Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A.

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.

Gene silencing of virus replication by RNA interference.

Small interfering RNAs (siRNAs) are as effective as long double-stranded RNAs (dsRNAs) at targeting and silencing genes by RNA interference (RNAi). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. They are also promising reagents for developing gene-specific therapeutics. The specific inhibition of viral replication is particularly well suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on the recent advances in siRNA-based clinical trials.

Inhibition of syncytium formation by antisense oligonucleotide phosphorothioates complementary to tax mRNA of human T-cell leukemia virus type 1 (HTLV-1).

HTLV-1 infection is known as the factor to cause adult T-cell leukemia (ATL). Antisense oligonucleotide phosphorothioates against tax gene and control oligonucleotide phosphorothioates were synthesized. Antisense oligonucleotide was complementary to the region of initiation codon of tax gene. Two control oligonucleotides were tax sense and random. HTLV-1-positive human T-cell line, C91/PL and HTLV-1 non-infected human glioma cell line, U251-MG were co-cultured in the presence of antisense or control oligonucleotides for 24 hours. Oligonucleotides used in this study were not toxic at 10 microM concentration. Antisense oligonucleotide against tax gene inhibited 59% the syncytium formation assay at 10 microM concentration.

Specific inhibition of human telomerase activity by transfection reagent, FuGENE6-antisense phosphorothioate oligonucleotide complex in HeLa cells.

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides.

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.

Inhibition of HIV-1 replication by a two-strand system (FTFOs) targeted to the polypurine tract.

Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA-binding proteins can be inhibited by the use of double-stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN-kappaB) and circular (CDODN-kappaB) dumbbell DNA oligonucleotides containing a NF-kappaB binding site to analyze the inhibition of the NF-kappaB-dependent activation of the human immunodeficiency virus type-1 (HIV-1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF-kappaB sequence were found to compete with the native strand for NF-kappaB binding. The circular dumbbell and double-stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF-kappaB binding proteins, while the nicked NF-kappaB dumbbell was a less effective competitor. In Jurkat T-cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV-NL4-3 Luc. The CDODN-kappaB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN-kappaB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non-specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti-HIV-1 or other therapeutic effects.

Difference in prognostic value between sialyl Lewis(a) and sialyl lewis(x) antigens in blood samples obtained from the drainage veins of the colorectal tumors.

Subtraction values, (i.e. values obtained by subtracting the serum titer of sialyl Lewis(a) (CA19-9) and sialyl Lewis(x) (SLX) antigens in peripheral venous blood from the serum titer of the same antigen in the tumor's drainage venous blood) were determined in order to clarify whether or not such values for these specific antigens (d-CA19-9 and d-SLX) are prognostic factors after resection for colorectal cancer. The blood samples were obtained from 144 colorectal cancer patients during surgical excisions of the tumors. Univariate and multivariate analyses revealed that d-SLX level was an independent prognostic factor, separate from stage, while d-CA19-9 level did not have any additional prognostic value. In conclusion, a high d-SLX level is a predictor of poor outcome after surgery.

Acquisition of HIV type 1 resistance by beta-chemokine-producing CD4+ T cells.

The CD4+ T cell is a major target cell type for human immunodeficiency virus type 1 (HIV-1) infection. In this study, we provide evidence that the susceptibility to HIV-1 infection is variable in individual CD4+ T cells. Five CD4+ T cell clones were isolated from an HIV-1-seronegative donor and were investigated for their susceptibility to HIV-1 infection. Four CD4+ T cell clones were resistant to infection by a macrophage-tropic (R5) HIV-1 isolate whereas one clone was fully permissive. The level of susceptibility to HIV-1 correlated inversely with beta-chemokine production, including RANTES (regulated on activation, normally T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. Resistance to HIV-1 infection was abrogated by the combined use of neutralizing antibodies against these three beta-chemokines. Interestingly, a complete inhibition of HIV-1 infection was observed in peripheral blood mononuclear cells on infection induced by adding the culture supernatant or a small number of HIV-1-resistant cell clones. Our results suggest the presence of a clonal self-defense mechanism within the CD4+ T cell population in vivo that involves the secretion of beta-chemokines.

Wear properties of Ti and Ti-6Al-7Nb castings for dental prostheses.

