Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo.

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers.

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers.

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.

Inhibition of Vesivirus infections in mammalian tissue culture with antisense morpholino oligomers.

Caliciviruses infect and cause disease in animals and humans. They are nonenveloped, positive-stranded RNA viruses with a genome of approximately 7.5 kb that encodes viral proteins in three open reading frames (ORF). Antisense oligomers targeting one of the three ORF of caliciviruses of the genus Vesivirus significantly inhibit viral replication in tissue culture. Porcine kidney and African green monkey kidney cells were infected with Vesivirus isolates SMSV-13 and PCV Pan-1. Phosphorodiamidate morpholino oligomers (PMO) with sequence complementary to the AUG translation start site regions of ORF1, ORF2, and ORF3 were evaluated for their effect on viral titer. Scrape-loading delivered PMO to 50%-70% of the cells of the two cell lines, as measured by fluorescence microscopy and flow cytometry. A PMO targeting ORF3 caused a significant increase in viral titer. A PMO targeting ORF2, a scrambled PMO control sequence, and an unrelated PMO antisense sequence did not alter viral titer. Various PMO sequences antisense to an upstream region of ORF1 were effective in reducing viral titer up to 80% in a dose-dependent and sequence-specific manner. The extent of viral titer reduction was proportional to the delivery of PMO to cells. These observations demonstrate that antisense PMO can disrupt caliciviral gene function in a nucleic acid sequence-specific manner and are potentially effective antiviral agents.

Arthroscopic stabilization of anterior shoulder instability: a historical perspective.

The role of arthroscopic procedures in the management of glenohumeral instability continues to evolve and represents an effective alternative for addressing the pathology associated with this condition. Patient selection criteria, operative techniques, and implants all continue to evolve and have resulted in improved rates of success. Arthroscopic procedures benefit patients by avoiding the common morbidities associated with the disruption of the anterior soft tissues, including a loss of external rotation associated with open procedures. Arthroscopic procedures remain technically demanding and require skills to address all of the existing pathology. The surgeon must be prepared to address many conditions beyond the Bankart lesions including glenoid bone lesions. capsular laxity, rotator interval lesions, and SLAP lesions. In addition to the documentation of recurrence, the success of this procedure must be evaluated within the context of retained ranges of motion, recovery time, proprioceptive control, and the return to prior levels of activity. Further studies are necessary to continue to validate the efficacy of arthroscopic stabilization.

c-Myc antisense limits rat liver regeneration and indicates role for c-Myc in regulating cytochrome P-450 3A activity.

Expression of c-myc protein is associated with cell proliferation. The present study uses antisense oligomers to inhibit c-myc expression in the regenerating rat liver after 70% partial hepatectomy (PH). Antisense phosphorodiamidate morpholino oligomers (novel DNA analogs) were administered i.p. immediately after surgery to block expression of c-myc within the first 24 h after PH. A 20-mer PMO complimentary to the c-myc mRNA at the translation start site was an effective sequence (AVI-4126, 5'-ACGTTGAGGGGCATCGTCGC-3'). A single i.p. dose of 0.5 mg/kg AVI-4126 caused reduction of the regenerating liver c-myc protein in a sequence-specific and dose-dependent manner. Inhibition of c-myc expression resulted in reduction of proliferating cell nuclear antigen and arrested cells in the G(0)/G(1) phase of the cell cycle. The ratio of G(2):G(0) cell populations in the regenerating liver 24 h after PH dropped from 29.1 in saline vehicle-treated rats to 18.0 in rats treated with 2.5 mg/kg AVI-4126. The expression of cell cycle checkpoint protein p53 was inhibited with increasing doses of AVI-4126, but expression of p21(waf-1) was unaffected. The activity of cytochrome P-450 3A2 (CYP3A2) was evaluated by immunoblot analysis and erythromycin N-demethylation. AVI-4126 did not alter CYP3A activity in nonhepatectamized animals but showed a dose-dependent decrease in PH rats. We conclude that AVI-4126, antisense oligomer to c-myc, can reduce cell proliferation in the regenerating rat liver. Furthermore, inhibition of c-myc may indirectly influence the expression of CYP3A.

[Significance of coronary thrombosis for chronic myocardial ischemia]. / Die Bedeutung der Koronarthrombose für die chronische Myokardischämie.

