Interaction of human papillomavirus type 11 E7 protein with TAP-1 results in the reduction of ATP-dependent peptide transport.

Human papillomaviruses (HPVs) cause benign and malignant epithelial tumors of the respiratory and genital mucosa. We previously reported that recurrent respiratory papillomas caused by HPV 6/11 express low levels of antibody-detectable TAP-1, the protein that transports peptides into the endoplasmic reticulum for assembly and presentation by MHC Class I, and that the extent of TAP-1 immunostaining is inversely related to the frequency of disease recurrence. We have now determined a mechanism for the reduction in TAP-1 detection. Anti-TAP-1 antibody immunoprecipitated very low amounts of protein from papilloma cells. However, immunoprecipitation of calreticulin, another member of the MHC I assembly complex, coprecipitated TAP-1 at levels comparable to those of uninfected cells. Immunoprecipitation of an HPV-positive cell line with either anti-TAP-1 or anti-calreticulin coprecipitated HPV E7 protein. Finally, purified HPV 11 E7 protein inhibited ATP-dependent peptide transport in vitro. We propose that the interaction of E7 with TAP-1 prevents TAP-1 antibody detection and efficient peptide transport, resulting in poor presentation of viral antigen on HPV-infected cells and thus failure to mount an effective immune-mediated prevention of disease recurrence.

Minimally invasive aortic valve replacement: echocardiographic and clinical results.

BACKGROUND: Port access has been described for mitral and bypass surgery. The purpose of this study was to review the clinical and echocardiographic outcomes of aortic valve replacement by use of port access. METHODS: Between 1996 and 1999, 153 port-access aortic valve replacements were performed at our institution. The mean age was 63 years (range 16-91 years); 58% were male. The New York Heart Association mean class was III; 18% were in class IV. Thirteen percent had diabetes, 42% hypertension, 7% prior transient ischemic episode or stroke, 7% lung disease, 3% renal failure, and 13% previous surgery. Echocardiograms were obtained after valve replacement in 125 patients (96 intraoperative transesophageal and 97 transthoracic echoes). RESULTS: Median length of stay was 8 days. There were no intraoperative deaths; 10 patients (6.5%) died in the postoperative period. Stroke occurred in 4 (2.6%), sepsis in 5 (3.3%), renal failure in 5 (3.3%), pneumonia in 3 (2%), and wound infection in 1 (0.7%). Tissue prosthesis was present in 83 and a mechanical prosthesis in 42. No or trace regurgitation was seen on 94 of 96 (98%) postbypass intraoperative echocardiograms and mild on 2. On follow-up echocardiograms, moderate regurgitation was seen in 4 of 97 (4.1%), mild-to-moderate in 2 (2.1%), mild in 18 (18.6%), and no or trace in 71 (73.2%). Of those who had aortic regurgitation on intraoperative or follow-up echocardiograms, it was paravalvular in 8. CONCLUSIONS: Minimally invasive aortic valve replacement with a port-access approach is feasible, even in high-risk patients. Small incisions, a low infection rate, and a short length of stay are attainable. However, the complications associated with traditional aortic valve replacement still occur. Echocardiography is valuable both for intraoperative monitoring and follow-up of this new procedure.

Minimally invasive versus sternotomy approaches for mitral reconstruction: comparison of intermediate-term results.

