Establishment and transformation diminish the ability of fibroblasts to contract a native collagen gel.

Cultures of established and transformed fibroblasts were less able to contract a hydrated collagen gel than normal precrisis cells. Postcrisis fibroblasts from different rodent strains and species underwent a further reduction in contraction ability and either spontaneous or simian virus 40 (SV40) transformation. Human precrisis fibroblasts contracted much more efficiently than two SV40-transformed human lines. Fibroblasts from a patient with Glanzmann's thrombasthenia were intermediate between all other human fibroblasts assayed and the SV40-transformed human lines. The absolute efficiency of contraction was dependent on temperature and serum concentration, but no conditions were found that resulted in equal efficiencies for the three types of cells. Precrisis cells were extremely sensitive to the passage procedures when assayed for collagen contraction.

Induction of c-Ha-ras gene expression by double-stranded RNA and interferon requirement.

The addition of double-stranded RNA (dsRNA) to NIH 3T3 cells led to an increase in the RNA levels of c-Ha-ras. The double-stranded configuration was required for the increase in c-Ha-ras mRNA levels, as heat-denatured dsRNA and single-stranded RNA did not have any effect. Nuclear run-on transcription experiments indicated that the increase in c-Ha-ras mRNA levels stimulated by dsRNA was due to transcriptional activation of the gene. The induction of c-Ha-ras gene expression by dsRNA was inhibited by anti-beta interferon antibodies, suggesting that interferon might mediate the induction.

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.

Laryngeal papilloma cells have high levels of epidermal growth factor receptor and respond to epidermal growth factor by a decrease in epithelial differentiation.

Laryngeal papillomas are benign epithelial tumors caused by human papillomaviruses. These tumors are characterized by hyperplasia of the spinous layer and abnormal differentiation. Many tumor cell lines over-express the epidermal growth factor (EGF) receptor on their surface, and EGF regulates normal cell growth. We have asked about the relationship of the EGF receptor and EGF response in laryngeal papilloma cells. Papilloma cells showed markedly greater immunohistochemical staining for the EGF receptor, compared to uninfected cells. Both cell types showed a 2-3-fold increase in nuclei incorporating bromodeoxyuridine when EGF was present. Removal of EGF from papilloma cells cultured on collagen rafts permitted normal stratification and differentiation, as determined by synthesis of keratin 13. Inclusion of EGF induced abnormal differentiation with minimal expression of keratin 13. Uninfected laryngeal cells cultured on rafts in the presence of EGF synthesize keratin 13 in all suprabasal cells. EGF reduced both human papillomavirus RNA levels in the papilloma cells and expression of a reporter gene linked to the human papillomavirus 11 enhancers and E6 promoter in uninfected cells. These results suggest that the phenotype of papillomas is induced, in part, by EGF binding to the abundant EGF receptors.

Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx.

Verrucous carcinoma of the larynx clinically resembles laryngeal papilloma in that both are wart-like masses on the vocal cords and may be characterized by multifocality and recurrence. Human papillomavirus (HPV) infection is an etiological factor in laryngeal papilloma, and recent evidence implicates HPV in squamous neoplasias. To determine whether HPV is also associated with verrucous carcinoma of the larynx, we analyzed tissue specimens from six patients with verrucous carcinoma of the larynx by Southern and DNA dot blot hybridization for HPV DNA. From three patients, specimens of normal laryngeal epithelium were also studied. All tissues showed evidence of HPV sequences related but not identical to HPV-16. They were negative for HPV-11. In contrast, four squamous cell carcinomas of the larynx and three normal laryngeal tissues were negative for HPV DNA. Histological sections of the six verrucous lesions were found to contain koilocytosis. Immunoperoxidase staining for HPV capsid antigens was negative in all these cases. The consistent and specific association of HPV with the verrucous carcinomas in this report suggests the possibility of a pathogenic involvement.

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.

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.

Papillomavirus. Effects upon mother and child.

Papillomaviruses are widespread, sexually transmitted agents with an increasing prevalence. They are associated with a significant risk of genital carcinoma in infected women. Because they can be transmitted to the fetus before or during birth, they are also a risk to the infant born to an infected woman. Laryngeal HPV infections, while presumably much less prevalent than genital tract infections, are associated with a high degree of morbidity and a significant degree of mortality when they cause laryngeal papillomas. Therefore, transmission of these viruses to the fetus is a major problem. Much more information regarding mode of transmission and possible cure for this infection is needed in order to reduce the risk of laryngeal papillomatosis in infants in the future.

Use of artificial intelligence to analyze clinical database reduces workload on surgical house staff.