Apart from the relevance of disorders of lipid metabolism for the clinical and morphological progression of coronary artery disease, coronary thrombosis has received increasing attention in recent years. It is undoubtedly the decisive factor in the pathogenesis of acute coronary syndromes, which is underlined by the therapeutic success of various antithrombotic interventions. Furthermore coronary thrombosis is regarded to be a key factor for morphological disease progression also in stable coronary syndromes, which eventually may lead to critical limitation of myocardial perfusion. This is caused by the formation of subclinical coronary thrombi, which either undergo endogenous lysis or become morphologically fixed as they are incorporated into the plaque. Besides local factors, systemic disturbances of hemostasis and endogenous thrombolysis are of relevance. The concept of thrombotic progression of coronary thrombosis is supported by data on the reduction of morphological disease progression or antiischemic effectiveness of anti-thrombotic interventions like aspirin, low-molecular weight heparin and low-dose intermittent urokinase therapy. Percutaneous transluminal coronary angioplasty results in deep mechanical injury of the vessel wall, which is accompanied by secondary coronary thrombosis in the majority of the cases, not necessarily leading to abrupt vessel closure. Particularly, dilatation of primary thrombus as it has been described as the substrate of the culprit lesion in unstable coronary syndromes, promotes release of thrombin and activation of platelets, which in turn furthers the proliferative processes in the pathogenesis of restenosis. Even though data on the reduction of the rate of restenosis by the use of platelet aggregation inhibitors like aspirin, ticlopidin and dipyridamole have not consistently supported this concept, the EPIC. Study has shown that even in patients with stable angina pectoris clinical restenosis rate may be reduced by a platelet-IIb/IIIa-antagonist.

Enhanced cancer growth in mice administered daily human-equivalent doses of some H1-antihistamines: predictive in vitro correlates.

BACKGROUND: Present studies of drug-induced tumor growth promotion have evolved from earlier investigations into the mechanism of action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy[ethanamine.HCl, a tamoxifen derivative which potently inhibits lymphocyte mitogenesis in vitro and stimulates tumor growth in vivo. It is thought that potency to bind to intracellular histamine receptors (HIC), some of which are on cytochromes P450, may correlate with tumor growth-promoting activity. PURPOSE: We assessed the effectiveness of five in vitro assays in predicting in vivo tumor growth stimulation by the H1-antihistamines loratadine, astemizole, cetirizine, hydroxyzine, and doxylamine. METHODS: Potency of each agent was ranked 1-5 in each of the following in vitro assays: 1) inhibition of [3H]histamine binding to microsomal HIC, 2) inhibition of histamine binding to microsomal P450, 3) inhibition of the P450-catalyzed demethylation of aminopyrine, 4) inhibition of lymphocyte mitogenesis, and 5) stimulation of tumor colony formation. An overall rank score was assigned to each drug and correlated with tumor growth stimulation in vivo. Two laboratories conducted in vivo studies in a blinded fashion. Female C57BL and C3H mice were given a subcutaneous injection on day 1 of syngeneic B16F10 melanoma cells (5 x 10(5)) or C-3 fibrosarcoma cells (1 x 10(5)), respectively. Mice were randomly assigned to treatment groups, then received a single, daily intraperitoneal injection of an estimated human-equivalent dose (or range of doses) of antihistamine or vehicle control for 18-21 days before being killed. Tumors were surgically removed and wet weights compared statistically among groups. RESULTS: The cumulative potency of each drug in affecting tumor growth or growth mechanisms in the five in vitro assays ranked as follows: Loratidine and astemizole ranked highest and were equally potent, followed in decreasing order by hydroxyzine, doxylamine, and cetirizine. A significant correlation (r = .97; P < .02) was observed between the rank order of potency of the antihistamines in all five in vitro assays and the rank order to enhance tumor growth in vivo: Loratidine and astemizole significantly (P < .001) promoted the growth of both melanoma and fibrosarcoma, hydroxyzine significantly (P < .001) promoted the growth of melanoma, while doxylamine and cetirizine did not promote the growth of either tumor. CONCLUSION: Data demonstrate that the in vitro assays predicted the propensity of each H1-antihistamine to stimulate cancer growth in vivo. IMPLICATION: These in vitro tests may prove valuable to screen potential tumor growth promoters.

Evaluation and management of seizures in the patient with cancer.

Seizures are common in patients with cancer. They can be caused by the tumor itself, metabolic disturbances, radiation injury, chemotherapy-related encephalopathies, cerebral infarctions, or central nervous system infections. Evaluation requires a meticulous history and search for the precipitating cause. Treatment is directed at the underlying etiology and entails the rational and precise use of anticonvulsant drugs.

Structural isoforms of a membrane transport protein from Leishmania enriettii.

A membrane transport protein of the glucose transporter superfamily from Leishmania enriettii is encoded by a family of tandemly repeated genes. The first gene in this tandem repeat codes for a structural isoform that contains a unique amino-terminal hydrophilic domain, probably located in the cytoplasm; the remainder of the protein is identical to the polypeptide encoded by the internal genes in the tandem repeat. The unique isoform is represented by a distinct stable RNA.

Deposits of crystalline material containing silicon in surgically excised human valves.

