Downregulation of Bcl-2 sensitises interferon-resistant renal cancer cells to Fas.

Interferon alpha (IFNalpha) is used to treat patients with advanced renal cell carcinoma (RCC) despite limited clinical benefit. IFNalpha can induce Fas receptor-mediated apoptosis by direct activation of pro-caspase-8 followed by activation of caspase-3. Alternative, indirect activation of caspase-3 via mitochondrial release of cytochrome c can occur and may explain the rescue from Fas-activated cell death by the antiapoptotic members of the Bcl-2 family. In this study, we examined G3139, a novel antisense compound targeting Bcl-2, in combination with IFNalpha. Human RCC lines (SK-RC-44 and SK-RC-07) were treated with IFNalpha, G3139 or a combination of the two. Fas-mediated cytotoxicity was induced by anti-Fas mAb, CH11. An analysis of Bcl-2, Fas and the cleavage of PARP was performed. IFNalpha induced Fas and Bcl-2 in SK-RC-44 and SK-RC-07. IFNalpha sensitised SK-RC-44 to anti-Fas and induced PARP cleavage confirming that IFNalpha has a cytotoxic effect on RCC lines by induction of the Fas antigen. Cytotoxicity was not evident in SK-RC-07 cells treated with IFNalpha. G3139 induced a specific downregulation of Bcl-2 in SK-RC-07 cells, which were then sensitised to anti-Fas after treatment with IFNalpha. Taken together, these results suggest that Fas-dependent pathways as well as alternative pathways, which can be inhibited by Bcl-2, exist in renal cell carcinoma. G3139 in combination with IFNalpha is a potential therapy in patients with metastatic renal cell carcinoma.

Tumor-suppressive effects of neutral endopeptidase in androgen-independent prostate cancer cells.

Expression of neutral endopeptidase (NEP) 24.11 is diminished in metastatic, androgen-independent prostate cancers (PCs; C. N. Papandreou et al., NAT: MED:, 4: 50--57, 1998). To determine the effects on androgen-independent PC cells of overexpressing cell-surface NEP, an inducible tetracycline-regulatory gene expression system was used to stably introduce and express the NEP gene in androgen-independent TSU-Pr1 cells generating WT-5 cells, which expressed high levels of enzymatically active NEP protein when cultured in the absence of tetracycline. TN12 cells, which contain the identical vectors without the NEP gene and do not express NEP, were used as control. Expression of NEP in WT-5 cells after removal of tetracycline from the media resulted in a >80% inhibition in cell proliferation over a 1-week period (P < 0.005) compared with control cells. Tumor formation occurred in the prostate glands of orthotopically injected athymic mice killed at 30 days in 4 of 5 mice that were given injections of 2 x 10(6) WT-5 cells and were fed doxycycline (NEP suppressed), and in all mice that were given injections of TN12 cells and were fed with or without doxycycline. In contrast, only 1 of 5 mouse prostates developed a tumor in mice that were given injections of WT-5 cells and that did not receive doxycycline. Analysis of the mechanisms of NEP-induced growth suppression revealed that NEP expression in WT-5 cells induced a 4-fold increase in the number of PC cells undergoing apoptosis, and increased the expression of p21 tumor suppressor gene protein and the level of unphosphorylated retinoblastoma protein as determined by Western blot. Flow cytometric analysis show that induced NEP expression in WT-5 cells resulted in a G(1) cell cycle arrest. These data show that NEP can inhibit PC cell growth and tumorigenicity and suggest that NEP has potential as therapy for androgen-independent PC.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

The efficacy of bioresorbable fixation in the repair of mandibular fractures: an animal study.

