Klara Abravaya

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Heat Shock-Induced Interactions of Heat Shock Transcription Factor and the Human hsp70 Promoter Examined by In Vivo Footprinting

Genomic footprinting of the human hsp70 promoter reveals that heat shock induces a rapid binding of a factor, presumably heat shock transcription factor, to a region encompassing five contiguous NGAAN sequences, three perfect and two imperfect matches to the consensus sequence. Arrays of inverted NGAAN sequences have been defined as the heat shock element. No protein is bound to the heat shock element prior to or after recovery from heat shock. Heat shock does not perturb the binding of factors to other regulatory elements in the promoter which contribute to basal expression of the hsp70 gene.

Transcription of the Human HSP70 Gene: cis-Acting Elements and trans-Acting Factors Involved in Basal, Adenovirus E1A, and Stress-Induced Expression

The eukaryotic genome encodes a large multigene family of proteins that share extensive sequence identity and biochemical properties with the major heat shock-inducible protein, HSP70. In human cells there are at least five distinct members of the HSP70 gene family that are constitutively expressed or induced in response to physiological stress. The genes that encode many of these HSP70-related proteins have been cloned, and comparison of their corresponding nucleotide sequences has revealed a high degree of evolutionary conservation among members of the HSP70 family within a single species and between species (Moran et al. 1982; Lowe et al. 1983; Hunt and Morimoto 1985; Mues et al. 1986). For example, comparison of heat shock proteins between species as separated as dnaK from E. coli and human HSP70 reveals 50% identity at the amino acid level (Bardwell and Craig 1984). Among the human HSP70-related proteins, GRP78 and HSP70 are 76% identical while P72 and HSP70 are 85% identical (Hunt and Morimoto 1985).

Molecular Beacons as Diagnostic Tools:Technology and Applications

Abstract Molecular beacons are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Molecular beacons allow multiplex detection of PCR products in real time in a homogeneous assay format. Real time detection is inherently quantitative and affords a greater dynamic range than end-point detection methods. Reactions in a homogeneous assay format are sealed before amplification takes place, providing improved contamination control. A single cycler/reader instrument, coupled with automated sample preparation, results in higher throughput and greater ease of use. A multiplex qualitative assay that detects Chlamydia trachomatis and Neisseria gonorrhoeae, along with an internal control, has been developed. High specificity is achieved through careful selection of primers, probes and assay conditions. Quantitative HIV, HCV, and HBV viral load assays, with sensitivities of 50 copies/ml, 20 IU/ml, and 50 copies/ml, respectively, are achievable. The viral load assays are designed to quantitate all subtype and genotype specimens equivalently. A molecular beacon assay has been designed to detect a single nucleotide polymorphism in the β2 adrenergic receptor gene.

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5′-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3′-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

A new ultrasensitive assay for quantitation of HIV-1 RNA in plasma

Nucleic acid-based diagnostic assays for the quantitation of plasma HIV-1 RNA levels are used to monitor disease progression and the response of patients to antiretroviral drug therapy. The LCx HIV RNA Quantitative Assay (Abbott Laboratories, North Chicago, IL) is an assay for the quantitation of HIV type 1 RNA in plasma that uses competitive reverse transcription PCR (RT-PCR) followed by Microparticle Enzyme Immunoassay, and includes an internal control for inhibition and RNA recovery, that is taken through the entire sample preparation procedure. The performance of the assay was assessed for 1 and 0.2 ml sample volumes. For a 1 ml sample volume, the lower limit of detection was found to be 50 copies/ml with a linear range from 50 to 1 million copies/ml. For a 0.2 ml sample volume, the lower limit of detection was found to be 178 copies/ml with a linear range from 178 to 5 million copies/ml. The assay is able to detect and quantitate HIV subtypes A-G and group O. LCx HIV RNA assay quantitation results are highly correlated to the standard and ultrasensitive Amplicor HIV-1 Monitor assay (Roche Molecular Systems) quantitation results. Assay performance is consistent with the use of this test for routine quantitation of HIV-1 RNA in plasma.

Principles and analytical performance of Abbott RealTime High Risk HPV test.

