Targeting transcription factor activity as a strategy to inhibit pro-inflammatory genes involved in cystic fibrosis: decoy oligonucleotides and low-molecular weight compounds.

The development of drugs able to inhibit the expression of pro-inflammatory genes is of great interest in the treatment of cystic fibrosis (CF). Chronic pulmonary inflammation in the lungs of patients affected by CF is characterized by massive intra-bronchial infiltrates of neutrophils. This process is initiated upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells. Consequently, they release cytokines, the most represented being the potent neutrophilic chemokine Interleukin (IL)-8 and the pro-inflammatory cytokine IL-6. The chronic inflammatory process is crucial, since it leads to progressive tissue damage and severe respiratory insufficiency. In order to reduce the adverse effects of the excessive inflammatory response, one of the approaches leading to inhibition of IL-8 and IL-6 gene expression is the transcription factor (TF) decoy approach, based on intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of TFs and causing inhibition of binding of TF-related proteins to regulatory sequences identified in the promoters of specific genes. Since the promoters of IL-8 and IL-6 contain consensus sequences for NF-κ B and Sp1, double stranded TF "decoy" ODNs targeting NF-κB and Sp1 can be used. Alternatively, screening of drugs targeting relevant TFs can be performed using drug cocktails constituted by extracts from medicinal plants inhibiting TF/DNA interactions. Finally, virtual screening might lead to identification of putative bioactive molecules to be validated using molecular and cellular approaches. By these means, low-molecular drugs targeting NF-κB and inhibiting IL-8 gene expression are available for pre-clinical testing using experimental systems recapitulating chronic pulmonary inflammation of patients affected by CF.

Bangladeshi medicinal plant extracts inhibiting molecular interactions between nuclear factors and target DNA sequences mimicking NF-kappaB binding sites.

Several medicinal plants can be employed to produce extracts exhibiting biological effects. The aim of this work was to verify the ability of extracts derived from different medicinal plants of Bangladesh in interfering with specific DNA-protein interactions. The rationale for this study is based on the observation that alteration of gene transcription represents a very promising approach to control the expression of selected genes and could be obtained using different molecules acting on the interactions between DNA and transcription factors (TFs). We have analysed the antiproliferative activity of extracts from the medicinal plants Hemidesmus indicus, Polyalthia longifolia, Aphanamixis polystachya, Moringa oleifera, Lagerstroemia speciosa, Paederia foetida, Cassia sophera, Hygrophila auriculata and Ocimum sanctum. Antiproliferative activity was assayed on different human cell lines, including erythroleukemia K562, B-lymphoid Raji, T-lymphoid Jurkat and erythroleukemia HEL cell lines. We employed the electrophoretic mobility shift assay (EMSA) as a suitable technique for the identification of plant extracts altering the binding between transcription factors and the specific DNA elements. We found that low concentrations of Hemidesmus indicus, Polyalthia longifolia, Moringa oleifera and Lagerstroemia speciosa, and very low concentrations of Aphanamixis polystachya extracts inhibit the interactions between nuclear factors and target DNA elements mimicking sequences recognized by the nuclear factor kappaB (NF-kappaB). On the contrary, high amount of extracts from Paederia foetida, Cassia sophera, Hygrophila auriculata or Ocimum sanctum were unable to inhibit NF-kappaB/DNA interactions. Extracts inhibiting both NF-kappaB binding activity and tumor cell growth might be a source for anti-tumor compounds, while extracts inhibiting NF-kappaB/DNA interactions with lower effects on cell growth, could be of interest in the search of compounds active in inflammatory diseases, for which inhibition of NF-kappaB binding activity without toxic effects should be obtained.

Peptide nucleic acids (PNA)-DNA chimeras targeting transcription factors as a tool to modify gene expression.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa.