PURPOSE: The efficacy of bioresorbable fixation has recently been described in the treatment of cranial vault deformities and in midfacial trauma. However, little to no data exist regarding its use in load-bearing areas. The purpose of this study is to analyze and compare the treatment of mandibular fractures by using a bioresorbable fixation system with a conventional titanium system in a canine model. MATERIALS AND METHODS: Four adult beagles constituted the experimental group (A), and 2 beagles constituted the control group (B). Both groups underwent extraoral iatrogenic left mandibular angle osteotomies/fractures and open reduction and internal fixation by use of a bioresorbable fixation system (A) or a titanium fixation system (B). All operated animals were allowed to function immediately. Lateral skull radiographs were obtained preoperatively, immediately postoperatively after reduction, and at 3- and 6-month intervals. Preoperative and 6-month follow-up bite registrations were taken. At the 3-month interval, 1 animal from the experimental group was killed, and at 6 months the remaining animals were killed for morphologic, radiographic, and histologic analysis of the fractured interface and screw sites. RESULTS: Morphologically, in the bioresorbable group, there was no clinical evidence of 1) intraoral/ extraoral incisional dehiscence of wound infection; 2) deviation of the occlusion from maximum intercuspation; 3) intraoral/extraoral palpability of the device; 4) mobility of the fractured segments on manual manipulation; or 5) malunion as visualized at the time of sacrifice. All bioresorbable plates were clinically absent after 6 months and associated with adequate fixation and healing. Adequate restoration of function was achieved in both groups, with all of the animals showing weight gain. Radiographically, good alignment of the inferior border was seen, and histologically bony union was apparent in all specimens. CONCLUSIONS: This bioresorbable fixation system is effective in the treatment of mandibular angle fractures in a dog model, despite being placed in a load-bearing region.

Comparison of two techniques of dermal graft harvest for temporomandibular joint reconstruction.

PURPOSE: The purpose of our study was to compare the healing characteristics of two common techniques of obtaining a dermal graft and the patient's perception of the aesthetics of the donor site scar. MATERIALS AND METHODS: Fifty-two female patients (60 sites) were evaluated by means of questionnaire, interview, and chart review. The mean follow-up time was 4.08 years after temporomandibular joint reconstructive surgery. Comparisons were made between the two primary harvest techniques: the dermatome (trapdoor) and the elliptical excision methods. Of the 60 sites, 32 involved the elliptical method and 28 the trapdoor method. RESULTS: Common findings in both groups included prolonged discomfort (6.6%), altered contour (50%), color change (58.3%), prolonged numbness (10%), and itching (21.6%). There were few healing complications (5.0%). Aesthetically, 45% of patients thought their scars were noticeable, and 8.3% thought that they were bothersome. The elliptical method showed significantly less discomfort, texture change, numbness, and itching. The scars were concealed better and less noticeable. Although 60.7% of the patients with the trapdoor method considered the wounds to be noticeable, only four patients reported that the scars were bothersome. CONCLUSIONS: Both techniques were used successfully with minimal complications. Although the elliptical technique appeared to have cosmetic advantages, larger grafts produced longer wounds, which may be more difficult to conceal. The trapdoor method is a simple technique that is used effectively with good patient acceptance. The results of this study are useful in properly informing patients about what to expect after having a dermal graft.

Blockade of smoking satisfaction using the peripheral nicotinic antagonist trimethaphan.

The present study was conducted to investigate the role of peripheral nicotinic receptors in mediating the rewarding effects of cigarette smoking. Twelve cigarette smokers rated cigarettes after intravenous infusion of the short-acting peripheral nicotinic receptor antagonist trimethaphan and after placebo (saline) infusions. Subjects were blinded to the infusion and cigarette conditions. Cigarette conditions included subjects' usual brand of cigarette, denicotinized tobacco cigarettes, and nicotine-injected cigarettes that had a tar delivery equal to that of the denicotinized cigarettes but with an enhanced nicotine delivery equal to that of subjects' usual brands. The latter cigarettes were rated as extremely harsh due to the high nicotine/tar ratio. Trimethaphan significantly attenuated the airway sensations associated with nicotine, and eliminated the difference in smoking satisfaction between the usual brand of cigarette and the other two cigarettes. These findings suggest that nicotinic receptors on peripheral nerve endings in the respiratory tract modulate smoking satisfaction and may be important in the maintenance of cigarette addiction.

Carbon dioxide laser ablation with immediate autografting in a full-thickness porcine burn model.