BACKGROUND: This study compares intermediate-term outcomes of mitral valve reconstruction after either the standard sternotomy approach or the new minimally invasive approach. Although minimally invasive mitral valve operations appear to offer certain advantages, such as reduced postoperative discomfort and decreased postoperative recovery time, the intermediate-term functional and echocardiographic efficacy has not yet been documented. METHODS: From May 1996 to February 1999, 100 consecutive patients underwent primary mitral reconstruction through a minimally invasive right anterior thoracotomy and peripheral cardiopulmonary bypass and Port-Access technology (Heartport, Inc, Redwood City, Calif). Outcomes were compared with those for our previous 100 patients undergoing primary mitral repair who were operated on with the standard sternotomy approach. RESULTS: Although patients were similar in age, the patients undergoing the minimally invasive approach had a lower preoperative New York Heart Association classification (2.1 +/- 0.5 vs 2.6 +/- 0.6, P <.001). There was one (1.0%) hospital mortality with the sternotomy approach and no such case with the minimally invasive approach. Follow-up revealed that residual mitral insufficiency was similar between the minimally invasive and sternotomy approaches (0.79 +/- 0.06 vs 0.77 +/- 0.06, P =.89, 0- to 3-point scale); likewise, the cumulative freedom from reoperation was not significantly different (94.4% vs 96.8%, P =.38). Follow-up New York Heart Association functional class was significantly better in the patients undergoing the minimally invasive approach (1.5 +/- 0.05 vs 1.2 +/- 0.05, P <.01). CONCLUSIONS: These findings demonstrate comparable 1-year follow-up results after minimally invasive mitral valve reconstruction. Both echocardiographic results and New York Heart Association functional improvements were compatible with results achieved with the standard sternotomy approach. The minimally invasive approach for mitral valve reconstruction provides equally durable results with marked advantages for the patient and should be more widely adopted.

Robotic-assisted instruments enhance minimally invasive mitral valve surgery.

BACKGROUND: The potential for totally endoscopic mitral valve surgery has been advanced by the development of minimally invasive techniques. Recently surgical robots have offered instrument access through small ports, obviating the need for a significant thoracotomy. This study tested the hypothesis that a microsurgical robot with 5 degrees of freedom is capable of performing an endoscopic mitral valve replacement (MVR). METHODS: Dogs (n = 6) were placed on peripheral cardiopulmonary bypass; aortic occlusion was achieved with endoaortic clamping and transesophageal echocardiographic control. A small left seventh interspace "service entrance" incision was used to insert sutures, retractor blade, and valve prosthesis. Robotically controlled instruments included a thoracoscope and 5-mm needle holders. MVR was performed using an interrupted suture technique. RESULTS: Excellent visualization was achieved with the thoracoscope. Instrument setup required 25.8 minutes (range 12 to 37); valve replacement required 69.3+/-5.39 minutes (range 48 to 78). MVR was accomplished with normal prosthetic valve function and without misplaced sutures or inadvertent injuries. CONCLUSIONS: This study demonstrates the feasibility of adjunctive use of robotic instrumentation for minimally invasive MVR. Clinical trials are indicated.

Impact of heparin bonding on pediatric cardiopulmonary bypass: a prospective randomized study.

BACKGROUND: Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass in adult patients; however, little is known about its effects in the pediatric population. Two studies were performed to assess this technology's impact on inflammation and clinical outcomes. METHODS: In a pilot study, complement and interleukins were measured in 19 patients who had either uncoated cardiopulmonary bypass circuits or heparin-bonded circuits. Subsequently, 23 additional patients were studied in a randomized fashion. Respiratory function and blood product utilization were recorded. RESULTS: In the pilot study, heparin-bonded circuit patients had less complement 3a (p < 0.001) and interleukin-8 (p < 0.05) compared with uncoated cardiopulmonary bypass circuit patients. The randomized study revealed that the heparin-bonded circuit was associated with reduced complement 3a (p = 0.02). Multiple variable analysis revealed that the following postoperative variables were increased with bypass time (p = 0.01) and diminished with heparin-bonded circuits: interleukins (p = 0.01), peak airway pressures (p = 0.05), and prothrombin time (p = 0.03). CONCLUSIONS: Heparin-bonded circuits significantly reduce cytokines and complement during cardiopulmonary bypass and lower interleukin levels postbypass; they were also associated with improved pulmonary and coagulation function. Heparin-bonded circuits ameliorate the systemic inflammatory response in pediatric patients from cardiopulmonary bypass.

Overexpression of PTEN/MMAC1 and decreased activation of Akt in human papillomavirus-infected laryngeal papillomas.