BACKGROUNDnAbbott RealTime High Risk (HR) HPV is a new automated, qualitative real-time PCR test for detection of DNA from 14 high-risk human papillomavirus (HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. The test can also differentiate between HPV 16, HPV 18 and non-HPV 16/18 types in a single reaction.nnnOBJECTIVESnThis article describes the principles of assay design and the analytical performance of Abbott RealTime HR HPV.nnnSTUDY DESIGNnThe analytical performance characteristics of Abbott RealTime HR HPV were evaluated in terms of its sensitivity for each of the 14 high-risk types included in the test, specificity (cross-reactivity), potential for interference by substances that may be present in cervical specimens, and reproducibility.nnnRESULTSnAbbott RealTime HR HPV provided sensitive detection of the 14 high-risk HPV types included in the test. It was also highly specific to the HPV types targeted by the test and did not show cross-reactivity with 15 low-risk HPV types tested, or non-specific reactivity with other common microorganisms that may be present in the female anogenital tract. Test results were not impacted by potential interfering substances evaluated in the study. The test generated highly reproducible results in an in-house study and in studies carried out at 13 external evaluation sites.nnnCONCLUSIONSnAbbott RealTime HR HPV demonstrated a robust analytical performance with reproducible and reliable results.

Clinical performance of Abbott RealTime High Risk HPV test for detection of high-grade cervical intraepithelial neoplasia in women with abnormal cytology.

BACKGROUNDnAbbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk oncogenic HPV types combined with the ability to concurrently identify genotypes 16 and 18.nnnOBJECTIVESnThe clinical performance of the Abbott RealTime HR HPV test was evaluated in comparison with the Hybrid Capture 2 (HC2) test for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+). The relative accuracy of the Abbott RealTime HR HPV to detect high-risk HPV was also determined.nnnSTUDY DESIGNnCervical specimens were collected from 702 patients with abnormal cytology who were referred for colposcopy, and were tested with liquid based cytology (LBC), Abbott RealTime HR HPV and HC2. Genotyping was done using the Linear Array (LA) method. Histological assessment was used as the gold standard for disease status. Clinical performance for detection of disease was evaluated for Abbott RealTime HR HPV in comparison with HC2 in the overall population and in each cytological grade. The relative accuracy for detection of high-risk HPV was assessed by concordance between the two tests and based on LA genotyping.nnnRESULTS AND CONCLUSIONSnThe Abbott RealTime HR HPV showed similar clinical performance for detection of CIN2+ when compared with HC2, for both the overall population and those with a cytological grade of atypical squamous cells of undetermined significance (ASC-US). The accuracy for detection of high-risk HPV was significantly higher with Abbott RealTime HR HPV than with HC2.

High-risk HPV detection and concurrent HPV 16 and 18 typing with Abbott RealTime High Risk HPV test

BACKGROUNDnHigh-risk human papillomavirus (HPV) is the causative agent of cervical cancer. Among the high-risk types, infection with HPV 16 and 18 is associated with significantly higher risk of disease progression, and consequently these two types together cause approximately 70% of invasive cervical cancer worldwide. Identification of HPV 16 and HPV 18 can provide valuable information for risk stratification and clinical management of patients infected with these two types in both ASC-US triage and primary screening in women over age 30. It may also be valuable in the assessment of HPV vaccine efficacy. Abbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk HPV types with the ability to concurrently identify HPV 16 and 18.nnnOBJECTIVEnTo evaluate the clinical performance of Abbott RealTime HR HPV test.nnnSTUDY DESIGNnAbbott RealTime HR HPV was evaluated with 253 cervical specimens obtained from patients with CIN 3 and 340 specimens from patients with cervical cancer to determine clinical sensitivity of the test and the prevalence of types 16 and 18. Additionally, 757 cervical specimens obtained from women 30 years of age or older with normal cytology in a general screening population were tested to determine high-risk HPV positivity rate.nnnRESULTSnThe Abbott RealTime HR HPV test detected 97.2% (246/253) of CIN 3 specimens and 98.5% (335/340) of cancer specimens. HPV 16 was the most prevalent type in both CIN 3 (72.8%) and cancer specimens (64.5%). HPV 16 and 18 combined were detected in 78.9% of high-risk HPV positive CIN 3 and 84.8% of high-risk HPV positive cancer specimens. In specimens from women 30 years of age or older with normal cytology in a screening population, the HPV positivity rate was 6.5% (49/757).nnnCONCLUSIONSnAbbott RealTime HR HPV is a highly sensitive test for detection of high-grade cervical disease and cancer. The HPV 16 and HPV 18 typing capability of the test offers the advantage of stratifying patients at greater risk of progression and may thus aid in better patient care and management.

A novel RealTime HIV-1 Qualitative assay for the detection of HIV-1 nucleic acids in dried blood spots and plasma

Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A-H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80 copies/mL of plasma using the plasma protocol, and 2469 copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).