Randomized clinical trial comparing collagen plug and advancement flap for trans-sphincteric anal fistula.

BACKGROUND: The role of a collagen plug for treating anal fistula is not well established. A randomized prospective multicentre non-inferiority study of surgical treatment of trans-sphincteric cryptogenic fistulas was undertaken, comparing the anal fistula plug with the mucosal advancement flap with regard to fistula recurrence rate and functional outcome. METHODS: Patients with an anal fistula were evaluated for eligibility in three centres, and randomized to either mucosal advancement flap surgery or collagen plug, with clinical follow-up at 3 and 12 months. The primary outcome was the fistula recurrence rate. Anal pain (visual analogue scale), anal incontinence (St Mark's score) and quality of life (Short Form 36 questionnaire) were also reported. RESULTS: Ninety-four patients were included; 48 were allocated to the plug procedure and 46 to advancement flap surgery. The median follow-up was 12 (range 9-24) months. The recurrence rate at 12 months was 66 per cent (27 of 41 patients) in the plug group and 38 per cent (15 of 40) in the flap group (P = 0·006). Anal pain was reduced after operation in both groups. Anal incontinence did not change in the follow-up period. Patients reported an increased quality of life after 3 months. There were no differences between the groups with regard to pain, incontinence or quality of life. CONCLUSION: There was a considerably higher recurrence rate after the anal fistula plug procedure than following advancement flap repair. Registration number: NCT01021774 (http://www.clinicaltrials.gov).

Expression and gene amplification of primary (A, B1, D1, D3, and E) and secondary (C and H) cyclins in colon adenocarcinomas and correlation with patient outcome.

BACKGROUND/AIMS: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3, and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle progression and have not previously been evaluated in colon cancer. This study examined cyclin protein expression and gene amplification in colon adenocarcinoma and the correlation with patient outcome. METHODS: Immunohistochemistry and real time quantitative polymerase chain reaction were used to determine cyclin expression and gene amplification in 219 tumours. The results were compared with clinical variables and patient outcomes. RESULTS: Cyclin H was overexpressed in all tumours, cyclin C in 88%, cyclin B1 in 58%, cyclin A in 83%, cyclin D3 in 36%, cyclin E in 25%, and cyclin D1 in 11% of the tumours. Extra gene copies of cyclin A were seen in 6.2% of the tumours, cyclin B1 in 9%, cyclin C in 26.9%, cyclin D1 in 55%, cyclin D3 in 20.5%, cyclin E in 19.1%, and cyclin H in 5.1%. A significant correlation between protein overexpression and gene amplification was seen for cyclin C only. High expression of cyclin A was independently associated with improved survival. Amplification of cyclin C was independently associated with an unfavourable prognosis. CONCLUSIONS: Amplification of the cyclin C gene was related to an unfavourable prognosis and high protein expression of cyclin A was associated with a better outcome in colon adenocarcinoma.

Presence of isolated tumour cells in mesenteric lymph nodes predicts poor prognosis in patients with stage II colon cancer.

AIM: Most patients with stage I and stage II colon adenocarcinomas do not have disseminated disease, and the group is not offered adjuvant therapy. However, more than 30% of stage II colon adenocarcinoma patients get metastases to remote organs. Thus, it is important to identify patients in this group at risk of disease relapse. PATIENTS AND METHODS: We have examined the prognostic value of isolated tumour cells (ITC) in mesenteric lymph nodes in a consecutive series of 156 colon carcinoma patients with stage II disease. Immunohistochemistry, using antibodies to cytokeratins, and morphology were used to identify presence of ITC. RESULTS: ITC were detected in 59 (37.8%) patients. Presence of ITC in mesenteric lymph nodes was independently associated with reduced relative survival both in univariate (p=0.0199) and in a multivariate analysis (p=0.041). CONCLUSION: The results strongly suggest that presence of ITC in mesenteric lymph nodes is associated with reduced relative survival in colon carcinoma patients stage II, and that detection of ITC may be important in treatment of these patients.

Intraoperative manometry to assess the esophagogastric junction during laparoscopic fundoplication and myotomy.