Laryngeal papillomas are benign, human papillomavirus-induced hyperplastic tumors of the respiratory tract. They are characterized by overexpression of the epidermal growth factor receptor, constitutive activation of mitogen-activated protein kinase, a low proliferative rate, and defects in differentiation. We have now found that phosphoinositol 3-kinase (PI 3-K) activity is significantly increased in papilloma tissue. However, phosphorylated Akt (also known as protein kinase B), a downstream effector of PI 3-K, is reduced when compared with normal tissue. The ratio of activated Akt to total Akt is much lower in papillomas than in normal laryngeal tissue, suggesting decreased Akt activation. PTEN/ MMAC1 is a tumor suppressor that dephosphorylates phosphatidylinositol 3,4,5-triphosphate, an intermediate in the PI 3-K/Akt signaling pathway. We have found that PTEN protein is overexpressed in laryngeal papillomas when compared with normal laryngeal tissues. On the basis of reverse transcription-PCR analysis, PTEN mRNA is more abundant in papillomas, suggesting transcriptional up-regulation. We postulate that negative regulation of the PI 3-K/Akt pathway by PTEN may modulate the effects of the hyperactive epidermal growth factor receptor/mitogen-activated protein kinase pathway, contributing to the low proliferation and dysfunctional differentiation of laryngeal papillomas.

Altered expression of TAP-1 and major histocompatibility complex class I in laryngeal papillomatosis: correlation of TAP-1 with disease.

Recurrent respiratory papillomatosis (RRP) is an insidious disease caused by human papillomavirus (HPV) infection. It is characterized by a variable clinical course that can include frequent disease recurrence, significant morbidity, and occasional mortality. The mechanisms responsible for the variability in the clinical course and the persistence of latent HPV infection remain unknown. Effective T-cell-mediated clearance of HPV-infected cells may be defective in patients with RRP, leading to recurrent disease and failure to suppress latent HPV reactivation. This study describes the down-regulation of the transporter associated with antigen presentation (TAP-1) and the major histocompatibility complex (MHC) class I protein expression in laryngeal papilloma tissue biopsies and cell culture of primary explants. There was a statistically significant correlation between reduction of TAP-1 expression in biopsy tissues and rapid recurrence of disease. Patients with RRP had less frequent recurrence if their papillomas expressed TAP-1 at levels close to that of normal tissue, compared with those with very low expression of TAP-1, who had frequent recurrence (32 versus 5 weeks to the next surgical intervention). These findings suggest that HPV may evade immune recognition by down-regulating class I MHC cell surface expression via decreased TAP-1 levels. Expression of TAP-1 could be used for prognostic evaluation of disease severity. Gamma interferon was able to restore class I MHC expression at the surfaces of laryngeal papilloma cells in culture. This up-regulation of class I MHC antigen at the cell surface potentially allows the infected cell to become a target for the immune system again. This finding provides some promise for nonsurgical treatment of laryngeal papillomas.

Recurrent respiratory papillomatosis: altered CD8(+) T-cell subsets and T(H)1/T(H)2 cytokine imbalance.

Human papillomaviruses (HPVs) cause benign papillomas and squamous cell carcinomas in the genital and respiratory tracts. Recurrent respiratory papillomas (RRP) generate a high level of morbidity and significant mortality because of their location, resistance to treatment, and relentless recurrence that can vary in frequency in a given patient and between patients. We have found that T-cells from these patients, when exposed to or isolated from autologous papilloma tissue, have an elevated percentage of CD8(+), CD28(-) T-cells, and that T-cells from many of these patients express an increase in T(H)2-like cytokine mRNA in response to autologous papilloma tissue. Furthermore, both of these immunologic findings correlate with disease severity. These observations suggest that patients with RRP, and possibly others with refractory HPV-induced lesions, are unable to manage their disease with an appropriate and effective HPV-specific, T-cell response. This immune imbalance may be responsible for the development and severity of HPV-induced respiratory papillomatosis.

Induction of E6/E7 expression in cottontail rabbit papillomavirus latency following UV activation.

