Chemokine therapy for anal sphincter injury in a rat model: a pilot study.

INTRODUCTION AND HYPOTHESIS: To determine whether delayed administration of CXCL12 alters anorectal manometric pressures and histology in rats following anal sphincterotomy compared to primary surgical repair alone. METHODS: Adult female rats were divided into three groups: A, a control group that did not undergo surgery; B, anal sphincterotomy with primary surgical repair; C, anal sphincterotomy with primary surgical repair and intra-sphincteric injection of CXCL12 at 6 weeks post-injury. All rats underwent anal manometry measurements at baseline and at 6 and 12 weeks post-injury. Histologic analysis of the anal sphincters was also performed. RESULTS: At baseline and 6 weeks, there were no statistically significant differences among D, Tmax and P∆ of Groups A, B and C. At 12-week manometry, the total duration of contractions on anal manometry was significantly less in Group C compared to Groups A and B (3.65, 5.5, 5.3 p < 0.01) as was time to peak of contraction at 12 weeks (1.6, 2.1, 3.1, p < 0.01); however, group C had a significantly higher P∆ at 12 weeks compared to Groups A and B (2.25, 1.4, 0.34, p < 0.01). There were no statistically significant differences in the ratio of muscle to collagen at the site of injury; however, muscle fibers were significantly smaller in group C and less per bundle than the other groups. CONCLUSIONS: Administration of chemokine therapy at 6 weeks post-repair using CXCL12 enhanced the magnitude of anal sphincter contractions in a rat model of anal sphincter injury but decreased overall duration of contraction. Increased anal sphincter contraction magnitude was not explained by histologic differences in explanted specimens.

The cost of chronic stress: impacts of a nonhabituating stress response on metabolic variables and swimming performance in sturgeon.

Metabolic scope for activity (MSA) and critical swimming velocity (U(crit)) were measured in green sturgeon exposed to two stressors daily for 28 consecutive days. The results were compared with unstressed fish in an effort to measure the "cost" of chronic stress. Chronic stress was simulated by exposing fish to a randomized order of acute stressors: a 5-min chasing stressor, a 10-min water depth reduction stressor, or a 5-min confinement stressor. The acute cortisol response to each stressor was initially determined, and the maintenance of that response was verified in 7-d intervals during the chronic stress regime. Exposure to the chronic stress regime resulted in a 25% reduction of MSA caused by significantly increased maintenance metabolic rate (0.27+/-0.01 vs. 0.19+/-0.02 mg O(2) h(-1) g(-1), chronic and control fish, respectively) but did not affect the U(crit) of sturgeon. In addition, a 50% reduction in liver glycogen levels and a twofold increase of resting plasma glucose levels were measured in chronically stressed fish. We conclude that our chronic stress regime resulted in a significant maintenance cost to green sturgeon, possibly because of their inability to habituate to the stressors, but did not decrease their swimming performance.

Radiotherapy for regionally localized hormone refractory prostate cancer.

PURPOSE: Patients with regionally localized hormone refractory adenocarcinoma of the prostate are often referred for radiotherapy to relieve local symptoms, prevent further local progression, or prevent impending urinary tract obstruction. However, the merits of radiotherapy for this patient population have not been documented. In this retrospective series, the results of 29 such patients treated at our institution between 1987-1992 are reviewed. METHODS AND MATERIALS: Prior to androgen ablation, the majority of these patients (79%) had Stage D0 or D1 disease. After androgen ablation, radiotherapy was given to 16 (55%) for progressive symptoms (mostly urinary obstructive), 11 (38%) for palpable local progression in the absence of symptoms, and 2 for a rising prostate specific antigen (PSA) profile without palpable disease. None of the patients had distant metastasis at the time of radiotherapy. The median dose to the prostate was 66 Gy and the median follow-up after radiotherapy was 43 months. RESULTS: Following local-regional radiotherapy, the actuarial rate of local failure at 4 years was only 39%. However, 80% had disease progression or a rising PSA in this time period. The actuarial survival at 4 years following radiotherapy was 39%. Univariate analyses of potential prognostic factors revealed that preandrogen ablation Gleason score, preradiotherapy PSA, and preradiotherapy prostatic acid phosphatase (PAP) were predictive of patient outcome. Most importantly, doses above 60 Gy to the prostate at standard fractionation were associated with symptom-free local control in 90% of patients at 3 years. The majority of the patients were treated using limited fields (n = 20). CONCLUSIONS: The regionally localized hormone refractory prostate cancer patients described benefited from high dose, continuous course, local radiotherapy in that excellent local control rates were obtained for an extended period. Because the majority of these patients fail with distant metastasis within 4 years, this treatment represents an aggressive approach to palliation that is justified by the maintenance of freedom from local symptoms.