In the present paper we show that extracts from Aegle marmelos Correa are able to inhibit the in vitro proliferation of human tumor cell lines, including the leukemic K562, T-lymphoid Jurkat, B-lymphoid Raji, erythroleukemic HEL, melanoma Colo38, and breast cancer MCF7 and MDA-MB-231 cell lines. Molecules present within the studied Aegle marmelos C. extracts were identified by gas-chromatography/mass-spectrometry analysis; three derivatives (butyl p-tolyl sulfide, 6-methyl-4-chromanone and butylated hydroxyanisole) were found to exhibit strong activity in inhibiting in vitro cell growth of human K562 cells. The antiproliferative activity of these compounds was found to be comparable to that of known antitumor agents, including cisplatin, chromomycin, cytosine arabinoside and 5-fluorouracil. In addition, the antiproliferative activity of butyl-p-tolyl sulfide, 6-methyl-4-chromanone and 5-methoxypsolaren was associated to activation of the differentiation pattern of K562 cells.

Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis.

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be "entrapped" in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using "lab-on-a-chip." In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them.

Molecular interactions with nuclear factor kappaB (NF-kappaB) transcription factors of a PNA-DNA chimera mimicking NF-kappaB binding sites.

The decoy approach against nuclear factor kappaB (NF-kappaB) is a useful tool to alter NF-kappaB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-kappaB specific cis-elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-kappaB transcription factors to PNAs was either very low (binding to PNA-PNA hybrids) or exhibited low stability (binding to PNA-DNA hybrids). The main consideration of the present paper was to determine whether PNA-DNA chimeras mimicking NF-kappaB binding sites are capable of stable interactions with proteins belonging to the NF-kappaB family. Molecular modeling was employed for the design of PNA-DNA chimeras; prediction of molecular interactions between chimeras and NF-kappaB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA-DNA chimeras and NF-kappaB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-kappaB PNA-DNA chimera and duplex NF-kappaB DNA-DNA. However, it was found that these differences do not prevent the duplex PNA-DNA chimera from binding to NF-kappaB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-kappaB DNA-PNA chimeras could be used for a decoy approach in gene therapy.

Aromatic polyamidines inhibiting the Tat-induced HIV-1 transcription recognize structured TAR-RNA.

We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.

Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine.

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.

Characterization of a major histocompatibility complex class II X-box-binding protein enhancing tat-induced transcription directed by the human immunodeficiency virus type 1 long terminal repeat.

The X-box element present within the promoter region of genes belonging to the major histocompatibility complex (MHC) plays a pivotal role in the expression of class II molecules, since it contains the binding sites for several well-characterized transcription factors. We have analyzed a randomly selected compilation of viral genomes for the presence of elements homologous to the X box of the HLA-DRA gene. We found that human immunodeficiency virus type 1 (HIV-1) shows the highest frequency of X-like box elements per 1,000 bases of genome. Within the HIV-1 genome, we found an X-like motif in the TAR region of the HIV-1 long terminal repeat (LTR), a regulative region playing a pivotal role in Tat-induced HIV-1 transcription. The use of a decoy approach for nuclear proteins binding to this element, namely, XMAS (X-like motif activator sequence), performed by transfection of multiple copies of this sequence into cells carrying an integrated LTR-chloramphenicol acetyltransferase construct, suggests that this element binds to nuclear proteins that enhance Tat-induced transcription. In this report we have characterized two proteins, one binding to the XMAS motif and the other to the flanking regions of XMAS. Mobility shift assays performed on crude nuclear extracts or enriched fractions suggest that similar proteins bind to XMAS from HIV-1 and the X box of the HLA-DRA gene. Furthermore, a UV cross-linking assay suggests that one protein of 47 kDa, termed FAX (factor associated with XMAS)-1, binds to the XMAS of HIV-1. The other protein of 56 kDa was termed FAX-2. In a decoy ex vivo experiment, it was found that sequences recognizing both proteins are required to inhibit Tat-induced HIV-1 LTR-driven transcription. Taken together, the data reported in this paper suggest that XMAS and nearby sequences modulate Tat-induced HIV-1 transcription by binding to the X-box-binding proteins FAX-1 and FAX-2. The sequence homology between XMAS and X box is reflected in binding of a common protein, FAX-1, and similar functional roles in gene expression. To our knowledge, this is the first report showing that transcription factors binding to the X box of the MHC class II genes enhance the transcription of HIV-1.

