Robert F. Rando

Mechanism for human papillomavirus transmission at birth

We attempted to investigate mechanisms, in addition to sexual contact, by which human papillomaviruses associated with anogenital tract lesions could be transmitted. Samples of exfoliated cervical cells were obtained from 45 pregnant women and were assayed by Southern blot hybridization analysis for the presence of human papillomavirus nucleic acids. Twenty-five of the 45 women had cells positive for human papillomavirus deoxyribonucleic acid. A neonatal nasopharyngeal aspirate was obtained at term and analyzed for the presence of human papillomavirus deoxyribonucleic acid. We documented the presence of human papillomavirus deoxyribonucleic acid in the oral pharyngeal cavity of the neonates in 15 of 45 nasopharyngeal samples analyzed. Amniotic fluid was obtained from 13 patients when their membranes were artificially ruptured. These samples were assayed for the presence of human papillomavirus deoxyribonucleic acid; two of the 13 amniotic fluid samples contained human papillomavirus deoxyribonucleic acid. The detection of human papillomavirus deoxyribonucleic acid in the oral cavity of neonates is indicative of a perinatal mechanism of viral transmission. The detection of human papillomavirus deoxyribonucleic acid in the amniotic fluid may suggest an in utero mechanism of transmission. However, problems encountered in collecting the amniotic fluid samples preclude us from definitive interpretation of these data.

Characterization of multiple molecular interactions between human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1)

In transient gene expression assays we observed an increase in expression of the bacterial chloramphenicol acetyl-transferase (CAT) gene, under the transcriptional control of the HIV-1 LTR (pLTR-CAT), when this plasmid was cotransfected into Vero or MRC-5 cells with a plasmid containing either the HCMV immediate early 1 and 2 (E1, IE2) genes (pRL43a) or just the IE2 gene (pMP18). When the HCMV IE1 gene (pMP12) was cotransfected with pLTR-CAT into Vero cells the level of measurable CAT gene activity was below the level observed when pLTR-CAT was cotransfected with a nonspecific carrier plasmid (pGEM3). The negative influence of the HCMV IE1 gene product on the HIV-1 LTR in Vero cells was also observed when the HIV-1 tat gene (pLTR-TAT) was contransfected into Vero cells with pLTR-CAT and pMP12. However, when the HCMV IE1 gene was cotransfected into rhabdomyosarcoma (RD) cells with proviral HIV-1 DNA, an increase in viral production, as monitored by measurement of HIV-1 reverse transcriptase activity, was observed. In electrophoretic mobility shift assays, nuclear extracts obtained 15 hr post-HCMV infection (hpi) were found to contain a lower level of interaction with an oligonucleotide which corresponded to the HIV-1 LTR Sp-1 binding motif. Nuclear extracts obtained 40 hpi of MRC-5 cells had a greater level of interaction with, and changed the mobility of, the Sp-1 oligonucleotide relative to the uninfected nuclear extracts. HCMV-infected MRC-5 cell nuclear extracts also contain a factor(s) which interacted with the HIV-1 LTR between nucleotide positions -15 to -2 relative to the HIV-1 mRNA start site.

Murine cytotoxic T cell response specific for human cytomegalovirus glycoprotein B (gB) induced by adenovirus and vaccinia virus recombinants expressing gB

A murine model of the cytotoxic T lymphocyte (CTL) response to glycoprotein B (gB) of human cytomegalovirus (HCMV) was developed based on the use of adenovirus (Ad) and vaccinia virus (Vac) recombinants expressing gB. Mice of different major histocompatibility haplotypes [CBA (H-2k), BALB/k (H-2k) and BALB/c (H-2d)] infected with the Ad-gB recombinant developed an Ad-specific CTL response. However, only the H-2k mice developed a significant HCMV gB-specific CTL response, as indicated by the major histocompatibility complex class I-restricted lysis of Vac strain Copenhagen (VacC)-gB recombinant-infected target cells by H-2k mouse immune spleen cells. The VacC-gB recombinant elicited only a weak gB-specific CTL response in these mice, indicating that the observed gB-specific CTL response in mice is dependent on the expression vector used for immunization. The gB-specific cytotoxicity observed in H-2k mice was mediated by the CD8 lymphocyte subset.

The N-terminal 303 amino acids of the human cytomegalovirus envelope glycoprotein B (UL55) and the exon 4 region of the major immediate early protein 1 (UL123) induce a cytotoxic T-cell response

We reported earlier that an adenovirus (Ad) recombinant expressing the full-length human cytomegalovirus (HCMV) glycoprotein B (gB) gene induces gB-specific cytotoxic T lymphocyte (CTL) responses in CBA (H-2k) mice (Berencsi et al., J. Gen. Virol. 74, 257-2512, 1993). Here we show that mice immunized with Ad recombinant viruses expressing truncated forms of the gB gene containing the first 700 (Ad-700), 465 (Ad-465) or 303 (Ad-303) amino acids of gB or an Ad construct containing exon 4 (E4) of the HCMV immediate early 1 (IEI) gene (Ad-IEI (E4)) demonstrate HCMV-specific CTL responses. These data suggest the importance of the first 303 amino acids of the gB polypeptide and the IEI E4 product in designing a vaccine to induce anti-HCMV CTL responses.