Titanium has been increasingly applied to dental prostheses because of its biocompatibility. However, application remains limited, due to the low strength and poor wear resistance of unalloyed titanium. The purpose of this study is to evaluate the wear resistance of high-strength Ti-6Al-7Nb alloy castings for dental application. Test specimens were cast from commercially pure titanium (CP-Ti grades 2 and 3) and Ti-6Al-7Nb alloy ingots, and subjected to a wear test simulating the occlusal loading pattern. Wear resistance was evaluated by the weight loss during the test. Ti-6Al-7Nb alloy was found to exhibit lower weight loss than CP-Ti. Moreover, scanning electron microscopic (SEM) observation after the test revealed that the worn surface of Ti-6Al-7Nb alloy is much smoother than that of CP-Ti. These results indicate that Ti-6Al-7Nb alloy castings can be used to produce dental prostheses of improved wear resistance and mechanical strength.

Non-pressure adhesion of a new adhesive restorative resin.

Non-pressure adhesion of a new adhesive restorative resin was investigated employing a new tensile test. The material was adhesive to both enamel and dentin as well as to carious dentin and showed strong adhesion to all substrates tested. Etching further increased the adhesion even to dentin.

Long-term, high dose interferon-alpha treatment in HTLV-I-associated myelopathy/tropical spastic paraparesis: a combined clinical, virological and immunological study.

The efficacy of long-term, high dose interferon-alpha (IFN-alpha) therapy was studied in seven patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). IFN-alpha was administered at a dose of 6 x 10(6) international units daily for the initial 2 weeks and thereafter 3 times a week for the following 22 weeks. Five patients showed a sustained improvement in motor performance during and up to 6 months after the completion of IFN-alpha. The other patient who responded to IFN-alpha initially dropped out at 3 months because of depression, while another patient first deteriorated and thereafter dropped out. In the six responders, the absolute number of peripheral blood lymphocytes (PBL) harboring the HTLV-I genome as evaluated by the quantitative polymerase chain reaction method decreased significantly during the therapy period (28.6 +/- 16.6% reduction, P = 0.0083), whereas the one deteriorated patient showed a 2.5-fold increase in HTLV-I-infected cells. The autoproliferation of CD4+ T clone cells from a single cell culture was markedly depressed even after the cessation of IFN-alpha in the responders who completed long-term IFN-alpha therapy. In addition, the CD8+DR+ T cells in the peripheral blood and soluble IL-2 receptor levels in the sera increased significantly during the therapy in all patients (P = 0.0431 and P = 0.0041, respectively). Therefore, the results of our study suggested that both the reduction of HTLV-I proviral DNA load and immunomodulation by long-term IFN-alpha therapy contributed to its sustained clinical benefits.

Circulating sialyl Lewis(x), sialyl Lewis(a), and sialyl Tn antigens in colorectal cancer patients: multivariate analysis of predictive factors for serum antigen levels.

Preoperative serum levels of sialyl Lewis(a) (CA 19-9), sialyl Lewis(x) (SLX), and sialyl Tn (STN) antigens in colorectal cancer patients were examined to establish predictive factors for serum levels of these antigens compared with carcinoembryonic antigen (CEA). A total of 308 patients who underwent resection for a colorectal cancer were divided into low and high antigen groups (higher or lower than a selected diagnostic-based cutoff value). The cutoff values were 37 U/ml for CA19-9, 38 U/ml for SLX, 45 U/ml for STN, and 2.5 ng/ml for CEA. The American Joint Committee on Cancer Classification and Stage grouping was used to classify the tumors. Statistical tests were conducted using univariate and multivariate logistic regression analyses. For CA19-9, 81 patients (26.3%) were assigned to the high antigen group: for SLX, 39 (12.7%); for STN, 33 (10.7%); and for CEA, 133 (43.2%). Multivariate logistic regression analysis revealed that predictive factors associated with high antigen levels were female sex (odds ratio [OR], 1.78 vs male sex), T4 (OR, 3.26 vs T1/T2), and M1 (OR, 3.35 vs M0) for CA19-9; M1 (OR, 6.40 vs M0) for SLX; mucinous carcinoma (OR, 8.45 vs well differentiated adenocarcinoma) and M1 (OR, 8.24 vs M0) for STN; and mucinous carcinoma (OR, 7.21 vs well differentiated adenocarcinoma), T3/T4 (OR, 3.84/4.18, respectively, vs T1/T2), and M1 (OR, 6.39 vs M0) for CEA. In conclusion, high serum levels of CA19-9, SLX, and STN are strongly associated with distant metastasis. In addition, high serum levels of CA19-9 may be an independent predictor for female gender and T4, and high serum levels of STN may be an independent predictor for mucinous carcinoma.

