Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa-2b.

Treatment with hepatitis C virus (HCV)-NS3-protease inhibitors lead to the selection of resistant variants. Viral kinetics and resistance profiles in patients who are re-treated with the same protease inhibitor are unknown. Viral kinetics and NS3-resistance mutations obtained by clonal sequencing of the NS3-protease were analyzed in nine HCV-genotype-1-infected nonresponder patients who were sequentially treated with boceprevir (400 mg t.i.d.) for 1 week, peginterferon-alfa-2b for 2 weeks and combination of the two for 2 weeks in varying order. In addition to predominant wild-type isolates, previously described boceprevir-resistant mutations (V36, T54, R155, A156, V170) were observed. Furthermore, two resistant mutations (Q41, F43) were detected for the first time in vivo. In three patients, mutations selected after initial treatment with boceprevir were re-selected during subsequent boceprevir exposure. However, mutational patterns after the first and second exposure to boceprevir were different in five patients. In one patient, a viral variant (V55A) known to reduce susceptibility to boceprevir was the predominant variant observed at baseline and throughout treatment and was associated with a shallow viral decline. Different resistance mutations were selected during treatment with boceprevir ± peginterferon. Sequential short-term dosing of boceprevir was not associated with accumulation of resistant variants but pre-existing variants may impair virologic response.

Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease.

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.

Quantitative genetics of age-related retinal degeneration: a second F1 intercross between the A/J and C57BL/6 strains.

PURPOSE: Previously, several quantitative trait loci (QTL) that influence age-related retinal degeneration (ageRD) were demonstrated in a cross between the C57BL/6J-c(2J) and BALB/cByJ strains (B x C). In this study, as a complementary approach to ongoing recombinant progeny testing for the purpose of identifying candidate quantitative trait genes (QTG), a second test cross using the A/J and the pigmented C57BL/6J strains (A x B) was carried out. The albino A/J strain was selected because it had the most amount of ageRD among several inbred strains tested, and the pigmented C57BL/6J strain was selected because along with its coisogenic counterpart C57BL/6J-c(2J) it had the least amount of ageRD. Thus, the effect of pigment on ageRD could be tested at the same time that the C57BL/6 genetic background was kept in common between the crosses from the two studies for the purpose of comparison. METHODS: A non-reciprocal F1 intercross between the A/J and C57BL/6J strains produced 170 F2 progeny. At 8 months of age after being maintained in relatively dim light, F2 mice, control mice and mice of other strains were evaluated for retinal degeneration by measurement of the thickness of the outer nuclear layer of the retina. The F2 mice were genotyped with dinucleotide repeat markers spanning the genome. Correlation of genotype with phenotype was made with Map Manager QTX software. RESULTS: Comparison of several strains of mice including the pigmented strains 129S1/SvImJ and C57BL/6J and the albino strains A/J, NZW/LacJ, BALB/cByJ and C57BL/6J-c(2J), showed significant differences in ageRD. The greatest difference was between the albino A/J strain and the pigmented C57BL/6J strain. However, there was no significant difference between the pigmented C57BL/6J and its albino coisogenic counterpart C57BL/6J-c(2J). Neither was there significant difference between the pigmented and albino F2 mice from the A x B cross. On the other hand, F2 males had a small but significantly lower amount of ageRD than females. Several QTL were identified in the A x B cross but surprisingly none of the 3 major QTL present in the original B x C cross (Chrs 6, 10, and 16) was present. There were minor QTL on proximal Chr 12 and proximal Chr 14 in common between the two crosses, and the proximal Chr 12 QTL was present in a previous light damage study involving the B and C strains. At least one sex-limited QTL was present on the X chromosome with a peak in a different location from that of a sex-limited QTL in the previous B x C study. In addition, the protective X allele was from the BALB/cByJ strain in the B x C cross and from C57BL/6J in the A x B cross. In both crosses, the C57BL/6J X-chromosome allele was recessive. CONCLUSIONS: Significant differences were observed in ageRD among several inbred strains of mice maintained in relatively dim light. AgeRD was not influenced by pigment but was influenced by gender, albeit to a small degree. The presence of the same QTL in one light-induced and two ageRD studies suggests at least partial commonality in retinal degeneration pathways of different primary cause. However, the three main QTL present in the B x C cross were absent from the A x B cross. This suggests that the genetic determinants responsible for the greater sensitivity to ageRD of BALB/cByJ and A/J relative to C57BL/6J are not the same. This is supported by the presence of sex-limited X-chromosome QTL in the two crosses in which the C57BL/6J allele is protective relative to the A allele and sensitive relative to the C allele. The findings in the two studies of differing allelic relationships of QTG, and differing QTL aid the identification of candidate genes mapping to critical QTL. Identifying natural modifying genes that influence retinal degeneration resulting from any causal pathway, especially those QTG that are protective, will open avenues of study that may lead to broad based therapies for people suffering retinal degenerative diseases.

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.

Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene.

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Combination surgery and nonviral interleukin 2 gene therapy for head and neck cancer.