BACKGROUND: The current quantity and diversity of hospital clinical, laboratory, and pharmacy records have resulted in a glut of information, which can be overwhelming to house staff. This study was performed to measure the impact of artificial intelligence analysis of such data on the junior surgical house staff's workload, time for direct patient care, and quality of life. METHODS: A personal computer was interfaced with the hospital computerized patient data system. Artificial intelligence algorithms were applied to retrieve and condense laboratory values, microbiology reports, and medication orders. Unusual laboratory tests were reported without artificial intelligence filtering. RESULTS: A survey of 23 junior house staff showed a requirement for a total of 30.75 man-hours per day, an average of 184.5 minutes per service twice a day for five surgical services each with an average of 40.7 patients, to manually produce a report in contrast to a total of 3.4 man-hours, an average of 20.5 minutes on the same basis (88.9% reduction, p < 0.001), to computer generate and distribute a similarly useful report. Two thirds of the residents reported an increased ability to perform patient care. CONCLUSIONS: Current medical practice has created an explosion of information, which is a burden for surgical house staff. Artificial intelligence preprocessing of the hospital database information focuses attention, eliminates superfluous data, and significantly reduces surgical house staff clerical work, allowing more time for education, research, and patient care.

Suppression of neointimal lesions after vascular injury: a role for polyclonal anti-basic fibroblast growth factor antibody.

BACKGROUND: Basic fibroblast growth factor (bFGF) is a potent local promoter of vascular smooth muscle cell migration and proliferation and may play a major role in the pathogenesis of intimal fibromuscular lesions. Preliminary studies have shown that exogenous bFGF localizes to injured rabbit aorta and suggest that this interaction might be inhibited by anti-bFGF immunoglobulin (Ig) G. This study was designed to evaluate the possible role of polyclonal anti-bFGF IgG in reducing intimal fibromuscular lesion formation in the injured rabbit aorta. METHODS: The abdominal aortic endothelium was subjected to balloon injury in 13 rabbits. Six rabbits received intravenous rabbit anti-bFGF IgG, and seven received irrelevant rabbit IgG (16 micrograms/kg) 30 minutes before injury and daily for 5 days after injury. At 14 days after injury the aorta was fixed and sectioned, and the intimal and medial areas were measured by computerized digital morphometry with the intimal/medial ratio as an index of neointimal lesion formation. RESULTS: In the control group the intimal/medial ratio was 0.538 +/- 0.046 (mean +/- SEM), which was significantly greater than the anti-bFGF-treated group value of 0.148 +/- 0.021 (p < 0.001). CONCLUSIONS: These results show that daily doses of intravenous polyclonal anti-bFGF IgG for 5 days after balloon aortic injury significantly inhibit intimal fibromuscular lesion formation at 14 days. The results suggest that the process of intimal fibromuscular lesion formation may be susceptible to modification by antagonists to bFGF.

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.

Mammary artery versus saphenous vein grafts: assessment of basic fibroblast growth factor receptors.

Neointimal hyperplasia limits the long-term patency of saphenous vein grafts (SVGs), but is notably absent from most internal mammary artery (IMA) grafts. Basic fibroblast growth factor (bFGF) is a local endothelial and vascular smooth muscle mitogen known to be involved in the pathogenesis of neointimal hyperplasia. This study used an animal model to compare the number of available high-affinity (HAR) and low-affinity (LAR) bFGF receptors in SVGs and IMA grafts and to determine whether distention injury causes an increase in receptor availability. The IMA and SVG specimens were harvested from 12 dogs and distended at 25 or 200 mm Hg for 15 minutes, and then the bFGF receptor uptake was measured in them using iodine 125-labeled bFGF. In the IMA conduits distended at low pressure, there were 2.54 +/- 0.10 (mean +/- standard error of the mean) HARs per mm2 of intimal surface area available and 5.19 +/- 0.40 LARs per mm2. High-pressure distention significantly (p < 0.001) increased the number of available HARs to 5.06 +/- 0.27 per mm2 and of LARs to 7.27 +/- 0.042 per mm2. At low pressure, the SVGs had significantly (p < 0.001) more HARs (9.14 +/- 0.84 per mm2) and LARs (18.2 +/- 0.57 per mm2) available than did the IMA conduits, and high pressure significantly (p < 0.001) increased the number of HARs available in SVGs to 24.1 +/- 2.43 per mm2 and the number of LARs to 44.7 +/- 2.34 per mm2.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe calcification does not affect long-term outcome of mitral valve repair.

