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. Therefore, antibodies hasten the accumulation of an expanded antigen-specific T-lymphocyte population (memory cells) at sites of bacillary lodgement. By 1-2 days, the primary and reinfection BCG lesions differed 400- to 500-fold in size. By 4-5 days, the size of the reinfection lesions had declined, while the size of the primary lesions had increased, so that, grossly, both types of lesion were similar. At 8 days in reinfection lesions and at 12 days in primary lesions, small secondary peaks in size occurred, which were probably caused by cell-mediated immune responses. In rabbits with primary BCG lesions, skin tests with Old Tuberculin were positive at 9 days, accompanied by a rise in the levels of antibodies to the secreted antigen, phosphate-specific transport protein 1, but the levels of antibodies to the constitutive antigens, purified protein derivative and heat-shock protein 65, did not increase appreciably until some time after 23 days. In tissue sections of reinfection BCG lesions, the percentage of mononuclear cells labelled, by in situ hybridization techniques, for the mRNA of monocyte chemoattractant protein 1 (MCP-1), a chemokine, peaked at 3 hr and then was down-regulated, whereas in primary lesions, this percentage was down-regulated only after 2 days. [The percentage in the tissue sections for the mRNAs of interleukins 1beta and 8, as well as the proteins of MCP-1 and tumor necrosis factor alpha (TNF-alpha), followed a somewhat similar time-course to that of MCP-1 mRNA.] A high percentage of mononuclear cells containing the MCP-1 mRNA 'factory' would favour enlargement of the lesions and a low percentage would favour their regression. At 5 days, the percentage of CD4 and CD8 lymphocytes, stained by immunohistochemical techniques, and the amount of microvasculature stained similarly for vascular cell adhesion molecule 1 were higher in the reinfection group, indicating that prior immunization caused a more rapid (antigen-dependent) up-regulation of these factors. Tuberculin reactions resembled early reinfection BCG lesions in almost every factor evaluated herein. In brief, the production of chemokines began soon after BCG reinfection, peaked within a few hours and was markedly down-regulated by 24 hr, a time at which the lesions of reinfection were of maximal size. Therefore, the amount of cell infiltration was tightly controlled, probably by the variety of mechanisms listed herein.

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. The percentage of mononuclear cells (MN) containing IL-1beta, TNF-alpha, MCP-1, and IL-8 mRNAs was highest in 1- to 3-day lesions, apparently because of the nonspecific inflammatory response caused by the tubercle bacilli in the BCG vaccine. At 5 days, this percentage was significantly reduced. With IFN-gamma, the peak and trough were delayed by 2 days. By 9 days, the percentage of MN containing the mRNAs of all five cytokines had again increased and the rabbits had become tuberculin-positive. In general, MCP-1 and TNF-alpha proteins and the vascular adhesion molecules, ICAM, VCAM, and perhaps ELAM, peaked at about 3 days. Many mononuclear cells surrounding the central areas of solid and liquefied caseous necrosis contained chemokine IL-8 mRNA. IL-8 is known to attract PMN, and PMN were present nearby. In contrast, MN containing chemokine MCP-1 mRNA were present more peripherally in areas rich in macrophages and lymphocytes. The early nonspecific cytokine response seems to be an adjuvant effect of the mycobacteria in BCG vaccine in that it causes a rapid entry of macrophages, lymphocytes, granulocytes, and probably dendritic cells into local sites of antigen deposition. This effect should be considered in developing improved vaccines for the prevention of tuberculosis, because BCG vaccines producing a strong early cytokine response should be more immunogenic than BCG vaccines with similar antigens producing a weak response.

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. Architecturally, the tumor showed papillary configuration reminiscent of squamous papilloma. Cytologically, the lesion showed morphologic features characteristic of severe squamous epithelial dysplasia. HPV infection was determined by DNA in situ hybridization using type-specific HPV-DNA probes. HPV-11 probes demonstrated homogeneous nuclear staining, suggesting productive viral replication. In contrast, HPV-16 probe produced a speckled pattern, suggesting HPV-16 DNA integration. Normal laryngeal epithelium did not yield specific hybridization. The presence of HPV-11 and HPV-16 was confirmed by PCR using HPV type-specific primers. Immunocytochemical staining was performed to detect Ki-67, a proliferation marker, and p53. Ki-67 expression was demonstrated throughout the whole thickness of epithelium. Staining for p53 was negative. This study suggests that multiple HPV infections can occur in the same lesion and that HPV-16 infection and its DNA integration may contribute to the occurrence of severe dysplasia in the lesion described.

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. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.

