Ex vivo reversal of chemoresistance by tariquidar (XR9576)

The expression of P-glycoprotein (P-gp) has been demonstrated to confer resistance to several anticancer drugs, including anthracyclines, taxanes and vinca alkaloids. Tariquidar is a novel inhibitor of P-gp that has been shown to reverse resistance to cytotoxic drugs in tumor cell lines and mouse xenografts. We have used an ATP-based chemosensitivity assay (ATP-TCA) to compare the activity of cytotoxic drugs in combination with tariquidar against a variety of solid tumors (n = 37). The expression of P-gp was determined in a subset of solid tumor samples by immunohistochemistry (n = 16). Resistance was seen in 20 of 37 (54%) tumors tested with doxorubicin, in 27 of 34 (79%) samples tested with paclitaxel and 17 of 31 (55%) with vinorelbine. Tariquidar alone showed no activity over a wide range of concentrations up to 2 microM (n = 14). The median IC90s for doxorubicin, paclitaxel and vinorelbine, alone were 2.57, 27.4 and 15.5 microM. These decreased to 1.67 (p<0.0005), 20.6 (p<0.05) and 9.5 microM (p<0.001), respectively, in combination with tariquidar. Tariquidar also significantly decreased resistance in 14 of 20 (70%), six of 27 (22%) and six of 17 (35%) samples tested with doxorubicin, paclitaxel and vinorelbine, respectively. Immunohistochemical staining for P-gp was positive in nine of 16 (56%) samples and in all of these cases addition of tariquidar improved the activity of the cytotoxic. The results show that tariquidar is able to decrease resistance in a number of solid tumors resistant to cytotoxic drugs known to be P-gp substrates. These data support the introduction of tariquidar in combination with chemotherapy to clinical trials of patients expressing P-gp.     

Heterogeneity of chemosensitivity of colorectal adenocarcinoma determined by a modified ex vivo ATP-tumor chemosensitivity assay (ATP-TCA)

Advanced colorectal cancer (CRC) has a poor prognosis with a 5-year survival of only 5% despite treatment with chemotherapeutic agents. Response rate and overall survival varies little between the commonly used single agents, although combinations achieve better outcomes. It is well established that considerable heterogeneity exists between cancers of the same tissue type, but it has been difficult to establish this for CRC. We therefore investigated the heterogeneity of chemosensitivity in CRC using a modified version of the ex vivo ATP-tumor chemosensitivity assay (ATP-TCA) capable of handling infected tumor tissue. Fifty-three specimens of primary solid or malignant effusions of CRC were tested, of which 46 (87%) were evaluable. There were considerable differences in sensitivities between individuals. The most active single cytotoxic agents in the assay were identified as 5-fluorouracil, irinotecan and mitomycin C (MMC). Cells were exposed to combinations of drugs added simultaneously at the same concentrations tested as single agents. All drug combinations achieved greater growth inhibition than drugs used alone. MMC+gemcitabine was found to be the most effective combination in 83% of specimens. The ATP-TCA has previously been shown to be a good predictor of response to chemotherapy in other tissue types. The degree of heterogeneity demonstrated from these results suggests that the ATP-TCA could be used to identify patients who might benefit from specific chemotherapeutic agents alone or in combination.     

Automated measurement of microaneurysm turnover

PURPOSE: An automated system for the measurement of microaneurysm (MA) turnover was developed and compared with manual measurement. The system analyses serial fluorescein angiogram (FA) or red-free (RF) fundus images; fluorescein angiography was used in this study because it is the more sensitive test for MAs. Previous studies have shown that the absolute number of MAs observed does not reflect the dynamic temporal nature of the MA population. In this study, almost half of the MAs present at baseline had regressed after a year and been replaced by new lesions elsewhere. METHODS: Two clinical datasets were used to evaluate the performance of the automated turnover measurement system. The first consisted of 10 patients who had two fluorescein angiograms acquired a year apart. These data were analyzed, both manually and using the automated system, to investigate the inter- and intraobserver variations associated with manual measurement and to assess the performance of the automated system. The second dataset contained FAs from a further 25 patients. This dataset was analyzed only with the automated system to investigate some properties of microaneurysm turnover, in particular the differing detection sensitivities of new, static and regressed microaneurysms. RESULTS: Manual measurements exhibited large inter- and intraobserver variation. The sensitivity and specificity of the automated system were similar to those of the human observers. However, the automated measurements were more consistent-an important condition for accurate turnover quantification. Regressed MAs were more difficult to detect reliably than new MAs, which were themselves more difficult to detect reliably than static MAs. CONCLUSIONS: The automated system was shown to be fast, reliable, and repeatable, making it suitable for processing large numbers of images. Performance was similar to that of trained manual observers.     

