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R D Burk

Publications and source records attributed to R D Burk.

At least 73 records · Page 4Linked to original sources
PubMed1996

Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents.

BACKGROUND & AIMS: Significant liver repopulation by hepatocyte transplantation will advance clinical applications. The aim of this study was to test the hypothesis that translocation of transplanted cells into liver plates will allow repeated cell transplantation for increasing the transplanted hepatocyte mass. METHODS: Hepatocytes were transplanted via spleen from either F344 rats into syngeneic recipients deficient in dipeptidyl peptidase IV or from transgenic hepatitis B surface antigen-producing G26 mice with hepatitis B virus into nontransgenic congeneic recipients. Portosystemic shunting was shown by radiological methods. RESULTS: Repeated hepatocyte transplantation led to progressively increased liver repopulation. Transplantation of 1.75 x 10(8) hepatocytes in three divided doses repopulated more than an estimated 5% of the host rat liver, with 3.8 x 10(6) +/- 0.1 x 10(6) transplanted cells/cm3 liver. This was a tenfold or threefold mean increase in transplanted cell number compared with recipients of 2.0 x 10(7) or 7.5 x 10(7) cells transplanted in single sessions, respectively (P < 0.001). Repeated hepatocyte transplantation interfered with neither cell integrations in liver parenchyma nor secretory function of transplanted cells. Portal hypertension, portasystemic collaterals, or intrahepatic shunting were not observed in cell recipients. CONCLUSIONS: Repeated transplantation of hepatocytes in large numbers is safe and effective and should advance strategies for liver repopulation.

Animals↗
PubMed1996

Phyllanthus amarus down-regulates hepatitis B virus mRNA transcription and replication.

The Phyllanthus amarus plant shows potential for treating hepatitis B virus. To define the mechanism of action of P. amarus, we used HepG2 2.2.15 cells, which support hepatitis B virus replication. P. amarus inhibited hepatitis B virus polymerase activity, decreased episomal hepatitis B virus DNA content and suppressed virus release into culture medium. To examine transcriptional control mechanisms, we used G26 hepatitis B virus transgenic mice, which produce serum HBsAg but neither HBcAg nor virion particles. When P. amarus was administered to transgenic mice, hepatic HBsAg mRNA levels decreased, indicating transcriptional or post-transcriptional down-regulation of the transgene. Increase in hepatitis B virus mRNA expression after stimulation of the glucocorticoid responsive element was also suppressed by P. amarus, suggesting involvement of the hepatitis B virus enhancer in this response. Disruption by P. amarus of hepatitis B virus polymerase activity, mRNA transcription and replication supports its role as an antiviral agent.

Animals↗
PubMed1996

Sexual behavior and partner characteristics are the predominant risk factors for genital human papillomavirus infection in young women.

Risk factors for cervicovaginal human papillomavirus (HPV) infection were investigated in 604 college women. HPV was detected in 168 (27.8%) of the subjects by L1 consensus primer polymerase chain reaction, Southern blot hybridization, or both. Significant independent risk factors for HPV (P < .05) included age (odds ratios [ORs]: 2.6 for 21-23 years old and 1.6 for > 23, vs. < or = 20), ethnicity (ORs: 3.2 for black, 2.2 for Hispanic, vs. white/other), number of lifetime male vaginal sex partners (ORs: 4.5 for 2, 5.8 for 3 or 4, 10.3 for > or = 5, vs. 1), living with smokers (OR: 1.9), male partner's number of lifetime sex partners (ORs: 2.1 for 2 or 3, 3.1 for 4-10, 2.7 for > or = 11, vs. 1), duration of sexual relationship for > 12 months (OR: 0.6), and male partner currently in college (OR: 0.6). These data demonstrate that the predominant risk factors for genital HPV infection in young women are related not only to their own sexual behaviors but also to those of their male partners.

Adult↗
PubMed1996

Immunologic distribution of an organic anion transport protein in rat liver and kidney.

A Na(+)-independent organic anion transport protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this protein is sufficient for cells to transport the organic anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane protein. This 80-kDa protein migrated as a 65-kDa protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the transporter migrated as an 83-kDa protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this transporter in liver and kidney may have important functional and regulatory consequences.

Adenosine Triphosphate↗
PubMed1995

Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells.

Recently we expression cloned a rat liver organic anion transport protein in Xenopus laevis oocytes (Jacquemin, E. Hagenbuch, B, Stieger, B., Wolkoff, A.W., and Meier, P.J.,(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 133-137). In the present study, we have stably transfected the cDNA encoding this protein into HeLa cells by using a vector containing a zinc-inducible promoter. The parent cells have virtually no baseline transport of [35S]sulfobromophthalein, whereas the induced transfected cells express a novel 74-kDa protein and avidly transport this ligand. Transport by these cells is saturable (Km = 3.3 microM, Vmax = 257 pmol/min/mg protein), bidirectional, and highly temperature-dependent. In the presence of albumin, uptake of [35S]sulfobromophthalein requires the presence of extracellular Cl, whereas in the absence of albumin, this C1- dependence is not seen. These studies indicate that cellular uptake of sulfobromophthalein does not result from direct interaction with the plasma membrane lipid bilayer but rather requires the presence of a specific plasma membrane transporter.