Latent human papillomavirus (HPV) infections are widespread in the genital and respiratory tracts and are a source of recurrent disease. This study used a cottontail rabbit papillomavirus (CRPV) model to determine the presence of E1, E6, and E7 transcripts in latent infection and to determine the temporal change in transcripts following UV activation. We found E1 transcripts in all latently infected sites but no detectable E6 and E7 transcripts, consistent with our earlier studies of HPV6/11 latency. These results suggest that this transcription pattern is broadly characteristic of latent papillomavirus infections. E6/E7 transcripts were detectable within 1 week of irradiation, with maximal induction (approximately 40% of sites) at 2 weeks postirradiation. Papillomas were induced in approximately 26% of irradiated sites after a 3- to 5-week lag. Sites that did not form papillomas by 3 months after irradiation were CRPV DNA positive but E6/E7 RNA negative. Thus, only a subset of latent infections can be induced to express E6/E7 transcripts and form papillomas. We propose that CRPV can be used to study the molecular processes regulating papillomavirus activation.

Mechanical endothelial damage results in basic fibroblast growth factor-mediated activation of extracellular signal-regulated kinases.

BACKGROUND: Endothelial damage, such as that associated with balloon angioplasty or preparation of veins for bypass grafts, results in intimal hyperplasia. Growth factors and cytokines that modulate endothelial cell functions through various intracellular signaling pathways mediate rapid endothelial repair, which may prevent or reduce restenosis. Here we investigated the effect of mechanical injury of endothelial cells on the mitogen-activated kinase signaling pathways, extracellular-signal-regulated kinases (ERKs), C-Jun N-terminal kinase (JNK/SAPK), and p38. METHODS: Confluent human umbilical vein endothelial cells or bovine aortic endothelial cells were wounded with a razor blade; mitogen-activated kinase activation was monitored by immunoblotting with antibodies to active ERK, JNK/SAPK, or p38. RESULTS: Wounding of human umbilical vein endothelial cell or bovine aortic endothelial cell monolayers resulted in rapid (5-minute) activation of ERK-1 and -2, which was abolished by monoclonal antibody to basic fibroblast growth factor (FGF-2). This antibody or an inhibitor of ERK activation, PD98059, also blocked endothelial cell migration after the wounding. Thus FGF-2-induced ERK activation mediates the endothelial response to wounding. CONCLUSIONS: ERK-1 and -2 are activated by FGF-2 released from endothelial cells in response to injury. Therapeutic strategies aimed at reducing FGF-2-induced intimal hyperplasia should preserve ERK activation in endothelial cells while abolishing it in smooth muscle cells.

Elevation of the epidermal growth factor receptor and dependent signaling in human papillomavirus-infected laryngeal papillomas.

Laryngeal papillomas are benign tumors caused by human papillomaviruses types 6 and 11. This study addressed alterations in levels of signal transduction from the epidermal growth factor receptor (EGFR) in papillomas and cultured papilloma cells compared to normal tissue and cells. Mitogen-activated protein kinase (MAPK) was activated to a greater extent, phosphotyrosine was more abundant, and EGFR was overexpressed in laryngeal papillomas compared to normal laryngeal epithelium by Western blot analysis. The EGFR was 3 times more abundant in cultured papilloma cells than in normal laryngeal cells by Scatchard analysis and Western blot, without gene amplification or an increase in steady-state levels of mRNA. Following stimulation with EGF, a significant portion of the EGFR was recycled to the surface in papilloma cells, whereas in normal cells, it was not. Tyrosine kinase activity and activation of MAPK was more responsive to epidermal growth factor stimulation in papilloma cells than in uninfected primary laryngeal cells. PD153035, a specific inhibitor of the EGFR, and an EGFR-specific antibody that blocks ligand binding completely abrogated basal MAPK activation by endogenous ligands in laryngeal papilloma cells. These results demonstrated that infection of laryngeal epithelium by low-risk human papillomaviruses elevates the EGFR by posttranslational mechanisms, increasing its responsiveness to ligand-mediated activation. They also showed that MAPK activation in laryngeal papillomas depends upon ligand-mediated EGFR stimulation.

Efficacy of DHE photodynamic therapy for respiratory papillomatosis: immediate and long-term results.

