Production and design of more effective avian replication-incompetent retroviral vectors.

Retroviral vectors have been invaluable tools for studies of development in vertebrates. Their use has been somewhat constrained, however, by the low viral titers typically obtained with replication-incompetent vectors, particularly of the avian type. We have addressed this problem in several ways. We optimized the transient production of avian replication-incompetent viruses in a series of cell lines. One of the optimal cell lines was the mammalian line 293T, which was surprising in light of previous reports that avian viral replication was not supported by mammalian cells. We also greatly increased the efficiency of viral infection. Pseudotyping with the vesicular stomatitus virus G (VSV-G) protein led to an over 350-fold increase in the efficiency of infection in ovo relative to infection with virus particles bearing an avian retroviral envelope protein. To further increase the utility of the system, we developed new Rous sarcoma virus (RSV)-based replication-incompetent vectors, designed to express a histochemical marker gene, human placental alkaline phosphatase, as well as an additional gene. These modified retroviral vectors and the VSV-G pseudotyping technique constitute significant improvements that allow for expanded use of avian replication-incompetent viral vectors in ovo.

Cell migration in the developing chick diencephalon.

We previously reported that retrovirally marked clones in the mature chick diencephalon were widely dispersed in the mediolateral, dorsoventral and rostrocaudal planes. The current study was undertaken to define the migration routes that led to the dispersion. Embryos were infected between stages 10 and 14 with a retroviral stock encoding alkaline phosphatase and a library of molecular tags. Embryos were harvested 2.5-5.5 days later and the brains were fixed and serially sectioned. Sibling relationships were determined following PCR amplification and sequencing of the molecular tag. On embryonic day 4, all clones were organized in radial columns spanning the neuroepithelium, which was composed primarily of a ventricular zone at this age. No tangential migration was seen in the ventricular zone. On embryonic day 5, most clones remained radial with many cells located in the ventricular zone; however, a few clones had cells migrating perpendicular to the radial column, in either a rostrocaudal or dorsoventral direction. The tangential migration began just beyond the basal limit of the ventricular zone. On embryonic days 6 and 7, many clones had cells migrating perpendicular to the radial column, which spanned from the ventricular to the pial surface. The migrating cells appeared to be aligned along axes that were perpendicular to the radial column. Using a combination of DiI tracing, immunohistochemistry and electron microscopy, we have determined that axonal tracts are present and are aligned with the migrating cells, suggesting that they support the non-radial cell migration. These data indicate that migration along pathways independent of radial glia occur outside of the ventricular zone in more than 50% of the clones in the chick diencephalon.

Bethesda classification of cervicovaginal smears: reproducibility and viral correlates.

Fifty-five cervicovaginal smears from women with squamous intraepithelial lesions (SILs) were independently evaluated on two separate occasions by four cytopathologists using a binary classification system (the Bethesda system). Smears were categorized as low-grade (LSIL) or high-grade (HSIL) using previously published criteria. All women had subsequent cervical biopsies containing human papillomavirus (HPV) DNA amplified with the polymerase chain reaction and typed by restriction fragment polymorphism analysis. Three or more observers agreed on classification in 49 of 55 cases (87%); unanimous diagnoses were rendered in 31 cases (56%). Interobserver and intraobserver reproducibility ranged from fair to near-excellent (kappa values 0.40 to 0.63; 0.63 to 0.74, respectively). HPV types included HPV 16 (27%), 18 (7%), 31 (9%), 35 (4%), 39 (4%), 6 (10%), 11 (2%), novel types (30%), and multiple types (4%). High-risk HPV types (16, 18, 31, 35, and 39) were significantly associated (P = .03) with consensus HSIL diagnoses (agreement of three or more observers). This was primarily because of the strong association of HPV 16 with HSIL (P = .001). After excluding HPV 16, the other high-risk HPV types (18, 31, 35, and 39) were no longer significantly associated with consensus HSIL diagnoses (P > .5). Conversely, LSIL diagnoses were significantly associated with non-high-risk HPV types (all HPV types except 16, 18, 31, 35, and 39; P = .006). Binary cytological classification of cervicovaginal SILs is reproducible among cytopathologists. Such classification correlates well with most low-risk HPV types and with the prototypic high-risk HPV 16 but not with other high-risk HPV types.

Restriction fragment length polymorphism analysis of isotype-labeled polymerase chain reaction-amplified human papillomavirus DNA combines sensitivity with built-in contaminant detection.

