Incidence of unintentional intraneural injection and postoperative neurological complications with ultrasound-guided interscalene and supraclavicular nerve blocks.

It is proposed that ultrasound guidance decreases the risk of intraneural injection and associated postoperative neurological complications. However, the incidence of unintentional intraneural injection with ultrasound is unknown. Two hundred and fifty-seven patients were enrolled in a prospective, single-blind observational study. All patients underwent a pre-operative neurological examination before ambulatory shoulder arthroscopy with sedation and ultrasound-guided interscalene or supraclavicular block. Patients were followed up at 1 week and at 4-6 weeks postoperatively. Two blinded anaesthesiologists viewed the same video of the ultrasound image during the block offline to determine intraneural trespass. Intraneural injection occurred in 42 patients (17%; 95% CI 12-22%). No patient suffered from postoperative neurological complications (0%; 95% CI 0-1.6%) at follow-up.

Sequence and activity of the rat PB-inducible aldehyde dehydrogenase promoter.

The 5' flanking region of the rat PB inducible aldehyde dehydrogenase gene was isolated and the sequence from +42 to -1339 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 and DBP as well as a GRE and several possible AP1 sites. A TATA and CCAAT motif were assigned at positions -26 and -53. A promoter construct containing the -1339 bp of the aldehyde dehydrogenase 5' flanking region was active when transfected into both H411 and HepG2 liver cell lines.

Sequence of the rat PB-inducible CYP2B1 promoter.

The 5' flanking region of the rat PB-inducible CYP2B1 gene was isolated and the sequence from +27 to -3878 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 as well as an AP1, two NF-kappa B and several possible STAT sites. The promoter sequence of the CYP2B1 gene was compared to that of the CYP2B2 sequence, published by Hoffman et al. ((1992) Gene Exp. 2, 353-362) and was found to be almost identical up to -2300 bp, beyond which it diverges significantly from the remaining published sequence of CYP2b2 gene. Transient transfection experiments in the differentiated hepatoma cell line, FGC4, showed that the 3.9 kb promoter was expressed, however, an increase in reporter activity was not observed in the presence of phenobarbital.

Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins.

Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 (CYP2B) genes. One site, which maps to a region in the 5'-flanking region between -2.2 and -2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between -0.05 and -0.15 kb. DNase I footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between -42 and -66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBP alpha bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBP alpha could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBP alpha.

Percutaneous technique for management of persistent airspace with prolonged air leak using fibrin glue.

A 68-year-old white man with lung carcinoma underwent removal of the lower lobe of the left lung. Four months later, the patient developed a hemothorax requiring exploratory thoracotomy, evacuation of hemothorax, and decortication. Postoperatively a persistent airspace with prolonged air leak developed. Bronchoscopic application of fibrin glue failed to seal the leak. Percutaneous transthoracic application of fibrin glue with CT scan guidance partially obliterated the space and completely sealed the leak. We describe this simple and effective technique for management of this special problem.

Optimization of chest films of equalization radiography (advanced multiple beam equalization radiography). Comparison by means of a receiver operating characteristic study of simulated nodular interstitial disease.

RATIONALE AND OBJECTIVE: To optimize screen-film combinations for equalization radiography (advanced multiple beam equalization radiography [AMBER]), five different film-screen-technique combinations were compared by receiver operating characteristics study of simulated interstitial disease. MATERIALS AND METHODS: The Ortho C-Lanex Regular and the Insight Thoracic Imaging HC system were compared in conventional nonequalized technique; T-Mat G-Lanex Regular and T-Max L-Lanex Regular were compared in conventional, nonequalized, and AMBER technique; and an experimental high-contrast, low-noise, near-zero crossover film-screen combination was compared in AMBER technique. Interstitial disease was simulated by superimposing birdseed on the back of a humanoid phantom. Twenty-five posterior-anterior radiographs were made with each technique. Seven observers scored the presence of interstitial disease in each of the quadrants on a 5-point scale following receiver operating characteristic methodology. RESULTS: The highest performance was found with the experimental film-screen-AMBER combination (Az = 0.92) and the lowest with the T-Mat L-Lanex Regular-AMBER combination (Az = 0.83) and the Insight Thoracic Imaging HC system-conventional combination (Az = 0.85). T-Mat L-Lanex Regular-conventional ranked second (Az = 0.90) while T-Mat G-Lanex Regular-conventional (Az = 0.89), T-Mat L-Lanex Regular-AMBER (Az = 0.88) and Ortho-C-Lanex Regular-conventional (Az = 0.87) scored lower. CONCLUSION: Higher contrast films in AMBER improve diagnostic performance, whereas a loss of information is found if the AMBER system is combined with lower contrast films.

