Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Colon carcinoma cell lines stimulate monocytes and lamina propria mononuclear cells to produce IL-10

Cytokines released from tumour cells may have function as signals to neighbouring immune and inflammatory cells. Several studies have shown that the immunoregulatory cytokines IL-10 and transforming growth factor-beta 1 (TGF-β1) as well as prostaglandin-E₂ (PGE₂) play an important role in tumour-induced immunosuppression. The aim of the study was to investigate the effect of colon carcinoma cell lines on IL-10 production in peripheral monocytes (PBMC) and lamina propria mononuclear cells (LPMC). We examined four colon carcinoma cell lines (HT-29, Caco-2, Colo-320 and HCT-116) and determined their production of TGF-β1, IL-10 and PGE₂. Peripheral monocytes were isolated by density gradient centrifugation and LPMC were isolated from surgical specimens using a collagenase digestion method. Monocytes and LPMC were cultured with colon carcinoma cell conditioned medium or in co-culture with colon carcinoma cells. Supernatants were then determined for the production of IL-10 by ELISA assays. All colon carcinoma cell lines stimulated peripheral monocytes as well as LPMC to produce markedly increased levels of IL-10. Colon cancer cells secreted negligible levels of IL-10, but high amounts of TGF-β1 and PGE₂. Neutralization of TGF-β1 by administration of anti-TGF-β as well as neutralization of PGE₂ with anti-PGE₂ antisera reduced the IL-10 production of monocytes markedly, indicating that tumour cell-derived TGF-β1 and PGE₂ are major factors for IL-10 stimulation. In vitro stimulation of monocytes with TGF-β1 and PGE₂ could confirm that TGF-β1 as well as PGF₂ at picogram concentrations were able to prime monocytes for enhanced IL-10 production. Our results demonstrate that colon carcinoma cell lines enhance the ability of monocytes and intestinal macrophages to produce IL-10. The stimulation of monocyte IL-10 by colon cancer cell-derived TGF-β1 and PGE₂ may act as a tumour-protecting mechanism by impairing the activation of anti-tumour cytokines.     

膨胀型肝癌光镜,电镜和免疫组化观察

对３０例膨胀型肝癌和２０例浸润型肝癌作病理组织学分析，并对２０例膨胀型和浸润型肝癌进行超微结构和免疫组化研究。结果显示，膨胀型肝癌的生物学特性和浸润型肝癌显著不同。膨胀型肝癌癌组织分化较好、侵袭性较小、增殖程度较低、癌巢周围基膜发育良好、瘤结节周围均有包膜而呈膨胀性生长，但包膜内浸润的微癌灶十分常见，多达７０％。提示此型有潜在的较强侵袭性和向浸润型肝癌转化的倾向。     

Bone growth in zebrafish fins occurs via multiple pulses of cell proliferation.

Fin length in the zebrafish is achieved by the distal addition of bony segments of the correct length. Genetic and molecular data provided evidence that segment growth uses a single pulse of growth, followed by a period of stasis. Examination of cell proliferation during segment growth was predicted to expose a graphical model consistent with a single burst of cell division (e.g., constant, parabolic, or exponential decay) during the lengthening of the distal-most segment. Cell proliferation was detected either by labeling animals with bromodeoxyuridine (during S-phase) or monitoring histone3-phosphate (mitosis). Results from both methods revealed that the number of proliferating cells fluctuates in apparent pulses as a segment grows (i.e., during the growth phase). Thus, rather than segment size being the result of a single burst of proliferation, it appears that segment growth is the result of several pulses of cell division that occur approximately every 60 microns (average segment length approximately 250 microns). These results indicate that segment lengthening requires multiple pulses of cell proliferation.     