Anti-human immunodeficiency virus type 1 of a two-strand-system targeted to the polypurine tract.

The polypurine tract (PPT) is highly conserved among the known human immunodeficiency virus (HIV)-1 strains, and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand-system (FTFOs) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In the observation of the FITC-DsDGloopT5-37 with MOLT-4 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm and nucleus. However, weak fluorescence was observed within the cytoplasm and nucleus of MOLT-4 cells treated with the antisense phosphorothioate oligonucleotides (S-ODN-gag-AUG). In HIV-1 infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Prognostic value of circulating sialyl Tn antigen in colorectal cancer patients.

To examine the prognostic value of assessing the concentration of circulating sialyl Tn antigen (STN) after surgery, we determined serum STN levels in peripheral venous blood (designated "p-") in 308 colorectal cancer patients and what we have termed the "d-p gradient" (obtained by subtracting the serum concentration in peripheral venous blood from that in the tumor's drainage venous blood) in 144 patients. The prognostic value of STN and carcinoembryonic antigen (CEA) was compared. Patients were divided into low or high p-antigen groups and low, intermediate, or high d-p gradient groups. Univariate and multivariate analyses revealed that high STN d-p gradient, high p-CEA, or high CEA d-p gradient were each independent variables for poor patient outcome after surgery, separate from stage. In conclusion, a high STN d-p gradient was a predictor of poor outcome after resection for colorectal cancer, while p-STN was not independent of stage.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Increased serum level of sialyl Lewis(x) antigen in blood from the tumor drainage vein in patients with non-polypoid growth type of colorectal cancer.

Two types of colorectal cancer with distinct morphologies have been described in recent studies: polypoid growth type (PG-type) and non-polypoid growth type (NPG-type). We hypothesize that the morphologic differences may correspond to additional biological distinctions. Ratios of sialyl Lewisa (CA 19-9), sialyl Lewisx (SLX), or carcinoembryonic antigen (CEA) in the venous blood drainage from the tumor to that of the respective antigen in the peripheral venous blood (d/p ratio) was examined in order to ascertain whether or not the ratio is correlated with either the PG-type or NPG-type colorectal tumor growth pattern. Blood samples from 118 patients with colorectal cancer were obtained from a peripheral vein and from the tumor drainage vein during surgical excision of the tumor. Statistical tests were conducted by univariate and multivariate (logistic regression) analyses. Among the cancers examined there were 17 PG-type (14.4%) and 101 NPG-type (85.6%). NPG-type cancers had a higher frequency of moderately differentiated adenocarcinoma cells and T3/T4 tumors than PG-type cancers (P<0.0001 and P<0.0001, respectively). NPG-type cancers had a more advanced stage than PG-type cancers (P=0.0007). The d/p ratio of SLX in NPG-type cancers was significantly higher than that in PG-type cancers (P=0.028). Multivariate logistic regression analysis showed that three variables, namely histologic type, T factor, and d/p ratio of SLX, were independently related to tumor growth patterns. In conclusion, NPG-type cancers are characterized by a high SLX d/p ratio, which may be at least partly responsible for a different tumor progression pattern compared to other cancer types.

Neuroblastoma in infants aged less than 6 months: is more aggressive treatment necessary? A report from the Pediatric Oncology Study Group of the Kyushu area.

Infants with neuroblastoma are known to have a better prognosis than older children. In Japan in 1985, mass screening for neuroblastoma in infants aged 6 months was introduced. With this policy, there has been an increase in the number of patients seen with neuroblastoma between 6 and 11 months of age. In a previous report the authors described the management and prognosis of infants with disease detected by mass screening, but there is still little information regarding the strategies of management for infants with neuroblastoma aged less than 6 months. The authors analyzed the data regarding 27 patients aged less than 6 months registered in their region (population 15 million) from 1985 to 1992, and compared it with that of the previous 8-year period. In the younger age group, there was a significantly higher rate of advanced disease stages (III and IV). In spite of the variation in treatment related to the choice of individual institutions, infants with stages I, II, and III disease had a good outcome, suggesting that aggressive chemotherapy is not necessary unless poor prognostic factors are present. One patient with stage IV disease died of disseminated disease and one with stage IVs and 22 copies of N-myc oncogene also died of tumor relapse in spite of aggressive chemotherapy. It is therefore concluded that the prognosis in infants with stage IV and IVs neuroblastoma under the age of 6 months is not as good as had previously been believed, and that such patients, therefore, require special consideration.