The clinical role of human papilloma virus typing

Abstract Patients with abnormal Pap smears underwent repeat Pap smear, colposcopy, biopsy, and human papilloma virus (HPV) typing to identify the presence of CIN and to assess the correlation of HPV type to grade of CIN and behavior of CIN. A total of 334 patients underwent evaluation and typing with Southern blot methodology. Fifty-five percent (185) of the patients demonstrated HPV viral sequences; 98 of the 185 positive patients demonstrated HPV types 16, 18. The presence of HPV sequences was not associated with a higher frequency of positive cytology of CIN II or III. High-grade CIN (II or III) was seen in 17.3, 13.5, 13.8, and 10.7% of patients with HPV 16,18; 6,11; 31,33,35; or no HPV sequences. Of 52 patients with normal cytology and biopsy revealing less than or equal to CIN I, no patients with types 6,11, 35 patients with 31,33,35, 315 patients with 16,18, and 223 patients with no HPV sequences progressed to greater than or equal to CIN II. These data do not support a role for HPV typing in predicting the initial histology. Typing may be of some value in monitoring patients with low-grade lesions.

Nucleic Acid Hybridization as a Diagnostic Tool for the Detection of Human Papillomaviruses

At this time nucleic acid hybridization tests are the most sensitive and reproduceable methods for the detection and differentiation of HPV types in clinical samples. The hybridization method of choice depends on the information desired and the availability of the proper diagnostic nucleic acid probes. Assuming most of the HPV nucleic acid probes become readily available in the near future, then the most sensitive test for screening clinical specimens--albeit the most laborious--will be the Southern blot procedure. As more information covering the involvement of HPV infections with the progression of lesions from benign to malignant is compiled, the need to know the particular subtype or status of HPV integration may become more or less important in the screening of clinical samples. If this information becomes less important, or unnecessary for a simple screening procedure, then dot blot hybridization may prove to be a much easier method for obtaining the information desired. If the sensitivity of in-situ hybridization using non-radioactive probes increases, then this method would become the fastest, easiest, and cleanest technique for the screening of a large number of clinical samples where limited information is desired. The ideal test for the future would be automated. In order to automate a test for HPV infections, the test design must become much simpler, and in order to design a simpler test, more information will be needed concerning the biology of HPV infections, how they cause benign or malignant cellular proliferation, and how the host immune system responds to HPV infections.

Studies on the Transmission of Viral Disease via the C02 Laser Plume and Ejecta

While recent reports have noted the presence of viral DNA sequences in the laser plume, no significant effort has been made to study transmission of the virus in vivo via airborne laser debris. Studies were undertaken to identify potential hazards to operating room occupants in gynecologic laser surgery. ACO2 laser in the continuous wave mode using a power density of 666 W/cm2 was fired through a 5-cm metal cylinder at virus-infected tissues. Airborne particulate debris, 100-200 microns, was removed from the cylinders inner surfaces. In one instance, deposition of the debris was found on the surgeons eyeglasses 1 m from the site of impact despite the use of a smoke evacuator. The first set of studies involved confirmed human papillomavirus (HPV) lesions of the human female lower genital tract. Specimens were collected for electron microscopy and Southern Blot viral hybridization. Additional cervical electron microscopy specimens were recovered from the speculum during pulsed CO2 laser treatment at 13 W average power during conization. Electron microscopy of the vulvar debris revealed only anucleate keratinized squamous epithelial cells. Cervical specimens demonstrated similar cells with nearly instantaneous vaporization of intracellular water and apparent condensation of cellular carbon. HPV Southern Blot testing revealed insufficient quantities of DNA for that technique. The second set of studies involved bovine papillomavirus lesions from dairy cattle. The debris was transmitted to susceptible animals. The bovine studies failed to demonstrate the transmission of disease in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The Clinical Role of Human Papilloma Virus Typing

Patients with abnormal Pap smears underwent repeat Pap smear, colposcopy, biopsy, and human papilloma virus (HPV) typing to identify the presence of CIN and to assess the correlation of HPV type to grade of CIN and behavior of CIN. A total of 334 patients underwent evaluation and typing with Southern blot methodology. Fifty-five percent (185) of the patients demonstrated HPV viral sequences; 98 of the 185 positive patients demonstrated HPV types 16, 18. The presence of HPV sequences was not associated with a higher frequency of positive cytology of CIN II or III. High-grade CIN (II or III) was seen in 17.3, 13.5, 13.8, and 10.7% of patients with HPV 16, 18; 6, 11; 31, 33, 35; or no HPV sequences. Of 52 patients with normal cytology and biopsy revealing less than or equal to CIN I, no patients with types 6, 11, 3/5 patients with 31, 33, 35, 3/15 patients with 16, 18, and 2/23 patients with no HPV sequences progressed to greater than or equal to CIN II. These data do not support a role for HPV typing in predicting the initial histology. Typing may be of some value in monitoring patients with low-grade lesions.