Albumins↗
PubMed1995

Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia.

BACKGROUND: Cervical dysplasia, also referred to as squamous intraepithelial lesion (SIL) in cytology or cervical intraepithelial neoplasia in histopathology, is thought to have the potential to advance in progressive stages to cervical cancer. However, not all cases of SIL progress, and most of the mild lesions spontaneously regress. Factors that govern regression, persistence, and progression of SIL are poorly understood. PURPOSE: Our analysis sought to identify factors that determined persistence or regression of SIL. METHODS: Seventy subjects with histopathologically confirmed cervical dysplasia were followed at 3-month intervals for 15 months. At each visit, the cervix was evaluated by Pap smear and colposcopy, and exfoliated cervicovaginal cells were analyzed for human papillomavirus (HPV) DNA. For each subject, data from every two consecutive visits were grouped as a pair. Persistent SIL was considered present if a lesion was detected at a visit (t) as well as at the next visit (t + 1) and absent if a lesion was detected at visit t but not at visit t + 1. A statistical model for time-dependent data correlated persistent SIL with various risk factors. RESULTS: Age, ethnicity, education, sexual behavior, smoking, and the use of oral contraceptives did not correlate with persistent SIL. The risk of persistent SIL was associated with continual HPV infection in visits t and t + 1 (HPV positive by Southern blot analysis: odds ratio [OR] = 3.91, and 95% confidence interval [CI] = 1.58-9.65; HPV positive by polymerase chain reaction [PCR]: OR = 2.42, and 95% CI = 1.03-5.67) and a persistent high viral load (OR = 4.07, and 95% CI = 1.35-12.30). When typed by PCR, individuals with type-specific persistent infection in visits t and t + 1, and particularly those with a continual high viral load (OR = 4.97; 95% CI = 1.45-17.02), had the highest risk for persistent SIL compared with those with a low level of type-specific persistent infection or non-type-specific persistent infection. The presence of persistent HPV infection in visits t-1 (the preceding time interval) was also predictive of persistent SIL in visits t and t + 1, although the strength of association was weaker, suggesting that persistent HPV and SIL occur synchronously. CONCLUSION: HPV infection and its associated cervical lesions tend to occur concurrently, and type-specific persistent HPV infection, particularly with a high viral load, produces chronic cervical dysplasia. IMPLICATIONS: The natural history of genital HPV infection directly influences the prognosis of cervical dysplasia as measured by persistence of the lesion. Testing for HPV infection may be valuable in the clinical management of women with cervical dysplasia.

Adult↗
PubMed1995

Genetic characterization of the human papillomavirus (HPV) 18 E2 gene in clinical specimens suggests the presence of a subtype with decreased oncogenic potential.

HPV 18 is associated with 2 divergent phenotypes: (i) aggressive cervical cancer and a preponderance of cancer relative to cervical intra-epithelial neoplasia (CIN) and (ii) benign warty lesions of the cervix. The E2 gene of HPV 18 encodes a regulatory protein that represses viral oncogene transcription and is involved in viral replication. Variation within the E2 gene of HPV 18 and its correlation with the morphologic grade of associated lesions were analyzed in a sample of 20 HPV 18-positive cervical specimens representing a spectrum of pathology from low-grade CIN to cervical cancer. An amplification HPV 18 E2 gene was present in 3 of 5 cancers, indicating that E2 disruption was not required for cancer development. Single-strand conformation polymorphism and PCR analyses revealed a high degree of polymorphism throughout the E2 gene. Direct DNA sequencing of both strands of a 154-bp fragment revealed a variability of 5.8%. Six intra-epithelial lesions contained alterations in common that account for 3.9% of the variation and appear to constitute a subtype. Within the 154-bp region, 2 of 3 cervical cancers and 0 of 12 intra-epithelial lesions were identical to the published HPV 18 sequence. DNA sequence analysis of a region extending into the E5 open reading frame revealed deletions in the E2/E5 intragenic region that were present in 50% of the members of the subtype. Our data demonstrate significant sequence variation within the E2 gene and suggest the presence of an HPV 18 subtype with decreased oncogenic potential.

Base Sequence↗
PubMed1995

Lack of efficacy of interferon-alpha therapy in recurrent, advanced cervical cancer.