OBJECTIVE: To compare the long-term clinical and histologic outcome of immediate autografting of full-thickness burn wounds ablated with a high-power continuous-wave CO2 laser to sharply débrided wounds in a porcine model. SUMMARY BACKGROUND DATA: Continuous-wave CO2 lasers have performed poorly as tools for burn excision because the large amount of thermal damage to viable subeschar tissues precluded successful autografting. However, a new technique, in which a high-power laser is rapidly scanned over the eschar, results in eschar vaporization without significant damage to underlying viable tissues, allowing successful immediate autografting. METHODS: Full-thickness paravertebral burn wounds measuring 36 cm2 were created on 11 farm swine. Wounds were ablated to adipose tissue 48 hours later using either a surgical blade or a 150-Watt continuous-wave CO2 laser deflected by an x-y galvanometric scanner that translated the beam over the tissue surface, removing 200 microm of tissue per scan. Both sites were immediately autografted and serially evaluated clinically and histologically for 180 days. RESULTS: The laser-treated sites were nearly bloodless. The mean residual thermal damage was 0.18+/-0.05 mm. The mean graft take was 96+/-11% in manual sites and 93+/-8% in laser sites. On postoperative day 7, the thickness of granulation tissue at the graft-wound bed interface was greater in laser-debrided sites. By postoperative day 180, the manual and laser sites were histologically identical. Vancouver scar assessment revealed no differences in scarring at postoperative day 180. CONCLUSIONS: Long-term scarring, based on Vancouver scar assessments and histologic evaluation, was equivalent at 6 months in laser-ablated and sharply excised sites. Should this technology become practical, the potential clinical implications include a reduction in surgical blood loss without sacrifice of immediate engraftment rates or long-term outcome.

Alteration of the aPA ELISA by UV exposure of polystyrene microtiter plates.

Interlaboratory inconsistencies in antiphospholipid antibody (aPA) solid phase assays have prompted controversy in clinical laboratory testing for aPA. We found that the aPA ELISA can be influenced by the type of microtiter plate utilized and by the conditions in which the plates are stored. By exposing 96-well, flat-bottom polystyrene microtiter plates to short wave UV light (254 nm), the aPA ELISA signal decreased in a UV dose-dependent manner. No effect was seen with long wave UV light (366 nm). These results were independent of the antibody isotype under study or the phospholipid (PL) antigen used: anionic phosphatidylserine (PS) and cardiolipin (CL), or zwitterionic phosphatidylethanolamine (PE). Purified human beta 2-glycoprotein I (beta 2 GPI), a known cofactor for anionic PL, and rabbit anti-beta 2 GPI antisera were used to demonstrate that beta 2 GPI bound equally to UV treated and untreated microtiter plates. In contrast, recognition of beta 2 GPI on an anionic PL surface was decreased on UV treated plates, suggesting that UV exposure alters the lipid binding properties of the microliter plate. To determine whether UV exposure inhibited PL binding directly or caused a change in the way the PL was bound, the amount of PL bound to UV treated and untreated plates was measured by using fluorescent labeled PS and a fluorimeter. PS binding was decreased by 53% in UV treated wells as compared to untreated wells. These data show that short wave UV exposure reduces PL binding to polystyrene microtiter plates, thereby reducing the amount of beta 2 GPI bound to PL coated ELISA plates. Thus by using UV exposed microtiter plates, decreased or false-negative a PA ELISA results may be obtained for aPA positive plasmas.

Alloantibody responses to antigens recognized by rabbit antitrophoblast antisera in trophoblast and mononuclear cell (MNC) membranes by primary aborting women before and after paternal leukocyte immunization.

PROBLEM: Because shared allogeneic antigens are expressed on peripheral blood lymphocytes, as well as trophoblasts, it has been proposed that lymphocyte transfusions may appropriately sensitize recurrent spontaneous aborters (RSA) to trophoblast and lead to pregnancy conservation. The degree to which these responses are affected by this treatment, however, has not been defined. METHOD: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were used to study the alloantibody responses in RSA patients both before and after immunization with paternal leukocytes (MNC) against membrane proteins isolated from peripheral blood mononuclear cells (MNC), first trimester and full term chorionic villi (CV). RESULTS: A distinct set of antigens with apparent molecular weights of 32, 36, 41, 63, 65, and 85 kDa was identified in both MNC and trophoblast membranes by rabbit anti-trophoblast antisera. In addition, a 55 kDa protein was recognized by MNC membranes. Thirty-eight percent of primary RSA sera recognized this 55 kDa protein. After paternal MNC immunization, all primary RSA samples displayed increased reactivity or produced antibodies to this 55 kDa protein when compared with preimmunization sera. The protein was identified as MCP using a rabbit polyclonal anti-MCP antisera. In contrast, reactivity to the other antigens present in the membrane preparations decreased after paternal MNC immunization. CONCLUSION: Changes in immune reactivity in RSA patients after paternal MNC immunization suggest that immunization alters serum immune reactivity to MNC and trophoblast shared antigens.

Diagnosis of obstructive airways disease from the clinical examination.