SUMMARY: Surgery for gastroesophageal reflux disease and achalasia is performed to alleviate symptoms by improving esophagogastric junction (EGJ) function. Intraoperative manometry was used to evaluate the pressure-length characteristics of the reconstructed EGJ during laparoscopic Nissen fundoplication and laparoscopic Heller myotomy. Intraoperative manometry was performed in 37 consecutive patients undergoing laparoscopic Nissen fundoplication (n = 22) or laparoscopic Heller myotomy (n = 15). Measurements were taken before surgery, after creation of the pneumoperitoneum, after completion of the myotomy in achalasia, and after creation of the fundoplication. Tracings were analyzed for pressure, length, and the integrated pressure-length relation (area under the curve [AUC]). Statistical comparison was made using paired t tests; intraoperative EGJ measurements did not correlate well with preoperative values for either pressure or length. Laparoscopic Nissen fundoplication significantly increased pressure, length, and AUC of the EGJ compared with prefundoplication values. Laparoscopic Heller myotomy significantly decreased EGJ pressure, length, and AUC. Creation of a Toupet fundoplication after myotomy did not significantly increase pressure, length, and AUC of the EGJ compared with postmyotomy values. Intraoperative manometry identified 2 of 15 achalasia patients (13%) with persistent areas of high pressure after initial myotomy that were corrected by extending the myotomy. Intraoperative manometry identifies mechanical changes created during EGJ surgery and may be a useful adjunct to improve outcomes of laparoscopic Nissen fundoplication and laparoscopic Heller myotomy.

Low-dose intravenous erythromycin: effects on postprandial and fasting motility of the small bowel.

BACKGROUND: Erythromycin is a motilin agonist and its effects on gastrointestinal motility are dependent on both dose and whether it is administered during the postprandial or fasting state. AIM: To study the motility response of the small bowel to a low dose of intravenous erythromycin after meal intake and during fasting. METHODS: Eighteen healthy subjects with mean age of 25 years were studied by small bowel manometry. Erythromycin was administered intravenously (0.75 mg per kg body weight) during 20 min in the postprandial (n=9) and the fasting state (n=9), and the motility response was recorded. RESULTS: Erythromycin significantly reduced the frequency of propagated contractions (P < 0.001) and the amplitude of contractions (P < 0.02) in the small bowel during established postprandial motility. During the fasting state, erythromycin invariably initiated a phase III-like activity, which was similar to the spontaneous nocturnal phase III and migrated significantly more slowly than the diurnal phase III (P < 0.01). CONCLUSIONS: A low dose of erythromycin administered intravenously during the postprandial state significantly inhibits small bowel motility, whereas administration during the fasting state initiates a phase III resembling the nocturnal rather than the diurnal phase III. These effects of erythromycin may indicate interference with vagal pathways. Due to its inhibitory effects, the clinical use of erythromycin in patients with hypomotility should be reconsidered, and the potential usefulness of these effects in patients with exaggerated intestinal motility deserves further attention.

Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems.

The BACTEC 460 system currently provides the most rapid detection of mycobacterial growth, but the system is radiometric and requires needles to inoculate specimens through the bottle's septum. The Mycobacteria Growth Indicator Tube (MGIT) system has a liquid medium, like the BACTEC system, and does not require needles when inoculating specimens. We compared mycobacterial growth from 510 specimens in the two systems. Average time to acid-fast bacillus (AFB) detection and identification to the species level was less with the BACTEC system, but this result was statistically significant only for AFB detection in specimens containing Mycobacterium avium-M. intracellulare complex. The contamination rate with MGIT was 29%; the BACTEC rate was 5%. To investigate MGIT contamination, we initiated a second study with changes in specimen processing. The MGIT contamination rate was reduced to 12%; the BACTEC rate was not significantly affected (5.5%). The most likely explanation for the contamination in MGIT is the richness of its medium compared to the BACTEC medium. Cost analysis for the two systems in a laboratory that processes 4,500 specimens a year is presented. The data suggest that the BACTEC 460 and the MGIT systems are approximately equivalent in cost and ability to support the growth of AFB. The MGIT system appears safer and easier to use and was preferred by laboratory personnel, but it cannot currently be used for blood specimens or antituberculosis susceptibility testing.

Acidic fibroblast growth factor: evaluation of topical formulations in a diabetic mouse wound healing model.