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. Periorbital abscess formation became grossly apparent by 2 months of age and Pasteurella pneumotropica was cultured from the harderian glands and conjunctival surfaces of many of the MLV/HIV animals but not their nontransgenic, cohabiting littermates. This gram-negative commensal bacterium has been previously associated with a similar disease phenotype in immunocompromised (e.g., nude mice) rodent colonies. MLV/HIV mice developed normally until 15 weeks of age, when weight loss and wasting occurred, culminating in premature death (as earlier as 6 months of age). The cachexia was associated with an initially focal and subsequently progressive myopathy, coinciding with age-related increases of HIV gene expression in muscle.

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. For the management of at least a subset of patients, the efficacy of adjuvant agents (interferon is a leading choice) should be investigated in a multi-institutional clinical trial. For the potential prophylactic benefit, cesarean sections in selected high-risk expectant mothers should be considered.

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). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.

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. Cells were transfected independently with expression plasmids containing the HPV-11gt E5a or E6 oncogenes or both plasmids simultaneously to examine potential interactions. Cells containing these plasmids were phenotypically transformed and had an accelerated doubling time, loss of contact inhibition of growth, and loss of anchorage dependence for cell division. Independent cell lines containing the HPV-11gt E6 gene exhibited variable levels of phenotypic transformation that correlated with the HPV-11gt E6 gene content. The degree of phenotypic transformation could be increased by elevating the level of transcription of the E6 gene, indicating that there is a dose response effect for transformation in this system. These results suggest that increased expression of E6 may be an important factor in malignant progression of naturally occurring tumors.

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. (Virology 171, 248. 1989). Comparative and phylogenetic analysis of amino acid sequences of the HPV-57b oncogenes E5, E6, and E7 were performed with HPV-6, 11, 16, and 18. Phylogenetic trees using the Jotun-Hein algorithm indicated a closer relationship of HPV-57b E5 and E7 with corresponding genes of HPV-18. Signature pattern analysis of these two oncogenes was also in agreement with a closer relatedness to HPV-16 and 18 oncogenes, which are associated with a high risk for malignant progression. Compared with 7861 bp of HPV-57a, HPV-57b had 7868 bp as well as differences in the restriction enzyme sites and the open reading frames, including at least five additional ones. To investigate the oncogenic potential of HPV-57b, NIH 3T3 and REF52 cells were cotransfected with two plasmids: pKP54. HPV-57b, which contains the HPV-57b genome, and pMT.neo.1, which confers resistance to G418. After selection in culture medium containing G418, 58% of the G418r NIH 3T3 colonies and 47% of the G418r REF52 colonies exhibited morphological transformation. These results indicate that the transcriptional regulatory elements and the oncoproteins of HPV-57b are active in vitro to induce cellular transformation, as are other high-risk HPV types.

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. These sites have the common histologic feature of a squamociliary junction. Papillomata also occur at the tracheostomy tract and at the midthoracic trachea in patients with tracheostomies. At the latter sites, abrasion injury to ciliated epithelium heals with metaplastic squamous epithelium and creates an iatrogenic squamociliary junction. The apparent preferential localization of papilloma at squamociliary junctions has at least 2 implications: first, that detection of occult asymptomatic papillomata is enhanced by careful examination of squamociliary junctions, and, second, that iatrogenic papilloma "implantation" is preventable by avoiding injury to nondiseased squamous and ciliated epithelia.

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. The presence of HPV-57b was confirmed with the polymerase chain reaction, which identified an additional 4 positive samples, bringing the total to 86% positive specimens. The results underscore the importance of HPVs in the aetiology of cancers at extragenital sites.

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. The median (and interquartile range) of the OD values in the controls and the cases was 0.078 (0.003, 0.101) and 0.230 (0.063, 0.725), respectively, a statistically significant difference (P = 0.001). Among the cases, there was no difference in seroreactivity between children with HPV-11-induced RRP and those with HPV-6-induced RRP (P = 0.31). Since HPV-11 viral particles do bind to the ELISA plate and remain intact and accessible to antibodies, we conclude that children with RRP, like adults with condylomata acuminata, develop antibodies directed at HPV-11 virions.

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. The extent of disease was measured by endoscopy when clinically indicated. RESULTS: Data on late-follow-up were obtained for 60 of the 66 patients. There were 22 complete remissions and 25 partial remissions; 13 patients had no response. The median duration of the complete remissions was 550 days, and 15 patients continued to be in complete remission. The median duration of partial remissions was 400 days and seven patients were still in partial remission. Thirteen of 28 patients responded to a second course of interferon after an interruption in treatment of at least one month. The rate of response in the 11 of 53 patients who had neutralizing antibody to interferon was the same as in the patients without the antibody. CONCLUSIONS: Patients with severe recurrent respiratory papillomatosis may have a sustained or repeated response to treatment with lymphoblastoid interferon alfa-n1. We recommend that patients with recurrent respiratory papillomatosis who require surgery every two to three months be given a six-month trial of interferon alfa-n1.

