Regulatory strategies to facilitate surgical innovation.

Phoebe Mounts, PhD, Esq, is a partner in the Food and Drug Administration Practice Group in the Washington, DC, office of Morgan Lewis & Bockius LLP. She counsels clients on regulatory issues for medical devices, drugs, and biologics. Prior to joining Morgan Lewis, she was on the faculty of the Johns Hopkins University School of Public Health in the Department of Molecular Microbiology, Immunology, and Infectious Diseases.

Immune responses in tuberculosis: antibodies and CD4-CD8 lymphocytes with vascular adhesion molecules and cytokines (chemokines) cause a rapid antigen-specific cell infiltration at sites of bacillus Calmette-Guérin reinfection.

Rabbit primary dermal bacillus Calmette-Guérin (BCG) lesions were compared with reinfection BCG lesions in order to gain insight into how immune responses protect against clinical tuberculosis. As early as 3 hr, a marked infiltration of macrophages and lymphocytes occurred in the reinfection group, while very little cell infiltration occurred in the primary group. It seems that only an antigen-antibody reaction could produce such an immediate pronounced antigen-specific chemotactic effect, because very few lymphocytes are normally present in the skin.

Nonspecific and immune-specific up-regulation of cytokines in rabbit dermal tuberculous (BCG) lesions.

To our knowledge, this is the first sequential study of cytokines in tissue sections of developing and healing tuberculous (BCG) lesions. In situ hybridization, immunohistochemical, and RT-PCR techniques were used. Cytokine mRNAs showed a biphasic pattern.

Coinfection of HPV-11 and HPV-16 in a case of laryngeal squamous papillomas with severe dysplasia.

Human papillomavirus (HPV) types 6 and 11 have been associated with benign laryngeal papilloma, while HPV-16 is occasionally associated with laryngeal carcinoma. In this study, a case of laryngeal squamous papillomas with severe dysplasia was evaluated for the presence of HPV infection. The biopsy specimens were taken from a 58-year-old female patient at two different time points 3 months apart.

Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.

HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined.

Myopathy and spontaneous Pasteurella pneumotropica-induced abscess formation in an HIV-1 transgenic mouse model.

In an effort to augment human immunodeficiency virus type 1 (HIV-1) gene expression in transgenic mice, an infectious proviral DNA clone was modified by deleting the two NF kappa B binding sites and some adjacent upstream LTR sequences and replacing them with the core enhancer of Moloney murine leukemia virus (MLV). Two independent lines of MLV/HIV transgenic mice were established that expressed HIV-1-specific RNA in lymphoid tissue, striated skeletal muscle, and the eye lens. Heterozygous animals from each transgenic line spontaneously developed an inflammatory disease of the eye associated with the production of copious amounts of purulent lacrimal secretions beginning at 2 weeks of age.

Recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis is an uncommon clinical disorder of the respiratory epithelium caused by HPV. It shares an identical viral etiology with genital condyloma and, in all likelihood, is transmitted at the time of birth (juvenile onset RRP) or through intimate sexual contact (adult onset RRP). Despite the precision of the surgical laser under magnification of the operating microscope, a substantial proportion of patients with RRP, adults as well as pediatric, require repeated operations at frequent intervals because of severe hoarseness and upper airway obstruction.

The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta, and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard.

Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM), the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hybridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine).

Transforming activity of the E6 gene of HPV-11gt in NIH 3T3 and REF 52 cells: correlation with the level of E6 transcription.

Among human papillomavirus (HPV) types, clinical association with benign vs malignant lesions correlates with the ability of the corresponding oncogenes to transform cells in vitro. However, even though HPV-11 is considered a low-risk type, we have reported previously that the E5a oncogene of HPV-11gt is capable of transforming NIH 3T3 cells in culture. In this study, we found that HPV-11gt E5a and E6 oncogenes have the ability to transform NIH 3T3 and the rat embryo fibroblast line REF 52.

Characterization of human papillomavirus type 57b: transforming activity and comparative sequence analysis as probes for biological determinants associated with high-risk oncogenic viruses.

The association of human papillomavirus type 57 (HPV-57) with premalignant and malignant tumors of the nasal cavity was previously reported (Wu et al., Lancet 341, 522, 1993). We determined the complete nucleotide sequence of HPV-57b (GenBank 37537), which was molecularly cloned from a benign fungiform papilloma, and compared it with other HPV types and HPV-57a, which was cloned from an inverted papilloma of the maxillary sinus by de Villiers et al.

Sites of predilection in recurrent respiratory papillomatosis.

Florid and widespread respiratory papillomatosis is a devastating disorder in a subset of patients with recurrent respiratory papillomatosis, and it poses a major dilemma for the patient and the surgeon. Contrary to common belief, the distribution of papilloma lesions is not random, but follows a predictable pattern, with lesions occurring at anatomic sites in which ciliated and squamous epithelia are juxtaposed. The predominant sites of disease in recurrent respiratory papillomatosis are the limen vestibuli, the nasopharyngeal surface of the soft palate, the midzone of the laryngeal surface of the epiglottis, the upper and lower margins of the ventricle, the undersurface of the vocal folds, the carina, and bronchial spurs.

Association of human papillomavirus with nasal neoplasia.

Since HPV-57b has been identified by two different techniques in benign, premalignant, and malignant lesions of the nasal cavity, but not in cases of chronic sinusitis, HPV-57 should be recognised as at least a co-factor in the aetiology of nasal neoplasia. Paraffin sections of 22 histologically confirmed nasal tumours were screened by in-situ hybridisation with riboprobes specific for HPV-57b. Virus was demonstrated in 6 of 7 fungiform papillomas, 6 of 8 inverted papillomas, 1 of 3 inverted papillomas with dysplasia, and 2 of 4 inverted papillomas with carcinoma.