The efficacy of topical formulations of acidic fibroblast growth factor (aFGF) in healing of full-thickness wounds has been studied in a diabetic db+/db+ mouse model. The effect of several formulation variables, dose, and application frequency was examined. It was found that wound healing in diabetic animals treated with aFGF or placebo was slower than in their nondiabetic littermates. The availability of aFGF from the viscous vehicle employed in this study (1% hydroxyethyl cellulose) was demonstrated in vitro using diffusion cells. The viscous formulation of aFGF was equally effective in wound healing as a nonviscous formulation in phosphate-buffered saline. A formulation containing heparin (necessary for full biological and conformational stability of aFGF) at a mass ratio of 3:1 to aFGF was more efficacious than formulations with lower heparin: aFGF ratios. Wounds treated with three doses of 3.0 micrograms/cm2 aFGF healed faster than those treated with a single dose of 3.0 micrograms/cm2 aFGF. Three applications of 3.0 or 0.6 microgram/cm2 a FGF were equally effective in accelerating wound healing.

Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens.

A clinical evaluation of the Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis in endocervical swabs (Roche Molecular Systems, Branchburg, N.J.) is described. This new clinical system used one-step sample preparation, amplification with biotinylated cryptic plasmid primer pairs (CP24-CP27), uracil-N-glycosylase (AmpErase), and a microtiter format for amplicon capture and detection. Culture with McCoy cells in duplicate 1-dram (3.697-ml) vials with fluorescent immunostaining was the reference system. Endocervical swab samples from 945 women provided 74 culture-positive specimens, of which PCR detected 71. The initial PCR result was positive for 12 additional specimens. Arbitration of the PCR-positive, culture-negative samples by PCR with major outer membrane protein primers, duplicate culture, elementary body direct fluorescent-antibody staining, and DNA extraction PCR showed that all 12 samples were positive for chlamydia, raising the number of truly positive samples from 74 to 86. After arbitration the true sensitivities of PCR and culture were 96.5 and 86%, respectively (P = 0.02). Specificities for both were 100%. For PCR, the positive and negative predictive values were 100 and 99.7%, respectively. Total test efficiency was 99.7%. A high-test-volume (121 samples) timing study with all items included in the College of American Pathologists work load method indicated that this PCR format took approximately 3 min per sample. Because of the high sensitivity, specificity, and improved ease of handling, we found PCR to be a good alternative to culture for detection of C. trachomatis.

Formulation design of acidic fibroblast growth factor.

The design of an aqueous formulation for acidic fibroblast growth factor (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spectrophotometric turbidity measurements were initially employed to screen the ability of polyanionic ligands, less specific compounds, and variations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raising the temperature at which the protein unfolds by 15-30 degrees C. Many commonly used excipients were also observed to stabilize aFGF in both the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperature of aFGF thermal unfolding by as much as 6-12 degrees C as shown by circular dichroism and differential scanning calorimetry. Other compounds were found which protect the chemically labile cysteine residues of aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integrity by binding to the protein and chelating agents (e.g., EDTA) to prevent metal ion-catalyzed oxidation of cysteine residues. While room-temperature storage (30 degrees C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are stable in vitro for at least 3 months at 30 degrees C. Three aFGF topical formulations were examined in an impaired diabetic mouse model and were found to be equally capable of accelerating wound healing.

A new approach for three-dimensional reconstruction of arterial ultrasonography.

This report describes a computerized approach that allows the creation of realistic three-dimensional arterial images from two-dimensional contiguous slices derived from a conventional ultrasound scan. Furthermore, the study assesses the method's feasibility and accuracy by performing in vitro cadaver artery three-dimensional reconstructions. Images are digitized into a computer, with a resolution of 512 x 480 pixels, and a dynamic range of 8 bits/pixel (256 gray scale). After edge enhancement with convolution filters through the original binary data, the intraluminal and outer edges are traced and converted to a polygon vector within a defined three-dimensional space. Serial cuts, 2 mm apart, are then "stacked" into a three-dimensional model, with interpolation of polyhedra between slices. Sixteen normal and arteriosclerotic distal aortic and common iliac arteries were obtained from fresh cadavers. Three-dimensional reconstruction models were compared with gross examination of the original artery. Anatomically accurate reconstructions were obtained, all with detailed surface information. Data analyzed included diameter, area, residual lumen, and percent stenosis. Pearson's coefficients determined for the pairs of data indicated excellent correlation (greater than or equal to 0.90) between separate measurements. With use of the described technology it is feasible to perform arterial three-dimensional reconstructions on a personal computer, with detailed and accurate surface information. The three-dimensional reconstruction method used can reliably and consistently reproduce the anatomic specimens.

Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography.

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.

Antiparkinsonian efficacy of a novel transdermal delivery system for (+)-PHNO in MPTP-treated squirrel monkeys.

We examined the ability of the antiparkinsonian agent (+)-4-propyl-9-hydroxynaphthoxazine (PHNO) to enter the systemic circulation in therapeutic concentrations after continuous transdermal absorption in squirrel monkeys rendered parkinsonian by MPTP. Direct subcutaneous administration of (+)-PHNO in the dose range of 2.5 to 20 micrograms/kg restored locomotor activity to levels seen in normal monkeys for approximately 1 hour. Application of transdermal patches capable of delivering, into an infinite sink, an estimated 2.6 micrograms/cm2/h of (+)-PHNO over a skin surface area of 4.78 to 19.12 cm2 also restored locomotor activity to the normal range during a 24-hour period. We suggest the use of transdermal application of PHNO as a novel drug delivery system for the improved management of Parkinson's disease.

The antiparkinsonian actions and pharmacokinetics of transdermal (+)-4-propyl-9-hydroxynaphthoxazine (+PHNO): preliminary results.

(+)-4-Propyl-9-hydroxynaphthoxazine (+PHNO) is a potent dopamine agonist that has been administered transdermally to four patients with Parkinson's disease and "on-off" fluctuations. Skin patches of increasing size were used to treat these patients, who also received infrequent doses of oral levodopa if required. The effect of +PHNO was measured as an increased duration of action of individual levodopa doses. The clinical effect measured in this way was directly proportional to the plasma concentrations of +PHNO achieved. The plasma concentrations of +PHNO began to rise 4-6 h after patch application and reached a steady state by 24 h. The final plasma concentration of +PHNO was proportional to the area of skin covered.

Duplex scanning versus conventional arteriography for the evaluation of carotid artery plaque morphology.

Examinations that are performed for the evaluation of the extracranial cerebrovascular system need to supply accurate information regarding the luminal surface of the carotid artery as well as the morphologic characteristics of the plaque. To compare conventional carotid artery angiography and Duplex scanning, 32 consecutive patients underwent both evaluations, the results of which were compared, in a blinded manner, to the plaque characteristics noted on direct examination by the attending surgeon and pathologist. The following observations were made: angiographic and Duplex scanning evaluations concurred with direct-specimen examination for luminal narrowing in 31 of 32 patients (97%) and 30 of 32 patients (94%), respectively, whereas Duplex scanning was more accurate for diagnosing ulcerative lesions, 13 of 14 patients (93%), than angiography, five of 14 patients (36%) (p less than 0.0001). Luminal surface irregularity was accurately described by Duplex scanning in 20 of 23 patients (87%) and by angiography in 11 of 23 patients (48%) (p less than 0.005), whereas plaque morphology or consistency was not commented on with angiography but was accurately described with Duplex scanning in 12 of 12 patients (100%). This series clearly demonstrates that both angiography and Duplex scanning are extremely accurate for determining the reduction in the diameter of the carotid artery, whereas Duplex scanning is more accurate for detecting important plaque characteristics such as irregularity, ulceration, and composition.

2'-Nor-2'-deoxyguanosine is an effective therapeutic agent for treatment of experimental herpes keratitis.

2'-Nor-2'-deoxyguanosine (2'NDG), previously reported by us to effectively treat acute herpes simplex infections in mice, was used therapeutically to significantly enhance healing of established herpetic corneal lesions and prevent stromal disease in rabbits. Treatment using 0.06% 2'NDG drops (5 times daily) starting 3 days after infection resulted in more rapid healing of corneal epithelial lesions, rapid resolution of conjunctival inflammation, and prevention of stromal clouding compared to placebo-treated animals. In comparative dose-response titrations, the relative potency of 2'NDG to acyclovir was 6.4, which was significant. In addition, soluble ophthalmic inserts were developed for delivery of 2'NDG. Once a day treatment using ophthalmic inserts which released 100 micrograms 2'NDG significantly enhanced corneal and conjunctival healing and prevented stromal disease; 2'NDG eye drops (100 micrograms) delivered once a day were also effective in inhibiting the progression of corneal lesions. These results indicate that 2'NDG may be therapeutically effective in treatment of herpes keratitis, and further suggest that for use as eye drops or in an ophthalmic insert, 2'NDG may be effective even if applied once per day.