Mass screening for neuroblastoma: quo vadis? A 9-year experience from the Pediatric Oncology Study Group of the Kyushu area in Japan.

Since 1985, a nationwide program of mass screening for neuroblastoma has been available for 6-month-old infants throughout Japan. From 1985 to 1993, the authors studied 285 patients with neuroblastoma among their regional population of 15 million. There was an increase in the total number of patients per year in comparison to the previous 6-year period (1979 to 1984). However, no significant difference was noted in the number of patients older than 1 year or in the incidence of advanced-stage (stages III and IV) unscreened cases. The majority of neuroblastomas in the screened group showed favorable biological factors, even in the advanced stages. However, there was a small group with histologically and/or biologically unfavorable factors; five of 115 had amplified N-myc oncogene, four of 74 showed unfavorable Shimada histological findings, and three of 33 had an unfavorable DNA ploidy pattern. One case from this group with unfavorable factors died of the tumor. 3) Thirty-eight cases were negative at the time of mass screening, but later presented with neuroblastoma. Most of them were diagnosed between 1 and 3 years of age, and 30 of the 38 cases (78.9%) were advanced stage with unfavorable prognostic factors. Thus, the authors conclude that mass screening at 6 months can detect a selected population of infants with neuroblastoma; some of the tumors may represent subclinical masses destined for spontaneous regression. However, some tumors with unfavorable factors have been detected by mass screening before progression and/or dissemination. Infants in this group are considered to benefit most from early diagnosis and treatment.

Improved survival for patients with advanced neuroblastoma after high-dose combined chemotherapy based in part on N-myc amplification.

BACKGROUND/PURPOSE: In spite of many different kinds of chemotherapy for neuroblastoma, the prognosis for advanced neuroblastoma remains unsatisfactory. In particular, the outcome of advanced neuroblastoma with high copies of the N-myc gene tend to be poor. Therefore, the new high-dosage combined chemotherapy regimens for advanced neuroblastoma based in part on the N-myc amplification status has been utilized in the Kyushu area of Japan since 1991. This study aims to investigate whether these new regimens based in part on N-myc amplification have improved the survival rate of stage III and stage IV patients in comparison with the old regimens. METHODS: Between 1983 and 1995, 77 patients over 1 year of age and with stage III or IV neuroblastoma were registered in the Kyushu Area. Between 1983 and 1990, 49 patients received 1 of 2 combined chemotherapy regimens consisting of cyclophosphamide, cisplatin plus VM-26, and Adriamycin plus DTIC. Since 1991, two new regimens (New A1 and A3) have been administered based on the N-myc amplification status in a total of 28 patients. The New A1 regimen, which consists of cyclophosphamide, cisplatin, Adriamycin, and VP-16 has been administered in cases of less than 10 copies of N-myc, whereas the A3 regimen, consisting of a higher dose of cyclophosphamide, cisplatin, Adriamycin, and VP-16, has been administered in cases of more than 10 copies of N-myc. The survival rate was then compared between the old regimens and the new regimens. RESULTS: The 3-year survival rate (61.5%) for patients treated by the new regimens was significantly higher than that (32.7%) for patients treated by the old regimens (P <.01). Regarding the 24 cases of more than 10 copies of N-myc, the 3-year survival rate (35.9%) of the 13 patients treated by the A3 regimen was higher than that (0%) of the 11 patients treated by the old regimens (P <.05). However, in the 19 stage IV patients treated by the new regimens, the 3-year survival rate (11.1%) of the 9 cases of more than 10 copies was significantly lower than that (77.8%) of the 10 cases of less than 10 copies of N-myc (P <.01). CONCLUSIONS: These results suggest that high-dose combined chemotherapy based in part on the N-myc amplification status significantly improved the prognosis of patients with advanced neuroblastoma. However, stage IV patients with N-myc amplification still require a more effective treatment modality.