Prostate-specific antigen cancer volume: a significant prognostic factor in prostate cancer patients at intermediate risk of failing radiotherapy.

PURPOSE: Although the pretreatment serum prostate-specific antigen level (PSAL) is the single-most significant predictor of local and biochemical control in prostate cancer patients treated with radiotherapy, it is relatively insensitive for patients with a PSAL in the intermediate range (4-20 ng/ml). PSA density (PSAD) has been shown to be slightly more predictive of outcome than PSAL for this intermediate risk group; however, this improvement is small and of little use clinically. PSA cancer volume (PSACV), an estimate of cancer volume based on PSA, has recently been described and has been purported to be more significant than PSAL in predicting early biochemical failure after radiotherapy. We report a detailed comparison between this new prognostic factor, PSAL, and PSAD. METHODS AND MATERIALS: The records of 356 patients treated with definitive external beam radiotherapy for regionally localized (T1-4,Nx,M0) adenocarcinoma of the prostate were reviewed. Each patient had a PSAL, biopsy Gleason score, and pretreatment prostate volume by transrectal ultrasonography. The median PSAL was 9.3 ng/ml and 66% had Gleason scores in the 2-6 range. The median radiation dose was 66.0 Gy and the median follow-up for those living was 27 months. PSACV was calculated using a formula which takes into account PSAL, pretreatment prostate ultrasound volume, and Gleason score. The median PSACV was 1.43 cc. Biochemical failure was defined as increases in two consecutive follow-up PSA levels, one increase by a factor > 1.5, or an absolute increase of > 1 ng/ml. Local failure was defined as a cancer-positive prostate biopsy, obtained for evidence of tumor progression. RESULTS: The distributions of PSACV and PSAL were similar and, when normalized by log transformation, were highly correlated (p < 0.0001, linear regression). There was a statistically significant relationship between PSACV and several potential prognostic factors including PSAL, PSAD, stage, Gleason score, and pretreatment prostatic acid phosphatase (PAP). In univariate analyses, PSACV, PSAL, and PSAD proved to be the most significant predictors of both biochemical and local control. In multivariate analyses using Cox proportional hazards models with PSAL, PSAD, PSACV, and PAP as continuous variables, PSAL, PSACV, and Gleason score were significant in predicting biochemical control. Only PSAL was significantly correlated with local control. However, when these analyses were restricted to patients with intermediate PSALs (4-20 ng/ml), only PSACV was significant for predicting both biochemical and local control. CONCLUSION: PSACV was highly correlated with actuarial local and biochemical control and was superior to both PSAL and PSAD in predicting these outcomes in patients with PSALs between 4 and 20 ng/ml.

Relationship of ultrasound staging and bilateral biopsy positivity to outcome in stage T1c prostate cancer treated with radiotherapy.

