A transatlantic perspective on the integration of immuno-oncology prognostic and predictive biomarkers in innovative clinical trial design.

Immuno-therapeutics aim to activate the body's own immune system against cancer and are one of the most promising cancer treatment strategies, but currently limited by a variable response rate. Biomarkers may help to distinguish those patients most likely to respond to therapy; they may also help guide clinical decision making for combination therapies, dosing schedules, and determining progression versus relapse. However, there is a need to confirm such biomarkers in preferably prospective clinical trials before they can be used in practice. Accordingly, it is essential that clinical trials for immuno-therapeutics incorporate biomarkers. Here, focusing on the specific setting of immune therapies, we discuss both the scientific and logistical hurdles to identifying potential biomarkers and testing them in clinical trials.

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones.

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression.

The emergence of a culture that promotes evidence based clinical decision making within an acute care setting.

Nursing research programs within acute care hospitals are essential to the development and integration of nursing knowledge, difficult to implement and rarely evaluated. The purpose of this paper is three fold: (1) to describe the development, structures, and processes of a nursing research program within an acute care teaching hospital and (2) to describe selected evaluation outcomes and (3) to discuss future directions.

Comparative safety of two recombinant hepatitis B vaccines in children: data from the Vaccine Adverse Event Reporting System (VAERS) and Vaccine Safety Datalink (VSD).

BACKGROUND: Preliminary review of data from the Vaccine Adverse Event Reporting System (VAERS), 1991-1994, revealed that more serious adverse events were reported in children who received a specific brand of recombinant hepatitis B (HepB) vaccine. OBJECTIVE: To compare the post-marketing safety experience of the two recombinant HepB vaccines licensed for use in infants and children in the United States. DESIGN: Review of a case series derived from passive surveillance data in the national VAERS. A retrospective cohort study using data from one health maintenance organization participating in Vaccine Safety Datalink (VSD), a computerized record linkage system. POPULATIONS STUDIED: U.S. children, ages birth-10 years for whom adverse events after HepB vaccine were reported to VAERS, 1991-1994. Children, ages birth-6 years, who received HepB vaccine at Kaiser Permanente Medical Care Program, Northern California, 1991-1994. MAIN OUTCOME MEASURES: VAERS reporting rates for each vaccine by manufacturer were calculated from the numbers of reported events occurring within 30 days of HepB vaccination and the number of doses distributed by the manufacturers. VSD event rates for each vaccine were calculated from the numbers of hospitalization or emergency room visits within 30 days of HepB vaccination and the number of vaccine doses administered to the cohort. RESULTS: In VAERS, higher rates of serious events (i.e., life threatening or resulting in hospitalization or permanent disability) were reported in children who received Vaccine A vs. Vaccine B (relative risk [RR]: 3.13-8.18, P < 0.01), particularly by those vaccinated in the private (RR: 7.62-28.58, P < 0.01), but not public sector (RR: 2.12, P = 0.19). Similar types of events were reported in recipients of both vaccines. In contrast, analysis of VSD data showed no significant difference in rates of hospitalization or ER visits in children who received either HepB vaccine (RR: 0.96-1.25, P > 0.05). CONCLUSIONS: Our investigation reveals that it is unlikely there is a true difference between rates of serious events temporally associated with the two HepB vaccines in children. This study demonstrates the dual roles played by VAERS and VSD in providing a more complete picture of the post-marketing safety profile of childhood vaccines, and underscores the importance of using other analytic studies to evaluate findings from passive surveillance systems of adverse events.

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.