OBJECTIVES/HYPOTHESIS: Recurrent respiratory papillomatosis is a potentially life-threatening disease that affects both children and adults and can result in complete respiratory obstruction. Conventional therapies cannot prevent multiple recurrences. The authors have been evaluating photodynamic therapy (PDT) to treat this disease since 1988. This study compared the efficacy of PDT with dihematoporphyrinether (DHE) with traditional therapy. STUDY DESIGN: This was a randomized prospective trial of DHE-PDT. Patients were randomly assigned to receive one of two doses of DHE--3.25 mg/kg or 4.25 mg/kg body weight. They were compared with a concurrent control group. Disease extent was evaluated by direct laryngoscopy before treatment and over a 1-year period following treatment. Results were also compared with two historical cohorts of patients treated with lower doses of DHE. METHODS: Eighty-one patients, ages 4 to 74 years, with moderate to severe recurrent disease were enrolled. Forty-eight received PDT and 33 in the control group were treated with conventional therapy. Both PDT groups received 50 J laser light to activate the drug. Patients received an intravenous infusion of DHE as outpatients 48 to 72 hours before treatment. During direct laryngoscopy, light (630 nm) was delivered by an argon-pumped dye laser. Tissue biopsies were analyzed for presence of human papillomavirus (HPV). RESULTS: There was notable improvement with either drug dose over the first year. Those receiving 4.25 mg/kg DHE experienced a significantly larger decrease in papilloma growth rate. Three-year follow-up of a subset of patients confirmed that improvement was maintained. There was no impact of DHE-PDT on persistence of HPV DNA. CONCLUSION: This therapy holds promise for the treatment of laryngeal papillomas.

Evidence for the separate regulation of the human papillomavirus type 11 E7 and E6 promoters by Viral cis sequences near the E6 promoter.

The human papillomavirus type 11 (HPV-11) E7 protein can modulate host cell functions and is required for papilloma formation, but little is known concerning the regulation of its expression. This study was designed to determine whether the viral upstream regulatory region controlled expression from the E7 promoter and whether cis sequences differentially regulated E6 and E7 expression in laryngeal mucosal keratinocytes, the natural target cells for this virus. Reporter constructs were designed to study expression of the luciferase gene from the HPV-11 E7 promoter in its natural position downstream of a functional E6 promoter. E7 expression, like E6 expression, required upstream regulatory sequences. However, E7 expression was less sensitive to repression by viral E2 protein and to mutation of the Spl binding site adjacent to the E2 binding site. Moreover, there was differential sensitivity of the two promoters to mutation of the E6 TATA box, with E7 expression more affected than E6 expression. These findings show that, in the normal host cells for this virus, the E6 and E7 promoters can be independently regulated by the cis regulatory region adjacent to the E6 promoter.

Comparative biodistribution of meta-Tetra(Hydroxyphenyl) chlorin in multiple species: clinical implications for photodynamic therapy.

BACKGROUND AND OBJECTIVE: To optimize photodynamic therapy, it is necessary to know the distribution of photosensitizer in normal tissue as well as tumors and to know how well animal models match human. This study measured the biodistribution of meta-Tetra(Hydroxyphenyl) Chlorin (mTHPC) in three species of animals and in humans. STUDY DESIGN/MATERIALS AND METHODS: mTHPC was injected intravenously into dogs, rabbits, and humans, and drug levels in various tissues were determined 6 days later. One dog was perfused with 3 L of saline to remove blood trapped within organs. RESULTS: Absolute and relative concentrations of drug in specific tissues varied between species and between individuals. There was a general pattern of distribution. Highly vascularized tissues had the highest levels of mTHPC, not simply due to trapping of blood. mTHPC did not localize in bone and did not cross the blood-brain barrier. Humans had much higher levels of drug in their plasma and tissues than did animals. CONCLUSIONS: First, drug retention varies from one tissue to another. Second, there is significant variability from one individual to another, whether animal or human. Third, current models cannot accurately predict from animal studies the optimum dose for humans. Measurement of photosensitizer level in plasma at time of treatment would allow optimal photodynamic dosing.

A possible role for human papillomaviruses in head and neck cancer.

Human papillomaviruses (HPVs) cause benign tumors in the respiratory tract. Mounting evidence suggests that they also play a role in the etiology of a subset of head and neck cancers. Carcinomas in patients with a history of recurrent respiratory papillomatosis clearly are caused by persisting HPV interacting with one of more carcinogenic agents. Verrucous carcinomas of the oral cavity, tonsillar and tongue carcinomas are strongly linked with HPVs, based on molecular epidemiologic data. Tonsillar cancer have been shown to express HPV RNA, presumed necessary to induce and maintain a carcinoma, supporting a viral etiology. This paper reviews the molecular and cellular basis for considering HPVs as causative agents of cancer, and reviews the literature that considers the possible role of HPVs in head and neck cancer.