Occult inguinal hernia, a cause of rapid onset of penile and scrotal edema in patients on chronic peritoneal dialysis.

From 1976 to 1993 we inserted 160 chronic peritoneal dialysis catheters for renal failure patients. Three of these patients developed sudden onset of penile and scrotal edema after the catheter had been in place for several months. The first patient was diagnosed by instilling technetium sulfur colloid in the peritoneal cavity, which showed the radioisotope flowing via the right inguinal canal. He was operated on and the processus vaginalis was tied off and the scrotal and penile edema resolved. Subsequently, two more patients were seen with similar problems and had their inguinal canals explored and the processus vaginalis in one and the hernia sac in the other were found and tied off, which resulted in resolution of the problem. This is an uncommon complication, reported to occur in 3 to 4% of patients.

A question-based approach to adopting pharmacogenetics to understand risk for clinical variability in pharmacokinetics in early drug development.

Understanding genetic variations that influence pharmacokinetics (PK) in humans is important for optimal clinical use of drugs. Guidances for making decisions on when to conduct pharmacogenetic research during drug development have been proposed by regulatory agencies, but their uniform adoption presents problems due to an inherent lack of flexibility. A questions-based approach (QBA) was developed to enable drug development teams at Merck to iteratively and flexibly evaluate the potential impact of pharmacogenetics (PGx) on clinical pharmacokinetic variability.

UGT2B17 genetic polymorphisms dramatically affect the pharmacokinetics of MK-7246 in healthy subjects in a first-in-human study.

MK-7246, an antagonist of the chemoattractant receptor on T helper type 2 (Th2) cells, is being developed for the treatment of respiratory diseases. In a first-in-human study, we investigated whether genetic polymorphisms contributed to the marked intersubject variability in the pharmacokinetics of MK-7246 and its glucuronide metabolite M3. Results from in vitro enzyme kinetic studies suggested that UGT2B17 is probably the major enzyme responsible for MK-7246 metabolism in both the liver and the intestine. As compared with those with the UGT2B17*1/*1 wild-type genotype, UGT2B17*2/*2 carriers, who possess no UGT2B17 protein, had 25- and 82-fold greater mean dose-normalized values of area under the plasma concentration-time curve (AUC) and peak concentration of MK-7246, respectively, and a 24-fold lower M3-to-MK-7246 AUC ratio. The apparent half-life of MK-7246 was not as variable between these two genotypes. Therefore, the highly variable pharmacokinetics of MK-7246 is attributable primarily to the impact of UGT2B17 genetic polymorphisms and extensive first-pass metabolism of MK-7246.

Coding of DNA samples and data in the pharmaceutical industry: current practices and future directions--perspective of the I-PWG.

DNA samples collected in clinical trials and stored for future research are valuable to pharmaceutical drug development. Given the perceived higher risk associated with genetic research, industry has implemented complex coding methods for DNA. Following years of experience with these methods and with addressing questions from institutional review boards (IRBs), ethics committees (ECs) and health authorities, the industry has started reexamining the extent of the added value offered by these methods. With the goal of harmonization, the Industry Pharmacogenomics Working Group (I-PWG) conducted a survey to gain an understanding of company practices for DNA coding and to solicit opinions on their effectiveness at protecting privacy. The results of the survey and the limitations of the coding methods are described. The I-PWG recommends dialogue with key stakeholders regarding coding practices such that equal standards are applied to DNA and non-DNA samples. The I-PWG believes that industry standards for privacy protection should provide adequate safeguards for DNA and non-DNA samples/data and suggests a need for more universal standards for samples stored for future research.

Current practices for DNA sample collection and storage in the pharmaceutical industry, and potential areas for harmonization: perspective of the I-PWG.

Collection and storage of DNA samples in clinical drug development programs are an important investment for the pharmaceutical industry to allow efficient evaluation of observed variability in drug response. To enable collection and future use of samples, individual companies must define (i) processes to collect specimens worldwide, (ii) whether collection is optional or mandatory, (iii) conditions and duration of sample storage, (iv) whether research data can be returned to subjects, and (v) other logistical aspects. To determine current industry practices for collection and storage of these samples, the Industry Pharmacogenomics Working Group (I-PWG) conducted a survey of the industry (21 respondents) to identify areas of commonality and divergence. On the basis of the survey results, the I-PWG details areas of focus for harmonization of the industry's sample collection practices. A more unified approach would facilitate DNA sample collection, thereby contributing to the advancement of personalized medicine and more efficient development of safe and effective drugs.