OBJECTIVE: To determine the operating characteristics of history and physical examination items for pulmonary airflow obstruction. DESIGN: Prospective observational study. SETTING: Medical Preoperative Evaluation Clinic at the Durham Veterans Affairs Medical Center. PATIENTS/PARTICIPANTS: Consecutive patients referred for outpatient medical preoperative risk assessment. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Number of years the patient had smoked cigarettes, patient-reported wheezing [LR+ (likelihood ratio for finding present) = 3.1; LR- (likelihood ratio for finding absent) = 0.58], and auscultated wheezing (LR+ = 12; LR- = 0.87) were independent predictors of obstructive airways disease from the history and physical examination. Forced expiratory time and peak expiratory flow rate, both measured by the clinician at the bedside, were additional independent predictors of airflow obstruction. A nomogram using patient-reported wheezing, number of years the patient had smoked, and auscultated wheezing was developed and validated (area under receiver operating characteristic curve = 0.78; p = 0.0001) for the bedside prediction of obstructive airways disease. Peak expiratory flow rate can be substituted for auscultated wheezing with similar predictive ability. CONCLUSIONS: The results of bedside clinical examinations predict the presence of obstructive airways disease. A nomogram based on a combination of four bedside findings predicts airflow obstruction as well as clinicians' overall clinical impressions.

Postoperative hypertension: a multicenter, prospective, randomized comparison between intravenous nicardipine and sodium nitroprusside.

OBJECTIVE: To compare the efficacy and safety of iv nicardipine with sodium nitroprusside in the treatment of postoperative hypertension after both cardiac and noncardiac surgery. DESIGN: Multicenter, prospective, randomized, open-label study. SETTING: Six tertiary referral medical centers (recovery rooms and surgical ICUs). PATIENTS: A total of 139 patients with postoperative hypertension: i.v. nicardipine (n = 71), sodium nitroprusside (n = 68). INTERVENTION: Administration of i.v. nicardipine or sodium nitroprusside. MEASUREMENTS: Vital signs (BP, heart rate), hemodynamic variables, medication dosage, total number of dose changes, and time to achieve BP control were recorded. MAIN RESULTS: Both medications were equally effective in reducing BP in both the cardiac and noncardiac surgical groups. Under the conditions of the study, i.v. nicardipine controlled hypertension more rapidly than sodium nitroprusside (i.v. nicardipine 14.0 +/- 1.0 mins and sodium nitroprusside 30.4 +/- 3.5 mins, p = .0029). The total number of dose changes required to achieve therapeutic BP response was significantly less in the i.v. nicardipine-treated patients (i.v. nicardipine 1.5 +/- 0.2 vs. sodium nitroprusside 5.1 +/- 1.4, p < .05). Adverse effects were observed with both drugs (i.v. nicardipine 7% [5/71] and sodium nitroprusside 18% [12/68] [NS]). CONCLUSIONS: Intravenous nicardipine is as effective as sodium nitroprusside in the therapy of postoperative hypertension. Specific advantages have been identified. The use of i.v. nicardipine should be considered in the therapy of postoperative hypertension.

Use of patient-controlled analgesia to compare the efficacy of epidural to intravenous fentanyl administration.

Fentanyl, unlike morphine, is highly lipophilic and rapidly diffuses out of the epidural space. Respiratory depression is, therefore, unlikely when fentanyl is given epidurally. However, much of fentanyl's analgesic effect is mediated by systemic rather than spinal receptor binding. To test this hypothesis, we performed a prospective, double-blind, cross-over study comparing epidural and intravenous (IV) administration of fentanyl in 16 patients for the first 12 h after lower abdominal or lower extremity surgery. To allow direct comparison of these two routes of administration, patient-controlled analgesia was used so patients could self-titrate their analgesia. Patients were randomized to receive fentanyl initially by an epidural (group A, n = 8) or IV (group B, n = 8) catheter for 6 h, after which they were crossed-over to the alternate route by means of a hidden three-way stopcock. The degree of analgesia was subjectively evaluated by a visual analogue scale, and by an observer rating patient's comfort and sedation. Cumulative dosage of fentanyl was recorded, and plasma fentanyl concentrations were measured. The onset of analgesia and increase in plasma fentanyl concentrations were more rapid with intravenous fentanyl. However, after 60 min, analgesia (visual analogue scale 2-4 cm) or plasma fentanyl concentrations (0.3-0.7 ng/mL) did not differ between the two routes of administration. There were also no significant differences in the cumulative dosage of fentanyl within each group (epidural vs IV) or between the groups. Thus, the analgesic effects of epidural fentanyl appear largely mediated by systemic absorption. Intravenous fentanyl achieves a similar degree of analgesia and a more rapid onset of effect without the need for epidural catheterization.