Ninety-seven surgically excised natural cardiac valves were examined by scanning electron microscopy and x-ray energy spectroscopy to assess the occurrence of crystalline deposits that contain the element silicon. Valves examined included 33 mitral valves, 63 aortic valves, and 1 tricuspid valve. To reduce the possibility of surface contamination, the deep layers of some valves were examined after exposure by fracture of the valve. Crystalline material containing silicon was observed in the deep tissue. Such crystalline material was sometimes entwined within subendothelial fibers. Crystalline deposits that contained silicon were associated with 34 of 97 of these valves (35%). Among the 34 valves that showed silicon, 24 (71%) also showed microdeposits of calcific material. In view of evidence that silicon may participate in the calcification of bone, and is found in the intima of arteries, a role for this element in ectopic calcification of valves may exist.

The prospective use of population pharmacokinetics in a computer-driven infusion system for alfentanil.

Maitre et al. recently evaluated the accuracy of a set of previously determined population pharmacokinetic parameters for the opioid alfentanil using data from an earlier study in which the drug had been administered using a computer-controlled infusion pump (CCIP). The present study evaluated the accuracy of these same parameters in a CCIP prospectively in two groups of clinically dissimilar patients: 29 healthy female day surgery patients and 11 relatively older and less healthy male inpatients. In addition, another set of pharmacokinetic parameters, previously determined by Scott et al. in the CCIP in 11 male inpatients was also evaluated. The bias and inaccuracy were assessed by the median performance error (MDPE) and the median absolute performance error (MDAPE) in which the performance error was determined as the difference between measured and target serum concentration as a fraction of the target serum concentration. Unlike Maitre et al., the current study found a consistent bias in both populations. The MDPE was +53% and the MDAPE was 53%, with no difference between patient groups. In the 11 patients studied using the Scott et al. pharmacokinetic parameters, the MDPE was +1% and the MDAPE was 17%. The parameters of Scott et al. were further tested by simulating the serum concentrations that would have been achieved had they been used in the CCIP in the first 40 patients; results indicated MDPE of +2% and an MDAPE of 18%. Therefore, reasonably reliable and accurate target serum concentrations of alfentanil can be achieved using the pharmacokinetic parameters of Scott et al. in a CCIP.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmentally regulated transporter in Leishmania is encoded by a family of clustered genes.

We have previously cloned a gene for a developmentally regulated transport protein from the trypanosomatid protozoan Leishmania enriettii. We demonstrate here that this transporter is encoded by a single family of tandemly clustered genes containing approximately 8 copies of the 3.6 kilobase repeat unit. Transcriptional mapping defines a contiguous 3.3 kilobase region of the repeat unit that encodes the mRNA. The 5' end of the mature mRNA contains the spliced leader or mini-exon previously identified in kinetoplastid protozoa, while the 3' ends of the mRNA are heterogeneous in sequence and in location of the polyadenylation site. We have identified genomic restriction fragments that flank the tandem repeat on the 5' and 3' sides and which may be linked to sequences required for expression of the gene family. Other species of Leishmania also contain sequences that hybridize to the cloned L. enriettii gene at high stringency.

Analysis of pleural effusions using automated flow cytometry.

Flow cytometry allows rapid and accurate analysis of the deoxyribonucleic acid (DNA) content of a large number of cells. In solid tumors, the presence of aneuploidy has been shown to correlate wall with the presence of neoplastic cells. Both cytologic examination and DNA analysis by flow cytometry were performed on pleural effusions from 33 patients. Results of the two examinations were in agreement in 10 of 12 malignant pleural effusion (two false-negatives) and in 20 of 21 benign effusions. One patient with cirrhosis, ascites and Nocardia pneumonia had hypodiploid cells (false-positive) in the pleural fluid. All patients who had a malignancy, but whose pleural effusion proved to be due to a benign cause, had cells with normal DNA content in their pleural effusion. DNA analysis using flow cytometry can be rapidly performed and is highly specific and sensitive. The finding of hyperdiploid cells is highly suggestive of malignancy.

Levels of angiotensin-converting enzyme in pleural effusion.

The value of determining the angiotensin-converting enzyme (ACE) in serum has been clearly established, particularly in sarcoidosis, but is significance in pleural effusions is practically unknown. In the present study, ACE level was determined in the pleural fluid of 18 hospitalized patients along with the values for total protein, lactic acid dehydrogenase (LDH), complete and differential cell counts and cytology. The ACE level was higher in exudates than in transudates and paralleled closely the total protein level in the pleural fluid. There was no correlation between ACE and LDH levels and either WBC or RBC counts in the effusions. The ACE levels failed to discriminate between benign and malignant diseases or to correlate with any specific etiology for the effusion. There was a significant gradient between ACE levels that were higher in the serum than in the effusion of all nine patients in whom both were measured. The filtration coefficient (ratio of pleural fluid to serum concentration of a protein) of ACE was less than that of total protein and was compatible with ACE penetration in the pleural fluid by a process of diffusion.