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. Tissue and vapor-plume specimens were collected from 22 patients undergoing CO2 laser excision of laryngeal lesions. Seven patients had adult-onset recurrent respiratory laryngeal papillomatosis (RRP), 12 had juvenile-onset RRP, two had laryngeal carcinoma, and one had nonspecific laryngitis. HPV-6 or HPV-11 was identified in 17 of 27 vapor-plume specimens from RRP and in none of three from non-RRP lesions. All but one RRP tissue specimen contained HPV-DNA, and none of the non-RRP tissues contained HPV-DNA. When HPV was present in vapor, the same HPV type was found in the corresponding tissue specimen. Identification of HPV-DNA in the laser plume raises concern regarding potential risks from exposure to the plume--particularly to the endoscopic surgeon and the operating team. The practical concerns and effectiveness of the plume scavenging systems are discussed.

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. The E5a open reading frame (ORF) was expressed under the control of the mouse metallothionein promoter in the expression vector pMt.neo.1, which also contains the gene for G418 resistance. Transfected cells were selected for G418 resistance and analyzed for a transformed phenotype. The transformed NIH 3T3 cells overgrew a confluent monolayer, had an accelerated generation time, and were anchorage independent. In contrast, E5a did not induce foci in C127 cells, but C127 cells expressing E5a did form small colonies in suspension. The presence of the 12-kilodalton E5a gene product in the transformed NIH 3T3 cells was shown by immunoprecipitation and was localized predominantly to nuclei by an immunoperoxidase assay. A mutation in the E5a ORF was engineered to terminate translation. This mutant was defective for transformation, demonstrating that translation of the E5a ORF is required for biological activity. This is the first demonstration of a transforming oncogene in HPV-6, and the differential activity of E5a in these two cell lines should facilitate future investigations on the mechanism of transformation.

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. Characterized antibodies generated against these fusion proteins were used in immunoperoxidase assays to identify the presence and distribution of HPV-6 E5 proteins in biopsy specimens of respiratory tract papillomata. The results showed that the E5a and E5b proteins were distributed throughout the thickness of the epithelium in the papillomata but not in the basal layer. The proteins were found in nuclei and in the cytoplasm of koilocytotic cells. Positive reactivity with a similar distribution in the epithelium and subcellular location was obtained in papillomata induced by other HPV-6 subtypes. This cross-reactivity was not unexpected, since nucleotide and amino acid (aa) sequence comparisons between HPV-6c and -6e demonstrated 79% sequence identity with 15 aa substitutions in the 91 aa of E5a. The E5b ORF of HPV-6c has the potential to encode a protein of 74 aa that differed at 28 positions compared with the 72 aa of HPV-6e.

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. Heteroduplex analysis among the three genomes demonstrated that they were colinear without apparent deletions or rearrangements and had greater than 90% sequence identity. In heteroduplex analyses with a different subtype (HPV-6e) that was molecularly cloned from a genital wart, the genomes were colinear with greater than 90% sequence identity over 90% of their length. The most divergent region had 75-80% sequence identity and was localized to the part of the genome containing the E5a and E5b open reading frames (ORFs). Comparison of the sequence of 1430 nucleotides in this region for two of the HPV-6c isolates did not identify any differences between them. Comparison with the published sequences of HPV-6b identified deletions/insertions and base changes with approximately 75% sequence identity, and comparison with HPV-11 identified only six base changes. Conservation of sequences in the E4-E5 region and similarity in the restriction enzyme maps demonstrated that HPV-6c and HPV-11 are independent isolates of the same HPV-6 subtype.

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. In respiratory tract lesions, immunoperoxidase assay detected only a few capsid antigen positive cells, while in the genital tract lesions, there were more capsid antigen positive cells. Southern transfer analyses and in situ hybridizations demonstrated the presence of more viral nucleic acids in genital tract papillomata than respiratory tract papillomata. Epithelial cells throughout the papillomata were infected by HPV-6 as evidenced by positive hybridization, with more viral DNA present in superficial cells. Our results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. Using stand-specific probes synthesized from subgenomic fragments of the HPV-6 genome in conjunction with nuclease digestions, we were able to demonstrate that HPV-6 transcripts specific to open reading frames (ORFs) E6, E7, E1, L1, and L2 occur in maturing superficial cells. In contrast, transcripts specific to ORFs E1, E2, E4, E5a, and E5b could be detected throughout the whole of the epithelium with more signals noted at the basal cell areas. In addition, the distribution of HPV-6 nucleic acids and protein in a carcinoma in situ of the larynx was analyzed. In comparison to benign respiratory tract papillomata, more viral DNA was found in the malignant lesion, but the pattern and distribution of transcription and capsid antigen was similar.

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. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.