OBJECTIVES: The strict definition of Stage T1c prostate cancer is that the tumor is not palpable on digital rectal examination (DRE) or seen on imaging studies such as ultrasound. The inclusion of ultrasound imaging was brought about without an understanding of the relationship between ultrasound upstaging and prognosis. We have also noticed that in clinical practice, treatment decisions are made on the basis of the finding of bilateral versus unilateral biopsy positivity. The objectives in this study were to determine the prognostic significance of upstaging by transrectal ultrasound (TRUS) to uT2 or uT3, and unilateral versus bilateral biopsy positivity in patients with Stage T1c cancer as determined by DRE (DRE-Stage T1c patients). METHODS: Between 1987 and 1995 there were 643 patients with DRE-Stage T1-T2 prostate cancer treated with external beam radiotherapy; 24 had T1a, 76 had T1b, 183 had T1c, 133 had T2a, 168 had T2b, and 59 had T2c. Of these, 135 DRE-Stage T1c patients underwent ultrasound staging and 122 underwent bilateral prostate biopsies. All had pretreatment prostate-specific antigen values (PSAs) available and no patient received adjuvant androgen ablation. The median pretreatment PSA was 9.1 ng/mL, median radiotherapy dose was 66.0 Gy, and median follow-up was 41 months. Post-treatment failure was defined as disease recurrence and/or two elevations in PSA on consecutive follow-up visits. RESULTS: The 5-year freedom from failure rate for DRE-Stage T1c patients (71%) was not significantly different from that of DRE-Stage T1b (65%) or DRE-Stage T2a (71%) patients. There was a trend (P = 0.1) toward a worse outcome for DRE-Stage T2b/T2c patients compared with DRE-Stage T1b/T1c/T2a patients. The distribution of DRE-Stage T1c patients by ultrasound staging was 29 with uT1c, 88 with uT2, and 18 with uT3 findings. Twenty percent of patients had bilateral positive biopsy specimens. In univariate and multivariate analyses, the only correlates of patient outcome were pretreatment PSA (P < or = 0.002) and isocenter dose (P = 0.03). TRUS upstaging had no effect on freedom from failure; uT1c patients had about the same risk of relapse or a rising PSA as uT2 or uT3 patients. Patients with bilateral positive prostate biopsy specimens had about the same prognosis as those with unilateral positive biopsy specimens. CONCLUSIONS: For patients with DRE-Stage T1c prostate cancer, the data indicate that ultrasound staging and bilateral biopsy positivity are not predictive of outcome for patients treated with external beam radiotherapy and treatment decisions should not be based on these parameters.

Motivational congruency and discrepancy between certified athletic trainers and noncertified student athletic trainers in the state of hawaii.

In March 1992, a survey to assess motivational preference was sent to all certified athletic trainers who were practicing in the State of Hawaii and all noncertified student athletic trainers who were enrolled in the athletic training curriculum at the University of Hawaii. The return rate was 80% for certified athletic trainers and 100% for student athletic trainers. The findings of the study indicated that a motivational discrepancy exists for the following motivational stems: freedom on the job, job growth, benefits and wages, being appreciated, helping the organization obtain goals, receiving raises, being an integral part of the work team, job security, and feedback on job performance (p <.05). Further, the study indicated differences in rating the importance of motivators between the certified and the student athletic trainers concerning freedom on the job, opportunity for advancement, benefits and wages, and job security (p <.05). The differences in motivational factors between the two groups indicated that the students are more concerned with intrinsic types of motivators and less concerned with extrinsic rewards. Further investigation needs to include mainland populations and students in approved/accredited curriculums.

Motivational congruency and discrepancy of hawaiian athletic trainers.

Motivation is an integral part of an effective organizational management scheme. In March 1992, we sent a survey designed to assess motivational preference to all certified athletic trainers in the State of Hawaii. The population included: 6 high school athletic trainers, 10 university athletic trainers, and 9 clinic athletic trainers. The surveys were completed and returned by 80% of the population. With the exception of being an integral part of a work team (p<.05), athletic trainers in the State of Hawaii showed little discrepancy in terms of motivation. Further, there are differences among the three groups of athletic trainers in rating the importance of motivators concerning being appreciated, receiving raises, and being an integral part of a team (p< .05) Differences in motivational factors among these three groups could be influenced by the organizational structure in which the athletic trainers operate. Further investigation should include a mainland population that includes athletic trainers in professional sports and the industrial setting.

Conducting a practice management review.

A practice management review can be used by a physician practice to identify problems within the practice, introduce operational improvements, and ensure that the practice is being run as profitably and efficiently as possible. Such a review involves scrutiny of nearly every aspect of a practice's operations--from reimbursement to personnel issues. Before deciding to have a practice review performed, the practice's management team must establish objectives for the review and must ensure that physicians and practice staff understand the review process. Once the review process is initiated, the steps that must be taken to identify and diagnose problems within a practice include conducting interviews, collecting data, analyzing data, and suggesting remedies.