We have developed a novel nonviral interleukin 2 (IL-2) gene therapy that demonstrates significant treatment-specific, antitumor efficacy in combination with subtotal surgical resection in a head and neck cancer murine model. Treatment of established head and neck tumors in immunocompetent mice was performed via direct injection with a cationic liposome composed of DOTMA and cholesterol formulation carrying DNA plasmid for human IL-2 (hIL-2) gene expression. ELISA assays of tumor extracts 24 h after treatment of hIL-2 gene therapy revealed increased local hIL-2 production as well as a formulation-specific secondary induction of murine IFN-gamma and IL-12. We hypothesize that the paracrine production of multiple cytokines after IL-2 single gene transfer is important for generating a therapeutic effect, and that this strategy will be well tolerated and effective in combination with surgery for head and neck cancer. In animal experiments where surgery was performed in conjunction with an operative site injection of hIL-2 plasmid formulation, no pre-, intra-, or postoperative toxicity or compromise to wound healing was identified. In murine experiments combining partial surgical resection with the nonviral gene therapy, significant antitumor efficacy was demonstrated in the hIL-2 plasmid formulation group compared with empty plasmid formulation and lactose-injected controls. In a separate experiment using smaller tumor sizes, we also demonstrated that treatment outcomes were dependent on the technical aspect of the actual treatment injection as well as visualization with surgical access. The hIL-2 plasmid formulation gene therapy induces local expression of multiple cytokines, results in treatment-specific antitumor effects, and circumvents many of the concerns and toxicity encountered with viral gene transfer. These data support the need for continued preclinical investigation and the consideration of human clinical trials for combination nonviral hIL-2 gene therapy and surgery for head and neck cancer.

Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors.

A new method of producing vesicular stomatitis virus (VSV) G protein pseudotyped retroviral vectors is described. In this method, stocks of VSV-G pseudotyped vector were reproducibly obtained by infecting an env-, human, retroviral vector producer cell line with a recombinant murine cytomegalovirus (CMV) which expresses VSV-G protein. The recombinant murine CMV, RMCMVG, expressed VSV-G protein under transcriptional control of the human CMV immediate-early promoter. RMCMVG, like murine CMV, can infect human cells, but the infection is limited to the expression of the viral immediate-early genes; no productive replication of murine CMV occurs. Recombinant murine CMV vector infection of non-permissive cells may be useful in situations where high levels of gene expression are desired without concomitant viral vector replication.

A systematic review and meta-analysis of elevated blood pressure and consumption of dairy foods.

Hypertension is a public health priority in developed countries and worldwide, and is strongly associated with increased risk and progression of cardiovascular and renal diseases. A systematic review and meta-analysis were conducted to examine the association between dairy food intake during adulthood and the development of elevated blood pressure (EBP), specifically comparing the association of EBP with consumption of low-fat dairy foods versus high-fat dairy foods, as well as cheese versus fluid dairy foods (milk or yogurt). Seven databases were searched and five cohort studies selected for inclusion, involving nearly 45,000 subjects and 11,500 cases of EBP. Meta-analysis of consumption of dairy foods and EBP in adults gave a relative risk (RR) of 0.87 (95% confidence interval (CI) 0.81-0.94). Separation of high- and low-fat dairy foods, however, indicated a significant association with low-fat dairy foods only (RR of 0.84 (95% CI 0.74-0.95)). Additional analyses showed no association between EBP and cheese, although fluid dairy foods were significantly associated with a reduced development in EBP (RR of 0.92 (95% CI 0.87-0.98)). Little heterogeneity was observed among the data presented. This meta-analysis supports the inverse association between low-fat dairy foods and fluid dairy foods and risk of EBP. Understanding these relationships can aid in the development of public health messages involving dairy foods, and supports current recommendations.

A peptide derived from hepatitis C virus E2 envelope protein inhibits a post-binding step in HCV entry.

The HCV envelope proteins E1 and E2 are required for virus binding to cellular receptors and pH-dependent fusion with endosomal membranes. Envelope protein interactions within this multistep process may provide novel targets for development of antiviral agents. To identify E1 and E2 regions involved in critical steps of HCV entry, we screened an E1E2 overlapping peptide library for inhibition of infection using a lentiviral reporter vector pseudotyped with E1E2 envelope proteins. A 16-residue polypeptide containing a portion of the E2 transmembrane domain (Peptide 75) inhibited HCV pseudoparticle infection with an IC50 of approximately 0.3microM and did not inhibit infection by VSV-g pseudoparticles at concentrations up to 50microM. Structure-activity analysis of Peptide 75 showed that antiviral activity was dependent upon L-configuration and hydrophobic character, and that the native sequence was required for maximal activity. Peptide 75 did not show virocidal activity against HCV pseudoparticles or other viruses. Temperature-shift experiments showed that the peptide acted at a post-binding step and that inhibition was further increased when used in combination with an anti-CD81 antibody previously shown to inhibit pseudoparticle entry at a post-binding step. These data suggest that interactions involving the C terminal region of E2 may play an important role in the HCV entry process.

Complementation of transforming domains in E1a/myc chimaeras.

The myc oncogene is functionally similar to adenovirus E1a in its ability to collaborate with activated ras oncogenes to transform primary fibroblasts. The transforming functions of E1a and myc have been mapped to two distinct regions in each protein. I investigated the functional similarities between E1a and myc by constructing E1a/myc chimaeras to discover whether the individual transforming domains of E1a could complement individual myc-transforming domains. Transformation assays in rat embryo fibroblasts demonstrated that the N-terminal transforming domain of E1a (CR1) could complement the C-terminal transforming domain of myc in cis, and that the reciprocal chimaera (N-terminal myc/C-terminal E1a) was also active. Chimaeras constructed using domains from transformation-defective mutants of either E1a or myc were inactive, indicating that both E1a and myc domains contribute to function. These experiments suggest that transformation by myc and E1a may involve interactions with common substrates.