Dissolution rate studies of compression-molded units made from hydroxypropyl cellulose films.

Hydroxypropyl cellulose films were prepared by compression molding of three different lots of hydroxypropyl cellulose powder at 149 degrees C, 188 degrees C, and 232 degrees C. Rectangular pieces were cut from these films and viscosity average molecular weight (Mv), degree of orientation, and rate of dissolution were measured. The viscosity average molecular weight (Mv) decreased with increasing processing temperature, while, as expected, the dissolution rate increased. Orientation in the thermoformed units was also evaluated. Correlation of these data with the Mv values suggests that orientation has some controlling influence on the dissolution rate. Because the samples possessing the least orientation were molded at the highest temperature, they also had the lowest Mv due to thermal degradation. Therefore, the effects of molecular weight were not fully separated from orientation effects with regard to control over the dissolution rate.

Initial evaluation of transdermal timolol: serum concentrations and beta-blockade.

The serum concentrations and beta-blockade after dermal application of timolol ointment were evaluated in six healthy men (21-31 years old; 74-82 kg). Two patches (25 cm2) containing placebo and either 30 (n = 2) or 60 mg (n = 4) timolol base were randomly applied to the chest for 30 h. Serial serum concentrations of timolol were measured by a radioligand receptor assay. Bicycle ergometry, at a predetermined workload, was performed before and at 3, 8, 24, and 48 h after patch application; mean +/- SD heart rates (beats/min) at these times were 167 +/- 2, 158 +/- 7, 125 +/- 7, 120 +/- 5, and 150 +/- 5 (last 3 values: p less than 0.05 from pretreatment), and beta-blockade was evident in all subjects. Measurable serum concentrations in the therapeutic range were achieved in all subjects. The change in exercise-induced heart rate (y) was closely related to log timolol serum concentration (x) (y = -36 X - 5.3; r = -0.92; p less than 0.001). Based on the amount of timolol in the residual ointment, 50-60% of the original timolol dosage was delivered from the patch. Skin irritation under the patch compared with placebo was minimal. Further studies are warranted to assess the potential clinical utility of transdermal timolol.

Comparison of two commercially available test methods with conventional coagulase tests for identification of Staphylococcus aureus.

The API STAPHase (Analytab Products, Inc., Plainview, N.Y.) and SeroSTAT Staph (Scott Laboratories, Fiskville, R.I.) tests were compared to the conventional tube coagulase test and a slide coagulase test by using fresh isolates of members of the family Micrococcaceae. The 4-h, 24-h, and combined readings of the tube coagulase test detected 94.5, 99.5 and 100%, respectively, of 219 Staphylococcus aureus isolates. The API STAPHase, SeroSTAT Staph, and slide coagulase tests detected 95.9, 95.4 and 95.9% of the isolates of S. aureus, respectively. There were no false-positive results with any of the systems when tested with 103 strains of members of the family Micrococcaceae other than S. aureus. We concluded that the STAPHase and SeroSTAT Staph tests were equal in accuracy to the slide coagulase and 4-h tube coagulase tests and were suitable for use in the clinical microbiology laboratory. However, SeroSTAT Staph gave faster results than the API STAPHase, and the test was easier to perform. Also, the false-negative rate was high enough with the STAPHase, SeroSTAT Staph, and the slide coagulase tests that all negative reactions should be confirmed with a tube test.

Efficacy of 2'-nor-2'-deoxyguanosine treatment for orofacial herpes simplex virus type 1 skin infections in mice.

2'-Nor-2'-deoxyguanosine (2'NDG), a new antiviral agent, conferred protection when given orally or topically to hairless mice after the mice were subjected to orofacial infection with herpes simplex virus type 1. The average severity of orofacial lesions was significantly reduced in mice receiving oral gavage treatments twice daily for 7 days beginning 3 h postinfection. The minimum effective dose of 2'NDG was 0.2 mg/kg per day. A minimum of eight treatments over 4 days resulted in a significant reduction in lesion severity. Topical treatment begun 3 h postinfection and continued four times daily for 3 days resulted in a minimum effective dose of 0.06%. Oral treatment with 2'NDG begun as late as 72 h postinfection or topical treatment begun as late as 48 h postinfection resulted in significantly reduced lesion severity compared with lesion severity among placebo-treated animals. In addition, significant prevention of ganglionic infection occurred when 2'NDG was administered either orally or topically within 24 h after infection.