H. James Hnatyszyn

Rapid and Specific Detection of Mycobacterium tuberculosis from Acid-Fast Bacillus Smear-Positive Respiratory Specimens and BacT/ALERT MP Culture Bottles by Using Fluorogenic Probes and Real-Time PCR

ABSTRACT A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.

The Role of SATB1 in Breast Cancer Pathogenesis

BACKGROUND SATB1 has been previously proposed as a key protein that controls the development and progression of breast cancer. We explored the potential of the SATB1 protein as a therapeutic target and prognostic marker for human breast cancer. METHODS We used aggressive (MDA-MB-231 and BT549) and nonaggressive (SKBR3 and MCF7) breast cancer cell lines to investigate the potential of SATB1 as a therapeutic target. SATB1 mRNA expression was silenced in aggressive cells by use of short hairpin RNAs against SATB1. SATB1 was overexpressed in nonaggressive cells by use of SATB1 expression vectors. We assessed the effect of modifying SATB1 expression on the transformed phenotype by examining anchorage-independent cell proliferation, acinar morphology on matrigel, and migration by wound healing in cultured cells. We examined tumor formation and metastasis, respectively, by use of orthotopic mammary fat pad and tail vein xenograft mouse models (mice were used in groups of six, and in total, 96 mice were used). SATB1 mRNA expression was compared with outcome for patients with primary breast cancer from six previous microarray studies that included a total of 1170 patients. All statistical tests were two-sided. RESULTS The transformed phenotype was not suppressed by SATB1 silencing in aggressive cells and was not enhanced by ectopic expression of SATB1 in nonaggressive cells. Modifying SATB1 expression did not alter anchorage-independent cell proliferation, invasive acinar morphology, or cell migration in cultured cells and did not affect tumor formation or metastasis in xenograft mouse models. In addition, SATB1 expression was not associated with decreased overall survival of patients with primary breast cancer in six previous independent microarray studies (overall odds ratio = 0.80, 95% confidence interval = 0.62 to 1.03, P = .10). CONCLUSION In contrast to previous studies, we found that SATB1 expression did not promote breast cancer progression and was not associated with breast cancer outcome.

Multicenter Evaluation of the VERSANT Hepatitis B Virus DNA 3.0 Assay

ABSTRACT The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 × 103 to 1.0 × 108 HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R2 = 0.900; Digene, R2 = 0.985; COBAS AMPLICOR, R2 = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment.

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequence-specific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4+ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4+ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4+ cells expressing the RNase P-associated U5 EGS (560) molecule.

Abstract 3916: Inhibition of GREB1 mRNA and protein limits ER+ breast tumor cell proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Discovering the mechanism by which estrogen regulates breast cancer growth will aid in the identification of patients who will benefit from endocrine therapy. Estrogen (E2) binding to the estrogen receptor (ER) stimulates the proliferation of normal mammary cells and some breast cancers. Preventing ER expression or receptor-ligand interaction in hormone-responsive breast cancers inhibits cell division and promotes tumor cell death. However, the mechanism of E2-regulated breast cancer cell growth remains unclear. Although E2-regulated genes have been identified, those critically involved in growth regulation remain elusive. Identification of critical E2-regulated genes involved in mammary cell proliferation would elucidate the key pathway(s) supporting hormone-mediated tumor growth as well as provide insight into mechanisms of resistance to endocrine therapies and potential prognostic and therapeutic targets for ER+ breast cancer treatment. Gene regulated in breast cancer 1 (GREB1) is an estrogen-regulated gene that has been implicated in hormone-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. GREB1 mRNA and protein expression correlate with ER expression in breast tumors and the addition of estrogen to ER+ breast cancer cell lines leads to an increase in GREB1 expression and cell proliferation. The function of GREB1 and its role in ER+ breast tumors remains undefined. Experiments were designed to elucidate the role of GREB1 in ER+ breast cancer cell lines. ER+ breast cancer cell lines were transfected with GREB1-specific siRNA and control species. These tumor cell populations were evaluated for GREB1 mRNA and protein expression, cell growth and proliferation as well as invasion and migration in vitro. Sequence-specific siRNA effectively inhibited GREB1 mRNA and protein expression for more than 96 hours in MCF7 and T47D ER+ breast tumor cell lines. Utilizing the real-time Xcelligence system to monitor cell growth in culture suggested tumor cells treated with siRNA targeting GREB1 had significantly retarded cell proliferation. These observations suggest that GREB1 is a critical E2-regulated gene involved in breast tumor cell proliferation and is an exploitable target for novel breast cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3916. doi:1538-7445.AM2012-3916