Human monocyte-derived dendritic cell (MoDC) have been used in the clinic with moderately encouraging results. Mouse XCR1+ DC excel at cross-presentation, can be targeted in vivo to induce protective immunity, and share characteristics with XCR1+ human DC. Assessment of the immunoactivation potential of XCR1+ human DC is hindered by their paucity in vivo and by their lack of a well-defined in vitro counterpart. We report in this study a protocol generating both XCR1+ and XCR1− human DC in CD34+ progenitor cultures (CD34-DC). Gene expression profiling, phenotypic characterization, and functional studies demonstrated that XCR1− CD34-DC are similar to canonical MoDC, whereas XCR1+ CD34-DC resemble XCR1+ blood DC (bDC). XCR1+ DC were strongly activated by polyinosinic-polycytidylic acid but not LPS, and conversely for MoDC. XCR1+ DC and MoDC expressed strikingly different patterns of molecules involved in inflammation and in cross-talk with NK or T cells. XCR1+ CD34-DC but not MoDC efficiently cross-presented a cell-associated Ag upon stimulation by polyinosinic-polycytidylic acid or R848, likewise to what was reported for XCR1+ bDC. Hence, it is feasible to generate high numbers of bona fide XCR1+ human DC in vitro as a model to decipher the functions of XCR1+ bDC and as a potential source of XCR1+ DC for clinical use.
Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining twothirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing ϳ600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.
Molecular analyses of large numbers of patient samples are increasingly used for diagnostic applications as well as for understanding cancer biology. Their accuracy depends on the quality and quantity of nucleic acids extracted from human cells or tissues. To optimize these preanalytical steps, we evaluated several automated technologies for nucleic acid purification from clinical samples. Three automated platforms were compared. DNA was extracted from peripheral blood leukocytes from five normal individuals, and its quality was assessed by D-HPLC and sequencing after PCR. Clinical samples from acute leukemia patients were used for automated RNA extractions; results were compared to our standard manual technique. RNA qualification was done using capillary gel electrophoresis and analysis of the Abelson gene transcript by real-time PCR. One robot produced higher total output for both DNA and RNA. While the quality of DNAs obtained from the three workstations allowed implementation of their analysis for detection of germinal mutations, important differences were observed in the quality of RNAs. One robot isolated RNA with similar quality and quantity to the manual technique, but the resulting products displayed low concentrations. A robust technique had therefore to be evaluated and validated to allow the implementation of this workstation within the daily diagnostic practices. This study is of interest at a time when hospital-based laboratories are bringing molecular signatures to the clinic.
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