Liposomes as carriers for DNA-PNA hybrids.

Peptide nucleic acids (PNAs) are DNA mimics composed of N-(2-aminoethyl)glycine units. This structure gives to PNAs (a) resistance to DNases and proteinases, (b) capacity to hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, and (c) capacity to form highly stable (PNA)(2)-RNA triplexes with RNA targets. Furthermore, DNA-PNA hybrid molecules are capable to reversibly interact with DNA-binding proteins, being therefore of interest for studies on regulation of gene expression by the decoy approach. The major conclusion of this paper is that cationic liposomes are able to efficiently complexate DNA-PNA hybrid molecules and mediate their binding to target cells. Our results are of some interest, since, unlike commonly used nucleic acids analogs, PNA oligomers are not taken up spontaneously into the cells. In addition, they are not suitable for an efficient delivery with commonly used liposomal formulations. Transfection of PNA-DNA hybrid molecules to in vitro cultured cells could be of great interest to determine the applications of these new reagents to experimental alteration of gene expression.

Computational procedures to explain the different biological activity of DNA/DNA, DNA/PNA and PNA/PNA hybrid molecules mimicking NF-kappaB binding sites.

Peptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigene PNAs have been synthetized and characterized. The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-kappaB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-kappaB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-kappaB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-kappaB binding proteins recognize both the NF-kappaB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-kappaB DNA/PNA hybrids are less stable than those generated with NF-kappaB target DNA/DNA molecules.

Interaction of the human NF-kappaB p52 transcription factor with DNA-PNA hybrids mimicking the NF-kappaB binding sites of the human immunodeficiency virus type 1 promoter.

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-kappaB p52 transcription factor. The binding of NF-kappaB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-kappaB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-kappaB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-kappaB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-kappaB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-kappaB p52 protein, although with an efficiency lower than DNA-DNA NF-kappaB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

Cardiologic complications of subarachnoid hemorrhage.

Cardiac complications are frequent in patients with subarachnoid hemorrhage (SAH). They include ECG abnormalities, cardiac arrhythmias, myocardial damage, and neurogenic pulmonary edema. The pathophysiology of these abnormalities is related to an imbalance of the autonomic cardiovascular control and to increased circulating and local myocardial tissue catecholamines. Cardiac involvement is more common in patients with severe neurological deficits and it may increase the morbidity associated with SAH because of the occurrence of life-threatening arrhythmias or pulmonary edema. Monitoring of cardiac events in patients with SAH might result in a better understanding of their clinical outcome, as well as providing a basis for specific treatment capable of preventing myocardial necrosis and cardiac arrhythmias.

Prophylaxis of thromboembolic events in congestive heart failure.

In patients with heart failure the incidence of thromboembolism is 0.9-5.5%/year (mean 1.9%/year), but no randomized studies are available to support the indication for anticoagulant therapy in those patients. Atrial fibrillation and previous thromboembolic events seem to be the major risk factors, whereas the effect of ventricular dysfunction has not been independently evaluated; nonetheless several studies suggest that thromboembolism is more likely among those patients with lower ejection fraction and lower peak exercise oxygen consumption. Anticoagulant therapy seems to be indicated also in patients with left ventricular aneurysm with mobile and protruding thrombi. Several studies of patients with dilated cardiomyopathy show that the incidence of thromboembolism ranges from 1.6 to 4.5%/year in patients not treated with anticoagulants, while it is virtually absent in anticoagulated patients. The clinical opportunity of long-term anticoagulant treatment in heart failure patients should be weighted not only on the clinical markers of thromboembolic risk, but also on the relative risk/benefit ratio of the single patient.

[Prognostic significance of normal exertion myocardial scintigraphy in asymptomatic subjects with abnormal ergometric test]. / Significato prognostico della scintigrafia miocardica da sforzo negativa in soggetti asintomatici con positività del test ergometrico.