Demonstration of biofilm in infectious crystalline keratopathy using ruthenium red and electron microscopy

OBJECTIVE: Bacterial biofilm formation has been implicated in the pathogenesis of infectious crystalline keratopathy. Biofilm cannot be visualized by electron microscopy without the addition of a fixative that stabilizes the polysaccharide-rich bacterial extracellular matrix that surrounds the bacterial colonies in a biofilm. We used ruthenium red as a fixative to evaluate corneal biopsy specimens for the presence of bacterial biofilm in three cases of infectious crystalline keratopathy (ICK) and five cases of chronic microbial keratitis without crystalline changes. DESIGN: Case series with clinicopathologic correlation. PARTICIPANTS: Eight patients underwent corneal biopsy or therapeutic keratoplasty as part of their management for chronic unresponsive microbial keratitis. METHODS: The corneal specimens removed were trisected for microbiology, pathology, and transmission electron microscopy (TEM). The TEM specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red. MAIN OUTCOME MEASURES: Demonstration of bacterial biofilm with TEM. RESULTS: TEM demonstrated organisms with a surrounding extracellular matrix consistent with a bacterial biofilm in the three cases of ICK but not in the five other cases of chronic microbial keratitis. CONCLUSIONS: The presence of biofilm in ICK can be demonstrated with TEM with appropriate fixation techniques that stabilize the bacterial extracellular matrix. Biofilm stains intensely with periodic acid-Schiff because of the polysaccharide-rich extracellular matrix and weakly with Gram stain because of the high proportion of nonviable organisms. Biofilm formation occurs in ICK but probably not in chronic bacterial keratitis without crystalline changes. Secretion of an extracellular matrix by bacteria to form a biofilm is a response to a nutrient-deprived environment in which growth and replication is depressed. The extracellular matrix of the biofilm may mask bacterial antigens, explaining the relative lack of inflammatory response in these infections. It may also be one of the mechanisms explaining the resistance to in vivo antimicrobial therapy when in vitro sensitivities have been proven.     

Histamine receptor--bearing peripheral T lymphocytes in patients with allergies.

The effect of histamine on the capacity of T lymphocytes to form E rosettes was tested in 10 healthy subjects and in 13 patients with allergies. Histamine had no effect on the capacity of T lymphocytes to form E rosettes in healthy subjects, but significantly inhibited the E rosette formation of T lymphocytes in patients with allergies.     

大连市乙肝疫苗查漏补种情况调查

[目的]了解大连市乙肝疫苗漏种的情况及其影响因素。[方法]2006年10月至2007年5月开展了大连市2002年7月1日至2006年9月3013出生儿童的乙肝疫苗查漏补种工作,对于在补种过程中新发现的漏种儿童,按照“随时发现,随时补种”的原则进行补种。[结果]本次“查漏补种”工作共摸底调查263727名儿童,查出漏种儿童2017人,漏种率为0．76％,应补种针次4198针次;实补种1933人,补种率为95．84％。大连市乙肝疫苗近5年平均接种率为99．9％,漏种率较低;流动儿童较本地常住儿童漏种率高（P〈0.05）,补种率低（P〈0．05）。[结论]流动儿童中仍存在免疫空白现象。     

双链断裂修复蛋白hKu70缺陷细胞株的建立及其生物学特性

目的 建立并鉴定DNA双链断裂(DSB)修复蛋白hKu70缺陷细胞株,并观察该缺陷细胞的某些生物学效应,用于AKu70基因功能及职业有害因素对DNA双链断裂修复影响的研究。方法 用构建的AKu70基因反义RNA绿色荧光蛋白真核表达载体(pEGFP—CI—K)转染人胚肺成纤维细胞(HLF),用蛋白兔疫印迹法鉴定转染细胞中AKu70基因的表达水平。同时观察转染细胞生长形态,绘制生长曲线,软琼脂培养法鉴定恶性程度。结果 pEGFP—CI—K载体在转染细胞内可较稳定表达,hKu70蛋白缺陷细胞株AKu70基因的蛋白表达水平下降了42％,转染后hKu70蛋白缺陷细胞生长形态、生长速度无明显变化,软琼脂培养未见细胞集落。结论 成功建立和鉴定了hKu70蛋白缺陷细胞株,该缺陷不足以单独引起可观察的某些生物学效应。     

铅对作业工人甲状腺功能的影响

目的　了解铅对作业工人甲状腺功能的影响。方法　选择暴露于铅作业环境下的人群 ,了解工人作业工龄 ,采用火焰原子吸收光谱法测定其作业环境中铅浓度 ,用原子吸收光谱法测定作业工人血铅 (PbB)浓度 ,用血液锌原卟啉测定仪测定血锌原卟啉 (ZPP)浓度 ,用放射免疫分析法检测血清中促甲状腺素 (TSH)、三碘甲状腺原氨酸 (T3)、甲状腺素 (T4 )、游离T3(FT3)、游离T4 (FT4 ) 5项甲状腺功能指标。结果　血铅 >2 .88μmol/L时 ,T3[(1 .54± 0 .39)nmol/L]、FT3[(5 .50± 1 .2 6)pmol/L]含量明显低于血铅 (1 .92～ 2 .88) μmol/L组 [T3(1 .71± 0 .45)nmol/L、FT3(6 .1 2± 1 .64)pmol/L] ,差异有显著性(P <0 .0 5)。铅作业工龄长短对甲状腺激素 (TH)含量未见明显影响。结论　高浓度血铅可能抑制了T4 的脱碘 ;铅作业工龄对甲状腺功能未见明显影响