The detection of low copy number DNA in archival tissue has been revolutionized by polymerase chain reaction (PCR), but widespread acceptance of this technique for diagnostic purposes has been hampered by problems with contamination. There is a technique, restriction fragment length polymorphism analysis for characterizing PCR-amplified human Papillomavirus (HPV) nucleic acids, that reportedly provides for increased sensitivity in detection of both target and contaminating sequences. This technique is a modification of standard restriction fragment length polymorphism and involves radiolabeled nucleotide incorporation during PCR and restriction enzyme digestion of the products followed by high resolution polyacrylamide gel electrophoresis. The amount of initial target DNA amplified by PCR that can be detected by isotope labeling or ethidium staining was compared, as was the sensitivity of the two methods for analyzing a PCR product of fixed amount. The sensitivity of ethidium staining or autoradiography was comparable for detection of initial target HPV DNA by PCR. However, for subsequent typing of amplified HPV DNA by restriction fragment length polymorphism, isotope-labeled products provided an approximately 125-fold increase in sensitivity over ethidium staining, with a maximum of 625-fold greater sensitivity with a 3-day exposure. The detection of low levels of potential contaminants in PCR-amplified HPV DNA was determined in a serial analysis of cervical biopsies. Contaminating DNA was identified in two gels where multiple samples yielded the same restriction pattern. On re-isolation of genomic DNA, these products of contaminating DNA were not seen. Extrapolating from the above experiments, the contamination of many samples would have escaped detection by ethidium staining alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Human papillomavirus type 18 and intraepithelial lesions of the cervix.

The conventional perception of HPV type 18 is that it is associated principally with invasive cancer of the cervix. However, in precursor lesions it is frequently identified in lesions of lower grade morphology, in contrast to the typical high grade lesions associated with HPV 16. To better characterize this uncommon relationship of low grade morphology and high risk virus, we studied four low grade and two high grade intraepithelial lesions of the cervix which were shown to contain HPV 18 by polymerase chain reaction/restriction fragment length polymorphism analysis. Immunohistochemistry and in situ hybridization identified capsid proteins and viral DNA in an epithelial distribution characteristic of episomal replication and late gene expression. Sequencing of the E2, E6, E7, and upstream regulatory regions revealed four silent mutations within these precursor lesions. Two of these sequence alterations were also noted in three of four HPV 18 positive squamous carcinomas, suggesting a viral subtype which was not unique to cervical precursors. The bland morphology of many HPV 18 related precursors contrasts with the high grade morphology of HPV 18 associated cancers. However, this diversity cannot be linked to functional differences in sequences encoding in vitro transforming potential, transcriptional regulation, or transactivation functions. Whether these differences in lesion phenotype relate to unique host variables remains to be determined.

A binary (Bethesda) system for classifying cervical cancer precursors: criteria, reproducibility, and viral correlates.

This study of cervical squamous precursors addressed the consistency with which pathologists could agree on diagnosis using a Bethesda system and the degree to which the classification system discriminated "high-risk" human papillomavirus (HPV) types. Four pathologists independently assessed biopsies of 75 squamous lesions; all contained HPV DNA amplified from archival fixed tissue with polymerase chain reaction (PCR) and typed by restriction digestion of the PCR product. Lesions were categorized as low or high grade using published criteria. In independently performed histologic evaluations a majority (three or more) of observers agreed on the classification of 63 of the 75 cases (84%) with good to very good interobserver (kappa values, 0.43 to 0.63), and fair to excellent intraobserver (kappa values, 0.32 to 0.83) agreement. A majority of the observers classified as high grade 15 of 17 (88%) HPV 16-positive lesions (P < .002), but only 15 of 21 (71%) lesions associated with other high-risk HPV types 18, 31, 35, and 39 (P = .089). Concurrence among observers also varied with HPV type; majority agreement between three or more observers was present for 100% and 94%, respectively, for lesions associated with HPV 6/11 and HPV 16 versus 82% and 76% for lesions associated with HPV 18/31/35/39 and other HPV types. A binary system for grading cervical precursor lesions was applied with good reproducibility among pathologists, and segregated as high-grade virtually all lesions associated with the prototype high-risk HPV (HPV 16). Conversely, other presumed high-risk HPV types, particularly type HPV 18, were not distinguished by this grading scheme and were segregated frequently with low-grade lesions. This finding suggests that variables other than HPV type alone will influence lesion grade. Resolution of these variables will determine whether lesion grade is a more potent biologic parameter than HPV type.

Papillary immature metaplasia of the cervix: a distinct subset of exophytic cervical condyloma associated with HPV-6/11 nucleic acids.

A subset of exophytic cervical precursor lesions are composed of immature metaplastic cells that differ from conventional condylomata by the virtual absence of koilocytotic atypia and the presence of slender filiform papillae. We evaluated a series of exophytic cervical lesions containing this morphology for HPV nucleic acids and compared the associated HPV types with conventional exophytic condylomata of the cervix. Six of six exophytic condylomata and five of six papillary immature metaplasias (PIM), respectively, contained HPV type 6/11 by in situ hybridization. Subtyping of three PIM by polymerase chain reaction combined with direct sequencing revealed nucleic acid sequences consistent with HPV 6/11. PIM were distinguished from high-grade squamous intraepithelial lesions by the rarity of mitoses and by the uniformity of nuclear size and staining intensity with multiple chromocenters. However, these lesions tended to involve the more cephalad region of the cervical transformation zone, and three cases extended deeply into the endocervix with two requiring conization for a definitive diagnosis. Although their bland morphology and association with HPV 6/11 nucleic acids suggest a benign process, their location within the endocervical canal implies that these variants of condyloma may differ biologically from conventional exophytic condylomas of the cervix. The differential diagnosis of PIM and potential explanations for their distinctive morphology, are discussed.