Human papillomavirus (HPV) is associated with 65-95% of in situ or early invasive squamous cell carcinomas of the cervix. A multiinstitutional, prospective phase II clinical trial was initiated by the Eastern Cooperative Oncology Group (ECOG) to study the activity of IFN-alpha 2b in women with metastatic or locally recurrent cervix cancer. The activity of IFN-alpha 2b was correlated with the presence of HPV as measured by Southern blot hybridization or polymerase chain reaction techniques in 17 patients. All patients had failed prior definitive therapy with surgery, radiation, and chemotherapy. IFN-alpha 2b was administered at 10 MU/m2 subcutaneously three times per week. Among 31 patients enrolled, 3 achieved a clinical response to treatment. Tumor was accessible for biopsy in 17 patients. The presence of HPV was assayed by Southern blot hybridization (2 of 17) and/or polymerase chain reaction (PCR) technology (15 of 17). Of the 17 assays, 16 were informative. HPV was detected in 5 of 16 patients. Of 5 HPV-positive women, 2 responded to treatment, versus 1 of 11 HPV-negative women, thus not permitting reliable statistical analysis. It is concluded that IFN-alpha 2b has only minimal activity against advanced, recurrent cervical cancer.

Adult↗
PubMed1995

Accuracy and interlaboratory reliability of human papillomavirus DNA testing by hybrid capture.

Epidemiologists and clinicians wishing to introduce human papillomavirus (HPV) testing into cervical cancer prevention programs need standardized, reliable, and accurate HPV DNA tests that can detect the full spectrum of pathogenic HPV types. The Hybrid Capture System assay from Digene (hybrid capture assay) is a nonradioactive kit designed to detect 14 HPV types in two groups: a mix of 9 high-risk types associated with anogenital cancer (HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56) and another group of 5 low-risk types associated with condyloma acuminatum (HPV types 6, 11, 42, 43, and 44). The assay yields quantitative data meant to reflect viral concentration. In a study of 199 cervical specimens from women with concurrent Pap smears, we assessed the reliability of the new assay by comparing the hybrid capture assay results from three laboratories. We assessed the accuracy of the hybrid capture assay in comparison with a reference standard of HPV DNA content (multiple testing by several methods in two reference laboratories). We also compared the hybrid capture assay results with the concurrent cytologic diagnoses on the basis of an independent review of each smear by five pathologists. Pairwise interlaboratory agreement rates on HPV positivity for either high-risk or low-risk types ranged from 87 to 94%, and kappa values ranged from 0.61 to 0.83. Among specimens positive for high-risk types (the most important clinical outcome), the interlaboratory correlations of the quantitative data ranged from 0.60 to 0.90. Test results from all three laboratories were strongly associated with those of the HPV DNA reference standard and with the concurrent cytopathologic diagnoses. The most common errors were sporadic, apparently false-positive results.

Cervix Uteri↗
PubMed1995

Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.

The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32-B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

Animals↗
PubMed1994

Analysis of short novel human papillomavirus sequences.

Consensus primer mediated PCR protocols have the potential to amplify previously uncharacterized human papillomavirus (HPV) genotypes. In a study on 500 cervicovaginal samples, we amplified four sequences (L1AE 1 to L1AE 4) that failed to hybridize to any of the available HPV type-specific oligonucleotide probes. Nucleotide sequencing revealed that the sequences were derived from the L1 region of hitherto unsequenced genotypes. Comparison of phylogenetic trees based on the amplified L1 sequences with E6-derived phylogenetic trees resulted in the identification of L1AE 1 and L1AE 2 as putative novel HPV PCR genotypes. L1AE 1 was related to HPV 39, whereas L1AE 2 was related to HPV 51. The L1AE 3 and L1AE 4 sequences occupied L1-phylogenetic branches equivalent to the positions of HPV 66 and HPV 61, respectively, in an E6-phylogenetic tree.

Amino Acid Sequence↗
PubMed1994

Risk of genital human papillomavirus infection in women with human immunodeficiency virus-induced immunosuppression.

This study examined the risk factors for genital human papillomavirus (HPV) infection, including HIV-related immunosuppression as well as demographic and behavioral factors. Subjects were 207 women with known HIV serologic status, the majority of whom were intravenous drug users recruited from a methadone treatment program in the Bronx, New York. A structured questionnaire interview, cervico-vaginal lavage, and CD4 cell measurements were obtained. HPV genomes in the cervico-vaginal cells were detected by Southern-blot hybridization. HPV prevalence was 23% among HIV seronegatives, 45% among HIV seropositives with CD4 percent > 20 and 61% among HIV seropositives with CD4 percent < or = 20. Multivariate analysis identified the following independent risk factors that were significantly associated with HPV positivity: HIV-related immunosuppression, < 35 years of age, and never used oral contraceptive pills. HIV-related immunosuppression was also associated with a stronger Southern-blot signal strength. HIV infection, however, did not increase the susceptibility to HPV infection with the oncogenic types (16, 18, 31, 33 and 35). Women with HIV infection or immunosuppression had a higher prevalence of HPV infection with the oncogenic types, but they also had an increased prevalence of infection with other HPV types as compared to the HIV-seronegative women. The results suggest that HIV-related immunosuppression increases the risk of genital HPV infection and has an effect on HPV replication.

