[Two case reports on resistant hypertension]. / Zwei Fallberichte zur therapierefraktären Hypertonie.

Primary aldosteronism (PA) is a frequent cause of resistant hypertension. The clinical presentation is heterogeneous, but a suppressed or low normal renin (especially with ACE inhibitors or sartans) should raise suspicion for primary aldosteronism, even when aldosterone levels are in the normal range. Diagnosis of unilateral hormone production from an adrenal adenoma (Conn syndrome), which is curable by surgery, requires adrenal vein sampling, which should be performed in experienced centers.

Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters.

Hepatitis C virus (HCV) is a main cause of chronic liver disease, which may lead to the development of liver cirrhosis and hepatocellular carcinoma. Therapeutic options are still limited in a significant proportion of patients. Small interfering RNAs (siRNAs) are an efficient tool to inhibit gene expression by RNA interference. As HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNA-directed antiviral strategies are likely to successfully block its replication cycle. In this study, a panel of siRNAs was used to target various important regions of the HCV genome [5' untranslated region (UTR), NS3, NS4A, NS4B, NS5B, 3' UTR]. Convergent opposing human H1 and U6 polymerase III promoters were used to generate siRNAs. Target genes in sense and antisense orientation were attached to a luciferase reporter system to test the inhibitory efficiency of both siRNA strands. Our data revealed effective RNA interference against the HCV(+)-strand, the HCV(-)-strand or both strands simultaneously up to 65%. Subsequently, active siRNAs were tested in HCV subgenomic replicon cells and suppression of HCV RNA and NS5B protein levels up to 75% was confirmed. Interestingly, siRNAs that were effective against the sense as well as the antisense strand revealed the greatest inhibitory effects on HCV subgenomic replicons. Additionally, combinations of siRNAs induced a greater inhibition of HCV subgenomic replication of up to 90% proving the potential of this combined antiviral approach.

Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication.

Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3'/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n = 53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection.

Expression of heat-shock protein 90 in glucocorticoid-sensitive and -resistant childhood acute lymphoblastic leukaemia.

Early reduction of leukaemic cells by chemotherapy is a strong predictor for treatment outcome in childhood acute lymphoblastic leukaemia (ALL). In ALL-(Berlin-Frankfurt-Münster) trials, early treatment response is assessed by the in vivo response to glucocorticoids (prednisone response, PR), the molecular background of which is unknown. The intracellular effects of glucocorticoids (GCs) are mediated by the glucocorticoid receptor (GR). In the absence of GC, the inactive GR resides within a multiprotein complex, consisting predominantly of the chaperone protein hsp90 (heat-shock protein 90). Until now, studies targeting GC resistance mainly focused on GR disorders and alterations of genes known to be associated with drug resistance. In addition, the GR multiprotein complex was associated with GC resistance in in vitro studies. We performed a case-control study for PR to investigate the association of in vivo GC resistance and hsp90 expression in childhood ALL. Hsp90 expression was assessed using a real-time PCR approach (Taqman technology) and Western blot technology. In this setting, we found no association of in vivo GC resistance and hsp90 expression. Therefore, we conclude that the expression of hsp90, the major component of the GR activating complex, is of minor importance for the in vivo GC resistance in childhood ALL.

Association of initial response to prednisone treatment in childhood acute lymphoblastic leukaemia and polymorphisms within the tumour necrosis factor and the interleukin-10 genes.

Plasma levels of TNF and IL-10 have been associated with therapy outcome in haematological malignancies and are influenced by genetic variation due to germline polymorphisms within the TNF and IL-10 genes. Different TNF and IL-10 genetic polymorphisms might therefore also correlate with clinical outcome in childhood acute lymphoblastic leukaemia (ALL). We analysed the association of TNF and IL-10 polymorphisms with response to initial treatment and risk of relapse in 135 children with ALL, treated according to Berlin-Frankfurt-Münster (BFM) protocols. Our data showed a protective effect from prednisone poor response in patients with the IL-10 G/G genotype, whereas no association of the risk of relapse and IL-10 genotype was found. In the total study group, subjects expressing the TNF2 allele neither showed a statistically significant general association with prednisone response nor with risk of relapse compared to subjects homozygous for the TNF1 allele. Nevertheless, we did find a higher risk of relapse in poor prednisone responders expressing the TNF2 allele compared to poor prednisone responders not expressing the TNF2 allele. We conclude that IL-10 genotype might influence prednisone response in patients with childhood ALL, whereas TNF genotype seems to influence the risk of relapse in high risk ALL patients.

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3'X) targeting the HCV minus-strand replication intermediate at position 40 within the 3'X tail. Surprisingly, Rz3'X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3'X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3'X tail sequences, the results indicate that Rz3'X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome alpha-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3'X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification.

A hairpin ribozyme, RzCR2A, directed against position 323 of the hepatitis C virus 5'-untranslated region (HCV 5'-UTR) was used to establish and validate a novel method for the detection of cellular target molecules for hairpin ribozymes, termed C-SPACE (cleavage-specific amplification of cDNA ends). For C-SPACE, HeLa mRNA containing the transcript of interest was subjected to in vitro cleavage by RzCR2A in parallel with a control ribozyme, followed by reverse transcription using a modified SMART cDNA amplification method and cleavage-specific PCR analysis. C-SPACE allowed identification of the RzCR2A target transcript from a mixture containing the entire cellular mRNA while only requiring knowledge of the ribozyme binding sequence for amplification. In a similar approach, C-SPACE was used successfully to identify human 20S proteasome alpha-subunit PSMA7 mRNA as the cellular target RNA of Rz3'X, a ribozyme originally designed to cleave the negative strand HCV 3'-UTR. Rz3'X was found to substantially inhibit HCV internal ribosome entry site (IRES) activity and PSMA7 was subsequently confirmed to be involved in HCV IRES-mediated translation. Thereby, C-SPACE was validated as a powerful tool to rapidly identify unknown target RNAs recognized and cleaved by hairpin ribozymes.

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach.

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an "inverse genomics" approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer.

Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach.

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bgamma) and 2 (eIF2gamma), respectively. The involvement of eIF2Bgamma and eIF2gamma in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes.

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.

The importance of subjective claims management.

This article discusses the causes and effects of "subjective disability" on today's workforce and employers. As employees feel out of control with both their careers and demands placed upon them, the number of claims characterized by self-reported symptoms are increasing. Subjective disabilities include chronic syndrome, fibromyalgia, psychiatric claims and chronic pain. The author discusses creative strategies in case studies that have helped employers contain the costs of disability claims, while empowering the employee to take control of their own situation and return to work sooner.