Using specific antisera to neutralize ACTH in sturgeon: a method for manipulating the interrenal response during stress.

Interrenal function and the magnitude of the stress response were assessed in green sturgeon (Acipenser medirostris) passively immunized with antisera directed against adrenocorticotropic hormone (ACTH). The nucleotide sequence encoding ACTH was determined using reverse transcriptase polymerase chain reaction (RT-PCR). We identified two isoforms of ACTH that differ at a single site (position 26) in the 39 AA peptide. Both forms of green sturgeon ACTH (gsACTH1-39) display 100% homology with both sequences of white sturgeon ACTH (wsACTH1-39). The N-terminal portion of gsACTH also shares absolute identity with the comparable portion of human ACTH (hACTH). However, we identified considerable sequence divergence in the C-terminal domain between gsACTH and hACTH. Species-specific anti-ACTH sera were generated by vaccinating sheep against the C-terminal portion of gsACTH (gsACTH26-39). The peptide was covalently linked to a carrier protein (keyhole-limpet-hemocyanin [KLH]) to further enhance its immunogenicity. The anti-gsACTH sera recognized gsACTH1-39 and the immunogenic peptide (gsACTH26-39), but did not interact with hACTH1-39. To assess the impact of the antisera, fish were passively immunized with anti-gsACTH26-39 sera or anti-KLH sera and challenged with a hACTH1-39 injection on day 1 followed by a 1-min air emersion stressor on day 2. The magnitude and duration of the secretory response induced by hACTH did not differ (P > .05) between groups. Conversely, the magnitude of cortisol secretion induced by air emersion was significantly attenuated (P < .05) in fish passively immunized against gsACTH26-39. Collectively, these data demonstrate that the targeted antisera used in this study can discriminate between mammalian and green sturgeon ACTH and moderate the in vivo response to a stressor.

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques.

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.

Changes in cytochrome oxidation in outer and inner stripes of outer medulla.

To examine regional cytochrome oxidation in the outer medulla, we developed fiber optic probes that allowed us to obtain localized reflectance measurements from the outer and inner stripes of the outer medulla. We measured directional changes in cytochrome oxidation in these two regions. In the outer stripe furosemide surprisingly caused a significant decrease in cytochrome oxidation. The decrease occurred concomitantly with a fall in outer medullary blood flow as measured by laser-Doppler flowmetry. Saralasin, an antagonist of angiotensin II caused a significant increase in cytochrome oxidation in the outer stripe. In the inner stripe furosemide tended to increase cytochrome oxidation, and saralasin had no effect. These results indicate that the two regions of the outer medulla may be affected differently by the same agent. They suggest that cytochrome oxidation in the outer stripe is predominantly influenced by outer medullary blood flow, whereas the inner stripe is predominantly influenced by the rate of oxygen consumption. The advantages and limitations of this methodology are discussed.

Cloning of canine cytochrome P450 2E1 cDNA: identification and characterization of two variant alleles.

Cytochrome P450 (CYP) 2E1 is a toxicologically important enzyme that inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Although cDNAs for the human, rodent, and rabbit forms of CYP2E1 have been isolated and studied extensively, there is an absence of information about canine CYP2E1, despite the fact that the dog is routinely used in drug safety studies. In this study, we isolated and sequenced a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced canine CYP2E1 amino acid sequence exhibited 75 to 76% identity with rat, mouse, and rabbit CYP2E1 sequences, and 77% identity with human CYP2E1. Two populations of clones, differing at a single nucleotide, were isolated from the unamplified library. The T1453C base change results in a Tyr485His amino acid substitution, which is well beyond the heme binding region but is possibly part of a beta-sheet structure. An allele-specific polymerase chain reaction-based restriction enzyme test was developed for genotyping individual dogs from genomic DNA samples. One hundred mixed breed dogs were genotyped, and the frequencies of the Tyr485 and His485 alleles were found to be 0. 85 and 0.15, respectively. The canine Tyr485 and His485 alleles and human CYP2E1 were expressed in Escherichia coli cells, and catalytic activities of the proteins were assessed using the substrate chlorzoxazone. Although the two canine enzymes had similar catalytic activity; significant kinetic differences were seen between canine and human CYP2E1s.