Antibody response to human papillomavirus (HPV) type 11 in children with juvenile-onset recurrent respiratory papillomatosis (RRP).

We previously established, using an ELISA, the presence of specific antibodies directed at human papillomavirus (HPV) type 11 virions in the sera of patients with condylomata acuminata, mostly a disease of young adults that, like recurrent respiratory papillomatosis (RRP), is caused by two closely related HPVs, types 6 and 11. The present study was done to investigate if children with RRP can make viral-specific antibodies to an infection that is acquired at birth. Using the same ELISA, we studied the sera of 32 children with biopsy-documented juvenile-onset RRP and compared them to the sera of 31 control children.

Long-term response of recurrent respiratory papillomatosis to treatment with lymphoblastoid interferon alfa-N1. Papilloma Study Group.

Background: We earlier reported that patients with recurrent respiratory papillomatosis responded to six months of treatment with lymphoblastoid interferon alfa-n1. Because another study of patients treated for one year with leukocyte interferon alfa-n3 found that the growth rate of papillomas was slowed in the first six months but returned to base line during months 7 through 12 despite persistent interferon treatment, we now report the long-term results in our original study patients who were followed for a median of four years after the original one-year crossover study.

Methods: After the patients in our study had completed the first study year, their physicians could continue or recommence treatment with lymphoblastoid interferon alfa-n1 in a dose of either 2 MU per square meter of body-surface area per day or 4 MU per square meter every other day.

Polymerase chain reaction identification of human papillomavirus DNA in CO2 laser plume from recurrent respiratory papillomatosis.

Human papillomavirus (HPV) DNA was identified in the plume produced during CO2 laser vaporization of respiratory tract papillomata. The plume produced from CO2 vaporization was collected on Gelfoam pledgets that were affixed to suction tips evacuating the vapor plume from the operative field. The Gelfoam pledgets were snap frozen in liquid nitrogen, processed, and examined for HPV-6 and HPV-11 DNA by a polymerase chain reaction technique.

Transforming activity of E5a protein of human papillomavirus type 6 in NIH 3T3 and C127 cells.

Human papillomavirus type 6 (HPV-6) is the etiologic agent of genital warts and recurrent respiratory papillomatosis. We are investigating the mechanism by which this virus stimulates cell proliferation during infection. In this paper, we report that the E5a gene of HPV-6c, an independent isolate of HPV-11, is capable of transforming NIH 3T3 cells.

Detection by antibody probes of human papillomavirus type 6 E5 proteins in respiratory papillomata.

We have demonstrated the expression of proteins arising from the E5a and E5b open reading frames (ORFs) of human papillomavirus type 6c (HPV-6c) in respiratory tract papillomata. Recombinant plasmids were constructed to express the ORFs in the bacterial vectors pATH and pRIT2T. Fusion proteins were purified and injected into rabbits to produce polyclonal antibodies.

Structural analysis of human papillomavirus type 6c isolates from condyloma acuminatum and juvenile-onset and adult-onset laryngeal papillomata.

The human papillomavirus type 6c (HPV-6c) genome was molecularly cloned from biopsy specimens of a juvenile-onset and an adult-onset respiratory-tract papillomata and a condyloma acuminatum of the cervix. To determine if the genital-tract isolate and respiratory-tract isolates contain divergent sequences that may account for a difference in tissue trophism or for a difference in the age of onset of the disease, fine-structure mapping, heteroduplex analysis by electron microscopy, and nucleotide sequencing were used to examine the sequence relationship among these HPV-6c isolates. No differences were found in the digestion patterns with 23 restriction enzymes.

Sensitive detection of nucleic acids and protein of human papillomavirus type 6 in respiratory and genital tract papillomata.

We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin sections and strand-specific 3H-labelled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution. This modified immunocytochemistry provides better sensitivity when compared to previous methods using paraffin-embedded materials.

Method for cloning single-stranded oligonucleotides in a plasmid vector.

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase.

Heterogeneity in mRNA of human papillomavirus type-6 subtypes in respiratory tract lesions.

We have analyzed the structure of viral transcripts in six HPV-6c-induced respiratory tract lesions, which included four benign laryngeal papilloma, one benign nasopapilloma, and one malignant tumor, in four benign laryngeal papilloma induced by HPV-6e, and in one benign laryngeal papilloma induced by HPV-6f. Northern analysis and S1 nuclease digestion with subgenomic RNA probes demonstrated that the major exon of 1050 bases had a 5' end in the E4 open reading frame and a 3' end in E5B. Primer extension from a synthetic oligonucleotide in E4 was used to examine sequences 5' to the major exon.

Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6.

We have identified three elements in the noncoding region of human papillomavirus type 6 (HPV-6) that regulate transcription when assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyltransferase. One was a silencer that reduced expression in both a species- and tissue-dependent manner. The second was an enhancer element that was tissue specific.

Interferon alpha-n1 (Wellferon) for refractory genital warts: efficacy and tolerance of low dose systemic therapy.

This multi-center trial compared two doses of parenterally administered interferon alpha-n1 (Wellferon) in men and women with recurrent/resistant genital warts. Patients received either 1 or 3 MU/m2 daily for 14 days, then 3 times weekly for 4 weeks; non-responders could receive an additional four weeks of treatment. A total of 107 patients were enrolled, and 102 were evaluable after six weeks of study.