MR imaging of the juvenile temporomandibular joint: preliminary report.

Thirty-two children were evaluated by means of medical history and physical examination for signs and symptoms of internal derangement (ID) of the temporomandibular joint (TMJ) and mandibular dysfunction. These children also underwent magnetic resonance (MR) imaging of the TMJs. The study was double blind. At clinical examination, 19 patients (59%) had at least one positive finding of ID of the TMJ and/or mandibular dysfunction. MR images of the TMJ obtained in 60 of the 64 TMJs demonstrated 57 normal joints (95%) and three abnormal joints (5%). Two of these three joints had a mild anterior-lateral disk displacement, and one joint had an anterior dislocated disk. There were no false-positive MR examinations. MR imaging failed to depict abnormalities in 16 patients who had positive findings at history and/or physical examination. Although MR imaging may fail to depict ID of the TMJ in some patients, clinical techniques commonly used in population surveys probably overstate the prevalence of ID of the TMJ in children.

Recurrence of an odontogenic keratocyst in a bone graft. Report of a case.

The recurrence of an odontogenic keratocyst in a bone graft after partial mandibulectomy and reconstruction is presented. The possible reasons for recurrence are discussed, and methods of treatment reviewed.

Nicardipine infusion for postoperative hypertension after surgery of the head and neck.

The therapy of postoperative hypertension (POH) after head and neck surgery was evaluated in a prospective, randomized, double-blind trial. Nicardipine hydrochloride, a Ca channel-blocker for iv use, was compared with placebo. Patients were initially randomized to receive nicardipine infusion or placebo. Those not responding to placebo were given nicardipine infusion on an open basis. Hypertension was significantly better controlled in patients treated with nicardipine infusion compared with placebo (83% vs. 22%, p less than .002). Subsequently, six (86%) of seven of the placebo failures were successfully treated with nicardipine. There were no significant complications in either group. We conclude that the titratable infusion of nicardipine is an effective and safe method for the control of POH after surgery of the head and neck. Further studies are now warranted comparing nicardipine with other drugs currently used to treat this condition.

Effects of unilateral premature fusion of the zygomaxillary suture on the growth of nasomaxillary complex.

Unilateral premature fusion of the zygomaxillary suture was produced in 2-week-old guinea pigs by immobilization with methyl-cyanoacrylate adhesive. The effects of sutural fusion on the growth of the nasomaxillary complex were evaluated by radiographic cephalometry with implants, and histologic assessment. The immobilization successfully constrained the anteroinferior displacement of the maxilla and zygomatic bone on the fused side. Compensatory responsive remodeling at adjacent sutures and in certain regions of the nasomaxillary complex was observed. Developmental asymmetry was found when the fused side was compared with the nonfused side. No gross anatomic asymmetry of the facial complex as a whole, however, was detected. Adaptive compensation involving periosteal bone deposition occurred at the junction of the lower maxillary process and the maxilla proper on the fused side. This, together with periosteal bone resorption in corresponding areas on the nonfused side served to offset any developmental asymmetry that might have occurred, thus leading to a compensatory preclusion of gross anatomic asymmetry.

Temporomandibular synovial fluid pressure response to altered mandibular positions.

Hydrostatic synovial fluid pressure within the superior aspect of the temporomandibular joint space of the growing pig, Sus scrofa domesticus, was examined in response to various acute and chronic alterations of mandibular position. Bilateral measurements of pressure were recorded with chronically implanted wick catheters in three 8-week-old pigs before and at the time of appliance placement and then at 2-day intervals until the animals were 20 weeks old. Besides confirming the observations of a previous study, we noted that forward positioning of the mandible caused an increase in synovial fluid pressure that decayed to baseline levels within 2 hours. Posterior positioning of the mandible effected a larger increase in pressure that partially decayed over 2 hours but did not return to baseline levels over the entire course of the experiment. These results may have significance in the understanding of the physiology of the temporomandibular joint, local control mechanisms involved in the normal and treatment-aided growth of the mandible, and disorders of the temporomandibular joint.