Effects of diltiazem on the disposition and metabolism of the enantiomers of propranolol in the dog during multiple oral dosing.

The intravenous and oral dose kinetics and metabolism of the enantiomers of propranolol were investigated in five dogs during steady-state oral racemic propranolol dosing (5 mg/kg, every 8 hr for 3 days). These results were compared with those obtained during concomitant administration of oral diltiazem (2.5 mg/kg, every 8 hr for 3 days) in the same animals. The oral and intravenous propranolol test doses consisted of a pseudoracemic mixture of equal amounts of hexadeuterated-(R-(+))- and dideuterated-(S-(-))-propranolol. Propranolol metabolism in the urine was evaluated by coadministering 150 muCi of [4'-3H]racemic propranolol HCl, along with the deuterium-labeled compounds. Plasma concentrations of the deuterated enantiomers were measured by HPLC-thermospray MS, using undecadeuterated racemic propranolol as the internal standard. Diltiazem coadministration had no significant effects on either the systemic clearance, renal clearance, the apparent volume of distribution, or the elimination half-lives of either enantiomer. On the other hand, concomitant diltiazem treatment significantly reduced the oral clearance of S-(-)- and R-(+)-propranolol by 58 and 61%, respectively. These reductions resulted in an increase in their respective apparent steady-state oral availabilities of 129 and 106%. The S/R enantiomeric ratio of the oral availability of propranolol was not significantly changed from control. The urinary propranolol metabolites were isolated and purified by solvent extraction and HPLC and quantitated by radioactivity. Twelve metabolites, including propranolol, were isolated and quantitated in the urine. A significant reduction in the percentage of ring oxidation products and a significant increase in the percentage of naphthoxylactic acid and propranolol glucuronide excreted in the urine occurred in the diltiazem-treated animals. The S/R enantiomeric ratios of urinary excreted propranolol, propranolol glucuronide, 4'-hydroxypropranolol glucuronide, and its sulfate were not altered by diltiazem. These results suggest that the decreased oral clearances of the enantiomers of propranolol by diltiazem is caused by a selective decrease in the formation of ring-oxidized products.

Renal tubular actions of ANF.

Many of the earliest investigations of the renal effects of atrial natriuretic factor (ANF) pointed to the glomerulus as a major site of the peptide's action. More recently, there have been many reports showing various effects of ANF on renal tubular epithelia, including collecting ducts, thick ascending limbs of Henle's loop, thin limbs of Henle's loops, and proximal tubules. The purpose of this review is to summarize the evidence for renal tubular actions of ANF and analyze it from the perspective of the specialized functions of the individual nephron segments, addressing the question: can renal tubule effects of ANF play a significant role in the precise day-to-day regulation of renal NaCl and water excretion? Based on these considerations, we propose that long-term renal tubular action of ANF may be distinct from its short-term natriuretic effect. The short-term action of ANF to accelerate salt and water excretion may play a role in the overall response to acute volume overload. This action of ANF appears to be largely due to an ANF-mediated increase in glomerular filtration rate accompanied by a blunting of the tubuloglomerular feedback mechanism, perhaps with some contribution from ANF-mediated inhibition of fluid absorption in the proximal tubule. In contrast, contributions of ANF to the precise day-to-day regulation of salt and water excretion are likely to be chiefly due to ANF-mediated inhibition of NaCl and water absorption in collecting ducts, but may also involve actions of ANF on the loop of Henle.

Pharmacokinetics of the enantiomers of verapamil in the dog.