Differential regulation of human papillomavirus type 6 and 11 early promoters in cultured cells derived from laryngeal papillomas.

Cells cultured from laryngeal papillomas contain episomal human papillomavirus type 6 or type 11 (HPV-6/11) DNA. We developed a sensitive RNase protection assay to simultaneously measure expression from the HPV E6, E7, and E1 promoters (P1, P2 and P3, respectively) in this manipulable culture system and found that P1, P2 and P3 transcript abundances could be independently modulated by culture medium composition and culture substrate. In undifferentiated cells grown in a low-calcium, serum-free medium, P1 transcripts commonly predominated over those from P2, P3 transcripts were often undetectable, and high concentrations of retinoic acid were able to selectively decrease P2 transcript abundance. When cultures were allowed to stratify and differentiate by growth on a collagen gel at he air-liquid interface, total HPV RNA increased up to sixfold because of selective increases in abundances of P1 and P3 transcripts. High-calcium submerged cultures also showed easily detectable P3 transcripts, and isolated suprabasal cells contained almost exclusively these transcripts. Growth arrest alone was not sufficient to induce P3 transcripts. Thus, in contrast to the HPV-6/11 E6 and E7 promoters, the E1 promoter was utilized primarily in a differentiation-specific manner. We also show that increased HPV gene dosage will not necessary bring about increased HPV transcript abundance, suggesting that other viral and cellular factors are responsible for regulation of total transcript levels as well as specific promoter usage.

Efficacy of intravenous delta-aminolaevulinic acid photodynamic therapy on rabbit papillomas.

Endogenously induced protoporphyrin IX (PPIX), a metabolite of delta-aminolaevulinic acid (ALA), has been evaluated as a photosensitising agent for destruction of papillomas in cottontail rabbit papillomavirus-infected Dutch belted and New Zealand rabbits. Three factors were evaluated: (1) relative retention ratio of drug in normal tissue, papilloma and plasma over time; (2) tissue tolerance to treatment factors; and (3) efficacy of treatment protocol. Three drug doses of ALA were examined: 50, 100 and 200 mg kg-1. Actual PPIX concentrations in tissue and plasma were determined spectrophotofluorometrically. The optimal treatment time occurred 3-6 h post ALA injection. The highest PPIX concentration ratio between papilloma and normal skin was 6:1. Different light doses were investigated, using an injection to exposure interval of 3 h and an irradiance of 100 mW cm-2 at a wavelength of 630 nm. Efficacy without risk of significant damage to normal skin was obtained using 100-200 mg kg-1 ALA and 40-60 J cm-2. A long-term (3 months) cure rate of 82% was obtained with a single treatment, provided that papilloma depth did not exceed 8 mm, volume was not more than 1000 mm3 and the plasma concentration of PPIX immediately before exposure was above 500 micrograms ml-1. The short time between injection and treatment and high efficacy, together with PPIX disappearance from plasma and tissue within 24 h, make injected ALA a highly attractive drug for photodynamic therapy.

Human papillomavirus type 11 transcripts are present at low abundance in latently infected respiratory tissues.

Respiratory tract tissues containing latent human papillomavirus (HPV) 11 were analyzed by reverse transcription-polymerase chain reaction for the presence of viral-specific RNA from the early region of the genome and compared to a similar analysis of laryngeal papillomas. Latently infected tissue contained low-abundance transcripts that could code for E1 and E2 proteins, but lacked evidence of spliced transcripts for the E6 and E7 proteins. Both latently infected tissue and papilloma tissue contained low-abundance antisense transcripts. Cultured cells infected with HPV 11 virions or transfected with HPV DNA, and cells derived from latently infected tissue, expressed transcripts similar to those seen in papillomas, but at a lower abundance. We postulate that latency is determined by the absence of or limiting levels of critical viral proteins.