AIM OF THE STUDY: ST-segment depression on exercise stress test (EST) is an independent predictor of future cardiac events. Nevertheless, in apparently healthy subjects without angina the occurrence of false positive results is frequent. Thallium myocardial imaging (TMI) may improve diagnostic and prognostic accuracy of EST. The aim of the present study was to assess the role of a normal exercise TMI for excluding a coronary artery disease in subjects with asymptomatic abnormal EST. METHODS: Subjects referred for TMI from 1/1980 to 5/1991 with an abnormal EST and without history of ischemic, congenital, or valvular heart disease or abnormal resting ECG were included into the study. 137 subjects (98 men, 39 women), mean age 53 +/- 8 yrs (range 37-74 yrs) were enrolled and followed-up for 6.4 yrs (range 3-13 yrs). Clinical indications for EST were: atypical chest pain in 56 (41%) cases, check-up in 52 (38%) cases, sport activity in 29 (19%) cases. All subjects had a maximal symptom-limited EST. Abnormal EST was defined by a horizontal or downsloping > or = 1 mm or upsloping > or = 1.5 mm ST-segment depression at 0.08 sec. from J-point, in at least 2 leads. EST was discontinued for fatigue in 129 (94%) cases, for ST-segment depression > or = 3 mm in 8 (6%) cases. None had chest pain during EST. All subjects selected for the study had normal TMI. Criteria for normal TMI were homogeneous Thallium uptake on postexercise images and a normal washout in the delayed images by qualitative analysis. Planar images were obtained in 118 (86%) cases, and tomographic SPECT images in 19 (14%). RESULTS: During the follow-up period no subject died for cardiac causes and only 9 subjects (1%/yr) had non fatal cardiac events: 4 (0.45%/yr) had a non fatal myocardial infarction (one subject had coronary angiography for postinfarction angina and subsequent 3 coronary bypass graft for multivessels disease), 2 subjects (0.2%/yr) became symptomatic for unstable angina (both had coronary angiography and subsequent PTCA for critical left main coronary artery stenosis) and 3 (0.34%/yr) developed stable angina (one had coronary angiography and subsequent bypass graft for a critical stenosis of left main coronary artery). Four further subjects died for non cardiac events. Comparing clinical data and TE results of subjects with and without coronary events, we found that some parameters were related to a higher incidence of cardiac events: hypertension (78% vs 31% respectively in subjects with and without cardiac events, p < 0.01), hypercholesterolemia (33% vs 4.7%, p < 0.01); > or = 2 conventional coronary risk factors (56% vs 17%, p < 0.02); and a slow regression of abnormal ST-segment depression during recovery (2.8 +/- 2 vs 1.5 +/- 1 min, p < 0.01). CONCLUSIONS: In conclusion, in subjects without typical chest pain and with abnormal asymptomatic EST, a normal exercise TMI identifies subjects with very low risk of future cardiac events (1%/yr). Our data suggest that subjects with abnormal asymptomatic EST should be routinely submitted to exercise TMI.

A rare case of type I aortic dissection.

Aortic dissection is the most common acute disease of the aorta. It can occur in different clinical ways depending on the particular anatomical segments involved. The sudden appearance of claudicatio in the lower limbs may be indicative.

Further characterization of virus obtained from herpes simplex virus type 1 recurrences and primary infections. Influence of the temperature of incubation upon glycoprotein synthesis and virus release. Brief report.

The virus contained in clinical isolates of herpes simplex virus type 1 (HSV-1) which have not undergone previous in vitro passages (new isolates) differs from HSV-1 prototype strains with respect to infected cell glycoprotein pattern, and, most probably efficiency of virus egress at 37 degrees C. The differences can be abolished by lowering the temperature of incubation to 33 degrees C. A few tissue culture passages cause the conversion of the original virus to a virus undistinguishable from HSV-1 prototype strains with respect to the parameters mentioned above.

Restricted replication of BK virus in human lymphocytes.

Human lymphocytes from either peripheral blood or continuous cultures (P3HR-1 cells) are able to support the replication of prototype polyomavirus BK (BKV) as well as its related strain BO15 virus (BO15V). Instead, human monocytes from peripheral blood, although able to bind and egulf BKV virions, do not express virus-specific antigens within a 50 day observation period. In the light of these results, a probable role is suggested for human mononuclear blood cells in the mechanism of natural infection by polyomaviruses.