The intravenous (0.5 mg/kg) and oral (5 mg/kg) dose kinetics of verapamil were studied in 6 dogs during steady-state oral verapamil dosing (5 mg/kg every 8 h for 3 days). Racemic verapamil and norverapamil, a metabolite of verapamil, were quantitated in plasma by HPLC-fluorescence detection. The verapamil peaks eluting off the column were collected and rechromatographed on an Ultron-OVM column, which resolved the two verapamil enantiomers. After intravenous administration, the systemic clearance and apparent volume of distribution of (-)-(S)-verapamil were nearly twice that of the (+)-(R)-isomer. There was no difference in the elimination half-lives between the two isomers. After oral administration, the oral clearance of (-)-(S)-verapamil was 20 times that of the (+)-(R)-isomer. The apparent bioavailability of (+)-(R)-verapamil was over 14 times that of (-)-(S)-verapamil. The plasma protein binding of the (+)-(R)-isomer was slightly higher by 5% than (-)-(S)-verapamil; however, this effect was not enough to account for the difference between the apparent volume of distribution of the enantiomers, indicating that the tissue binding of (-)-(S)-verapamil was greater than that of the (+)-(R)-isomer. This data on the disposition of the enantiomers of verapamil in the dog is similar to that reported for man and demonstrates that the dog may be an appropriate animal model for man in future studies on the disposition of the enantiomers of verapamil.

The effects of chronic oral diltiazem and cimetidine dosing on the pharmacokinetics and negative dromotropic action of intravenous and oral diltiazem in the dog.

The kinetics and negative dromotropic action of intravenous (1 mg kg-1) and oral (5 mg kg-1) diltiazem were studied in dogs after acute doses, after treatment for 3 days with oral diltiazem (5 mg kg-1, t.i.d.), and after 3 days' treatment with oral diltiazem (5 mg kg-1 t.i.d.) and cimetidine (200 mg t.i.d.). Plasma concentrations of diltiazem and two of its metabolites, desacetyldiltiazem and desmethyldiltiazem were measured by HPLC. Chronic oral dosing significantly lowered both the systemic and oral clearance of diltiazem, with no changes in either the volume of distribution or blood binding of diltiazem. Cimetidine treatment resulted in a significant reduction in diltiazem oral clearance from chronic control with no effect on its systemic clearance. The AUCs of both metabolites increased by greater than threefold from acute to chronic oral dosing; however, the ratio of each metabolite's AUC to that of diltiazem AUC was not significantly altered. Cimetidine treatment significantly lowered these ratios. The negative dromotropic potency of diltiazem after the acute oral dose was three times greater than that after intravenous or chronic control dosing. Cimetidine treatment resulted in further lowering chronic oral diltiazem potency. These data indicate that the disposition and negative dromotropic action of diltiazem is dependent both on the route of administration and the duration of treatment, and can be altered by co-administration with cimetidine.

Effects of propranolol on the disposition and negative dromotropic activity of diltiazem in the dog during multiple dosing.

The effects of propranolol coadministration on the disposition and negative dromotropic action of intravenous and oral diltiazem were studied in six dogs after 3 days pretreatment with diltiazem alone (2.5 mg/kg, every 8 hr) and with coadministration of oral propranolol (5 mg/kg, every 8 hr). Diltiazem and two of its metabolites, desacetyldiltiazem and demethyldiltiazem, were measured by HPLC. Propranolol coadministration had no significant effects on either the systemic clearance, the apparent volume of distribution, elimination half-life, or the blood binding of diltiazem. On the other hand, the oral clearance of diltiazem was significantly reduced by 51%, and its oral bioavailability was significantly increased by 48% during propranolol coadministration. The area under the plasma demethyldiltiazem concentration-time curve after oral diltiazem increased significantly during propranolol coadministration. This increase was in proportion to the increase in the plasma diltiazem area under the concentration-time curve, such that the ratio of the areas of demethyldiltiazem to that of diltiazem remained the same between control and propranolol coadministration. Propranolol coadministration increased the area under the negative dromotropic activity-time curve after both intravenous and oral diltiazem by 37 and 48%, respectively. Using a log-linear pharmacodynamic model to analyze the data, there were no significant effects on either the slope, y-intercept, or the estimated diltiazem concentration needed to increase the PR interval by 20% of either intravenous or oral diltiazem with propranolol coadministration. These data suggest that propranolol coadministration can result in an increase in the pharmacological activity of diltiazem due to a kinetic drug interaction by increasing its oral bioavailability.