios for identifying exudative pleural effusions(*)

STUDY OBJECTIVES: To determine multilevel likelihood ratios for pleural fluid tests that are commonly used to discriminate between exudative and transudative pleural effusions. DESIGN: Meta-analysis of patient-level data. PATIENT DATA: Selected studies included patients with diagnoses of exudative or transudative pleural effusions who underwent thoracentesis and laboratory analysis of their pleural fluid. MEASUREMENTS AND METHODS: Studies were identified by searching MEDLINE and related bibliographies. Data were obtained for 1,448 patients from seven primary investigators or extracted from dot plots in published reports. Likelihood ratios were calculated from extracted data stratified across ranges of test result values. RESULTS: Sufficient data were available to calculate multilevel likelihood ratios for the elements of Light's criteria (pleural fluid lactate dehydrogenase [LDH], ratio of pleural fluid to serum LDH, and ratio of pleural fluid to serum protein), pleural fluid protein, ratio of pleural fluid to serum cholesterol, pleural fluid cholesterol, and gradient of pleural fluid to serum albumin. Each of these tests provided levels of likelihood ratios through the most clinically relevant range (0 to 10). CONCLUSION: Multilevel likelihood ratios combined with a clinician's estimation of the pretest probability of an exudative effusion improve the diagnostic accuracy of discriminating between exudative and transudative pleural effusions. Likelihood ratios avoid the use of confusing terms, such as "pseudoexudates," that derive from the use of single cutoff points for pleural fluid tests.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

B-cell lymphoma after angioimmunoblastic lymphadenopathy: a case with oligoclonal gene rearrangements associated with Epstein-Barr virus

We describe a patient with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), who subsequently developed large-cell immunoblastic lymphoma of B-cell immunophenotype. At the time of the initial diagnosis, histologic examination of an inguinal lymph node showed typical features of AILD, and there was no evidence of a monoclonal B-cell population by immunohistochemical analysis. In situ hybridization and Southern blot analysis for Epstein-Barr virus (EBV) were negative. At autopsy 2 years later, the patient had widespread lymph node and organ involvement by large-cell immunoblastic lymphoma of B-cell immunophenotype. Southern blot analysis performed on DNA extracted from lymph nodes, liver, and spleen showed two patterns of Ig heavy chain and kappa light chain gene rearrangements. The T-cell receptor beta chain gene was in the germline configuration. Analysis with an EBV terminal repeat region probe showed two clonal populations that paralleled the Ig gene rearrangement studies. Double-labeling immunohistochemistry and in situ hybridization confirmed the presence of EBV within the neoplastic B cells. The data support the hypothesis that EBV was not etiologically related to AILD in this case, and that EBV proliferation may occur after the onset of the disease. Further, the data suggest that some B-cell lymphomas that arise in the setting of AILD resemble EBV-associated B-cell lymphomas that arise in other immunodeficiency states.     

End-of-life care preferences of patients enrolled in cardiovascular rehabilitation programs

STUDY OBJECTIVES: The study assessed the interests of ambulatory cardiac patients in advance planning and their willingness to participate in rehabilitation program-based end-of-life education. DESIGN: Observational survey study. SETTING: Fourteen outpatient cardiac rehabilitation programs in 11 states. PARTICIPANTS: Four hundred fifteen subjects enrolled in cardiac rehabilitation. MEASUREMENTS AND RESULTS: A questionnaire determined patient preferences for advance planning, completion of advance directives, completion of patient-physician discussions on end-of-life care, and effects of health status on patient acceptance of life-sustaining interventions. Seventy-two percent of patients wanted to direct their own end-of-life care, 86% desired more information on advance directives, 62% wanted to learn about life-sustaining care, and 96% were receptive to advance-planning discussions with their physicians. Seventy-two percent of patients had considered that they might require life-sustaining care in the future; acceptability of resuscitative care depended on health status and probability of survival. However, only 15% had discussed advance planning with their physicians, and 10% were confident that their physicians understood their end-of-life wishes. Physicians and cardiovascular rehabilitation programs were considered desirable sources of information on advance planning. CONCLUSIONS: Cardiac patients enrolled in rehabilitation programs want to learn more about end-of-life care and need more opportunities to discuss advance planning with their physicians. Patients consider cardiovascular rehabilitation programs to be acceptable sites for advance planning education.     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.