肝素酶和碱性成纤维细胞生长因子与非小细胞肺癌转移和预后的关系

目的 探讨肝素酶(heparanase)和碱性成纤维细胞生长因子(bFGF)在人非小细胞肺癌(NSCLC)组织中表达的临床意义及其与肺癌转移和预后的关系。方法 采用免疫组织化学、原位杂交和Western blot方法,检测115例人NSCLC石蜡切片和45例新鲜肺癌及对应癌旁正常组织中肝素酶和bFGF的表达情况。采用χ^2检验、t检验、生存曲线和Cox比例风险回归等方法分析肝素酶和bFGF分别表达及共表达的意义。结果 免疫组织化学染色证实肝素酶(91／115)和bFGF(89／115)主要表达在癌细胞质和(或)细胞膜中,在正常肺泡上皮和支气管上皮中则呈阴性表达。Western blot也证实肝素酶在肺癌中的表达明显增高(P=0．041)。统计分析结果显示：肝素酶和bFGF的表达具有明显的一致性(P：0．0001),二者单独表达和共表达均与肺癌的分期、血管侵袭、淋巴结转移、微血管密度和预后有关,其中,二者共表达时与分期和微血管密度的相关性更显著;另外,bFGF还与肺癌的分化程度有关。多因素分析结果显示,肺癌的分化程度、血管浸润、淋巴结转移和bFGF的表达可以作为判断肺癌预后的危险因素,但肝素酶不是影响预后的独立因素。结论 肝素酶和bFGF均与肺癌的转移、血管生成和预后密切相关。     

Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1 of Schizosaccharomyces pombe

The Schizosaccharomyces pombe rad51⁺ and dmc1⁺ genes code for homologues of the Escherichia coli recombination protein RecA. Deletion of rad51⁺ causes slow growth, retardation of cell division and a decrease in viability. rad51 cells have a defect in mating-type switching. The DNA modification at the mating-type locus required for mating-type switching contributes to slow growth in the rad51 mutant. Cell mating is reduced in crosses homozygous for rad51. Ectopic expression of the dmc1⁺ gene allowed us to demonstrate that the reduction in meiotic recombination in dmc1 mutants is not caused by a disturbance of rad24 expression from the dmc1-rad24 bicistronic RNA. We describe the functional defects of terminally epitope-tagged Dmc1 and Rad51 and discuss it in terms of protein interaction. Presumptive Rad51 and Dmc1 foci were detected on spreads of meiotic chromatin.     

Safety of nasal budesonide in the long-term treatment of children with perennial rhinitis

BACKGROUND: Intranasal budesonide is an efficacious treatment for perennial allergic rhinitis. Long-term effects on safety, particularly in children, need further investigation. OBJECTIVE: To investigate the long-term safety of intranasal budesonide in children. METHODS: In an open trial, 78 children (5-15 years) with perennial rhinitis were treated with intranasal budesonide pressurized metered dose inhaler 200 microg twice daily (delivered daily dose 256 microg) for 12 months; 43 children stayed in the study for 12 additional months and were switched to aqueous suspension (400 microg delivered daily dose) for 6 months. Statural growth, bone age, ophthalmologic and rhinoscopic status, cortisol and biochemical analyses in blood and urine were monitored during the first and second years, and adverse events (AEs) were continuously recorded. RESULTS: No significant effects on statural growth and bone age, compared with reference values, were observed. Morning plasma cortisol and 24-h urinary cortisol were not changed during treatment. Patients reported 195 AEs, most commonly nasal dryness (30%), blood-tinged secretions (21%) and, among non-nasal AEs, headache (13%). Rhinoscopy revealed no signs of mucosal atrophy, ulceration, or candidiasis but some nasal dryness. No treatment-related ophthalmological or biochemical aberrations were found. Reduction of blood eosinophils and nasal symptom scores, compared with pre-treatment values, indicated the efficacy of budesonide treatment. CONCLUSION: Long-term treatment for 1-2 years with intranasal budesonide 256-400 microg daily in children with perennial rhinitis revealed no negative effects on growth or endogenous cortisol production. Local side-effects were mild and patient symptoms decreased.     

Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF-1, FGF-2, and FGFR-1

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of fibroblast growth factors (FGF-1 and FGF-2) and their receptor, FGFR-1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = 15) and without COPD (FEV1 > 85%; n = 16). FGF-1 and FGFR-1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF-2 in bronchial epithelium (0.35 +/- 0.03 vs 0.20 +/- 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR-1 in ASM (p < 0.005) and of FGF-1 (p < 0.04) and FGFR-1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF-1 and/or FGF-2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady-state mRNA levels of FGFR-1 were elevated in human ASM cells treated with either FGF-1 or FGF-2. The increased bronchial expression of fibroblast growth factors and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR-1 system in the remodelling of bronchial airways in COPD.     

Liver metastasis from rectal cancer with prominent intrabile duct growth

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived from rectal cancer is reported. The patient was a 62-year-old man who had received a low anterior resection for rectal cancer in March 2000. He was re-admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well- to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.