Some surgeons have suggested that the presence of severe calcification in the mitral valve annulus or leaflets precludes successful repair. Our institution has attempted to repair these calcified valves when good annular and leaflet mobility could be achieved by annular debridement and leaflet resection. From June 1979 through June 1993 558 mitral valve repairs were performed using Carpentier's techniques. When calcified valves were encountered, these techniques were modified to include annular debridement and mechanical leaflet decalcification. Calcification was identified preoperatively in 49 patients (8.8%) by either left ventricular fluoroscopy or echocardiography and was debrided in 64 patients (11.5%). This included 24 annular debridements, 28 leaflet debridements, and 12 annular and leaflet debridements. Patient ages ranged from 13 to 83 years (mean age, 62.3 years), and 25 patients (39.1%, 25/64) had concomitant cardiac procedures. Operative mortality was 6.2% (4/64) overall and 2.6% (1/39) for isolated mitral valve repairs. Calcium debridement was performed in 19.3% (23/119) of patients with a rheumatic cause compared with 9.3% (41/439) of the nonrheumatic patients (p < 0.01). Long-term follow-up revealed the necessity for reoperation in 7.8% (5/64) in patients with calcium debridement as compared with 7.7% (38/494) with no debridement (p = not significant). Cumulative freedom from reoperation at 10 years was 83.3% for all patients, 88.1% for debrided patients, and 82.6% for nondebrided patients (p = not significant). Cox proportional hazards analysis revealed that the presence of rheumatic disease significantly increased the risk of reoperation (odds ratio = 3.28; p < 0.001), whereas calcium debridement had no significant effect. These results demonstrate that when good annulus and leaflet motion can be achieved in calcified mitral valves, calcium debridement allows durable repairs.

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.

Heparin bonding of bypass circuits reduces cytokine release during cardiopulmonary bypass.

BACKGROUND: Heparin bonding of the cardiopulmonary bypass (CPB) pump circuit decreases complement activation and fibrinolysis. It is not known whether inflammatory cytokines produced during CPB can also be modulated by the more biocompatible heparin-coated circuit. METHODS: This initial study evaluated the impact of heparin-bonded CPB circuits on production of the cytokines interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-a), IL-6, and IL-8 in adults undergoing complex cardiac operations with prolonged CPB. Twenty patients had blood samples drawn immediately before and at hourly intervals after the start of CPB using either a conventional oxygenator and circuit (n = 14) or a covalently bonded heparin oxygenator and circuit (n = 6). Levels of IL-1, TNF-a, IL-6, and IL-8 were measured in all serum samples using a "sandwich" enzyme-linked immunosorbent assay. RESULTS: The levels of IL-6 and IL-8 increased in a time-dependent fashion in both groups, but the response was significantly less over time in the heparin-bonded group (p < 0.05) for both IL-6 and IL-8. Levels of IL-1 and TNF-a were not significantly elevated with lengthening bypass interval in either group. CONCLUSIONS: These data indicate that the use of heparin-coated bypass pump circuits results in lower serum levels of the inflammatory cytokines IL-6 and IL-8 than standard circuits. Biocompatible materials that decrease the inflammatory response to CPB may ultimately reduce the morbidity associated with cardiac operations.

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.

Cytogenetic analysis of head and neck carcinomas.

Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "hotspots" of rearrangements.

Laryngeal papillomatosis: clinical, histopathologic and molecular studies.

The clinical course and pathology of 57 patients with laryngeal papillomatosis were reviewed. Tissues from 26 patients were analyzed for human papillomavirus (HPV) DNA by Southern blot hybridization. Histopathologic evaluation of the papillomas showed no correlation with age of onset or clinical pattern of remission and recurrence. The pathology was characterized by abnormal squamous maturation with parakeratosis, retardation of superficial cell maturation, papillomatosis, and basal hyperplasia. HPV DNA was present in all lesions, with 92% containing either HPV-6 or 11. Latent HPV DNA was detected in clinically uninvolved tissues of 11 of 14 (78.5%) patients studied. There was no correlation between HPV type, histopathology and/or clinical pattern. Despite homogeneity of pathology, the clinical expression of laryngeal HPV infection varied widely. A mechanism for the pathogenesis of laryngeal papillomatosis, based on the concept of maturational arrest, is proposed.

Clinical effects of alpha-interferon dose variation on laryngeal papillomas.

Achieving optimal clinical control of recurrent respiratory papillomatosis with prolonged treatment with human leukocyte (alpha) interferon appears to be dose-related and often requires individualized dosage elevation. Six of eight patients in this study needed a maximum dose of 18 x 10(6) IU/week for part of the therapy period to achieve better disease control. The strong correlation found between dosage and response suggests that it is the interferon causing the effect on disease expression, not just the unpredictable nature of the disease. An effect on papilloma growth was observed in all patients during IFN therapy. Three patients were able to be tapered off IFN with only minimal recurrence seen in one patient. No toxic side effects were observed.

Tracheal reconstruction: in vitro and in vivo animal pilot study.

In order to further address the problem of tracheal stenosis, an animal model was created to simulate both local and regional tracheal injury. An epithelial equivalent created from a fibroblast-collagen matrix was used to attempt to either inhibit or lessen the degree of tracheal stenosis that was evident in animal models. Preliminary data appears to show that the epithelial equivalent is effective in limiting and perhaps even abolishing the development of tracheal stenosis.