Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.     

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency

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Strain delineation and epidemiology of Candida (Clavispora) lusitaniae.

Electrophoretic karyotype (EK) patterns, determined by using contour-clamped homogeneous pulsed-field electrophoresis, and isoenzyme (IZ) profiles were evaluated as methods for strain delineation among 35 isolates of Candida lusitaniae recovered from 15 patients. All isolates were identified to the species level by using conventional morphologic and physiologic criteria, and the identification was confirmed by gas-liquid chromatography analysis of the cellular fatty acids. The isolates were then typed without knowledge of the patient source. The IZ profiles showed all isolates to be closely related. Fifteen EK patterns were found; each pattern was restricted to isolates recovered from a single patient. In contrast, on the basis of heterogeneity in phosphatases, beta-glucosidases, esterases, and catalases, 10 IZ profiles were found; 4 were shared by isolates recovered from more than one patient. Multiple isolates from six patients were analyzed, and for each patient, a single EK- and IZ-defined type was found. The types of isolates obtained from two patients, after the emergence of resistance to amphotericin B, remained the same as the types of isolates obtained earlier. The data suggest that a patient becomes colonized by a single strain of C. lusitaniae which may disseminate to multiple sites, that the colonizing strain can persist during the patient's hospitalization, and that it may develop resistance to amphotericin B. Both EK patterns and IZ profiles can be used to delineate strains of C. lusitaniae, but the EK pattern provides more discriminatory power.     

Cockayne''s syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation.

Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features.     

Multiple forms of glycoproteins in the secretory product of the bovine subcommissural organ--an ancient glial structure

The glial subcommissural organ (SCO) is a conserved structure of the vertebrate brain that secretes a glycoprotein-rich product into both the extracellular matrix and the cerebrospinal fluid of the third ventricle that forms Reissner's fibre (RF). In order to identify specific secretory proteins of the subcommissural organ, a panel of antigen- and epitope-specific monoclonal antibodies was raised against bovine RF to study the distribution of epitopes in Western blots of bovine RF. Six groups of epitopes that were specific for SCO secretion were distinguished on the basis of their phylogenetic conservation and their different grades of resistance against chemical denaturation. The monoclonal antibody aRFME 4 recognised a carbohydrate-containing epitope that was strongly conserved in vertebrates and unique for SCO secretion. All epitopes showed essentially the same distribution pattern over 15 bovine RF glycoprotein fractions of different molecular masses in immunoblots indicating that the different RF fractions are closely related. They may represent multiple forms of SCO spondin.     

Synergy between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset confirmed Alzheimer's disease

The allelic frequency of the gene for the K variant of butyrylcholinesterase (BCHE-K) was 0.17 in 74 subjects with late-onset (age > 65 years) histopathologically diagnosed Alzheimer's disease (AD), which was higher than the frequencies in 104 elderly control subjects (0.09), in 14 early-onset cases of confirmed AD (0.07) and in 29 confirmed cases of other dementia (0.10). The association of BCHE-K with late-onset AD was limited to carriers of the epsilon 4 allele of the apolipoprotein E gene (APOE), among whom the presence of BCHE-K gave an odds ratio of confirmed late-onset AD of 6.9 (95% C.I. 1.65-29) in subjects > 65 years and of 12.8 (1.9-86) in subjects > 75 years. In APOE epsilon 4 carriers over 75 years, only 1/22 controls, compared with 10/24 confirmed late-onset AD cases, had BCHE-K. We suggest that BCHE-K, or a nearby gene on chromosome 3, acts in synergy with APOE epsilon 4 as a susceptibility gene for late-onset AD.