Adult↗
PubMed1994

Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: correlation between Southern blot hybridization, polymerase chain reaction, and serology.

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic peptide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences.

Adolescent↗
PubMed1994

The Mastomys natalensis papillomavirus: nucleotide sequence, genome organization, and phylogenetic relationship of a rodent papillomavirus involved in tumorigenesis of cutaneous epithelia.

Mastomys natalensis is a rodent of African origin afflicted with a very high incidence of skin tumors (keratoacanthomas and squamous carcinomas), which are associated with a papillomavirus, M. natalensis papillomavirus (MnPV). We have determined the genomic sequence of MnPV, which has a size of 7687 bp. The genomic organization is similar to that of other papillomaviruses, with open reading frames E6, E7, E1, E2, and E4 in the early and L2 and L1 in the late region. Due to an unusually large hinge region, the transcriptional activator E2 has a size of 542 amino acids rather than 400 to 460 amino acids, as in other papillomaviruses. An open reading frame E5 coding for a small hydrophobic membrane protein is missing, as is the case for some cutaneous human papillomaviruses (HPV). This fact, together with the composition of cis-responsive elements in its long control region and phylogenetic evaluation of segments of its E6, E1, and L1 genes, indicates a relationship of MnPV to the cottontail rabbit papillomavirus and several HPV types found in lesions of cutaneous epithelia, in particular to those that are associated with epidermodysplasia verruciformis. MnPV may be a useful model system for tumorigenesis of cutaneous epithelia in humans.

Amino Acid Sequence↗
PubMed1994

Hepatocytes exhibit superior transgene expression after transplantation into liver and spleen compared with peritoneal cavity or dorsal fat pad: implications for hepatic gene therapy.

For hepatic gene therapy or applications of hepatocyte transplantation in liver failure, survival and function of transplanted cells is critical. Insights into site-specific gene regulation will significantly facilitate development of appropriate strategies for transplanting hepatocytes. To assess the function of transplanted cells, we used a transgenic hepatitis B virus (HBV) hepatocyte system, which allowed analysis of cellular gene expression with HBV surface antigen (HBsAg) mRNA expression, as well as secretion of HBsAg into peripheral circulation. When congeneic HBV hepatocytes were transplanted into the liver (via spleen), serum HBsAg promptly appeared in circulation and persisted for the entire duration of the studies. In contrast, transplantation of hepatocytes into the peritoneal cavity or dorsal fat pad resulted in serum HBsAg levels that were either significantly lower or gradually rose after a lag period. HBsAg mRNA expression was several-fold greater in transplanted hepatocytes in liver or spleen versus in peritoneal cavity or dorsal fat pad. Despite persistence of transplanted hepatocytes in peritoneal cavity or dorsal fat pad, serum HBsAg was cleared by antibody to HBsAg (anti-HBs) but this was not observed after hepatocyte transplantation into spleen. As the function of transplanted hepatocytes is optimally regulated in the liver, hepatic reconstitution with cell transplantation will be most appropriate for gene therapy.

Adipocytes↗
PubMed1994

Risk factors for squamous intraepithelial lesions on Pap smear in women at risk for human immunodeficiency virus infection.

To determine risk factors for squamous intraepithelial lesions (SIL) on Pap smear, 253 women at risk for human immunodeficiency virus (HIV) infection had Pap smear, HIV antibody testing, CD4+ cell measurements, and human papillomavirus (HPV) genome detection by Southern blot hybridization. Associated with SIL (P < .05) on univariate analysis were genital HPV (SIL prevalence in HPV-positive subjects, 36.3%; odds ratio [OR], 9.3; 95% confidence interval [CI], 4.2-20.3) and HIV infection (SIL prevalence in seropositive subjects, 21.9%; OR, 2.5; 95% CI, 1.2-5.1). No demographic or behavioral variables were associated with risk for SIL. Multivariate analyses identified genital HPV infection (OR, 6.78; 95% CI, 2.9-15.7), detection of known high-risk HPV types (OR, 11.8; 95% CI, 4.1-34.1), strong HPV Southern blot signal strength (OR, 10.8; 95% CI, 3.5-33.7), and severe HIV-related immunosuppression (OR, 3.1; 95% CI, 1.04-9.5) as independent risk factors associated with SIL. Thus, severe immunosuppression due to HIV infection increases the risk for SIL mediated by HPV infection.

Adult↗