Teaching the doctor-patient relation to medical students. Learning experience in a behaviour therapy clinic.

The results of a 2-year trial of teaching medical students in a phobia clinic are described. This has been found a useful experience by the students, particularly as an introduction to the doctor-patient relation. The success rate and attitude of patients justified this experiment, and there might be an indication for extension to other teaching and possibly regional hospitals. The long-term benefits to the medical student are discussed.

A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.

Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

Hepatocyte nuclear factor 3 is a major determinant of CYP2C6 promoter activity in hepatoma cells.

Cytochrome P450 2C6 (CYP2C6) is a developmentally regulated, constitutively expressed form of rat liver microsomal cytochrome P450 that in the liver of adult male rats is induced to a limited extent by phenobarbital. The gene is not expressed at detectable levels in the lung, kidney, or brain. It is expressed and inducible by phenobarbital in differentiated Reuber hepatoma cells that express many hepatocyte-specific genes but not in dedifferentiated derivatives lacking the majority of hepatocyte-specific functions. A 505-base pair proximal segment of the CYP2C6 promoter is highly efficient in driving transcription of a linked chloramphenicol acetyltransferase reporter gene in the differentiated rat hepatoma cell line FGC4, is much less effective in a related dedifferentiated variant H5, and has no measurable activity in nonhepatic C33 human cervical carcinoma cells. The activity of the CYP2C6 promoter in the differentiated hepatoma cells is strongly dependent on hepatocyte nuclear factor (HNF)3, which acts at a complex site just upstream of the TATA motif. Transactivation experiments show that the D-site-binding protein (DBP) may also contribute to CYP2C6 promoter activity, via a site that is adjacent to the proximal HNF3 site. A substantial contribution to promoter activity by the base pair -505 to -316 segment is observed in FGC4 and H5 cells but not in HepG2 cells; deletion of this segment causes a marked diminution in promoter activity only in the former two cell lines. Although footprinting experiments have permitted the definition of three protein binding sites in this region (two HNF3 and one unidentified), mutation of these sites does not diminish promoter activity. The functionally important cis sequences in this region therefore remain to be defined. In HepG2 cells the distal region does not contribute to promoter activity. This most likely accounts for the low promoter activity in HepG2 and implies a deficiency in the relevant trans-acting factor(s).

Social phobia: a comparative clinical study.

Eighty-seven people with the symptom of social phobia were compared with 57 people with the symptom of agoraphobia to determine whether these symptoms were part of distinct syndromes. Comparisons were made on demographic, clinical and questionnaire data. Significant differences were found on important variables. The social phobics were younger and more often male, unmarried, and from social classes I and II. The pattern of phobic situations was different in the two groups and so was the pattern of autonomic symptoms experienced in these situations. Symptoms visible to others were more frequent among social phobics. Fainting was more frequent among the agoraphobics.

The phenobarbital-induced transcriptional activation of cytochrome P-450 genes is blocked by the glucocorticoid-progesterone antagonist RU486.

Several of the hepatic microsomal cytochromes P450 can be induced by various drugs and xenobiotics, among them the barbiturate phenobarbital. Rat hepatoma cells (Fao and its derivatives) respond to phenobarbital or dexamethasone treatment with an increased accumulation of CYP2C6 mRNA and thus provide a culture system to investigate the mechanisms involved. Examination of the kinetics of CYP2C6 mRNA induction revealed that the response to dexamethasone is rapid, whereas induction by phenobarbital occurs only slowly after an 8-10-hr lag. Run-on transcription measurements demonstrated that phenobarbital treatment led to a 3-4-fold increase in CYP2C6 gene transcription. Surprisingly, induction by phenobarbital of both accumulation of CYP2C6 mRNA and transcription of the gene was blocked by the antiprogestin-antiglucocorticoid RU486, suggesting the involvement of a steroid receptor in the induction process. Transfection of promoter constructs containing a reporter gene whose expression is driven by a 1.4-kilobase 5' flanking segment of the CYP2B1 or CYP2B2 genes, which are highly inducible by phenobarbital in rat liver, led to > 3-fold increases in reporter gene activity in the presence of the drug. Again, phenobarbital induction was prevented by RU486. The RU486 inhibition of the phenobarbital induction of both the endogenous CPY2C6 gene and the transfected CYP2B1 and CYP2B2 promoter constructs leads us to propose a model whereby the drug acts indirectly to cause the accumulation of an endogenous steroid, and this molecule, acting via its receptor, would be the direct inducer of cytochromes P450. Whether or not this model proves to be correct, the results presented here provide the first evidence of the involvement of a steroid receptor in phenobarbital induction.

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.

Adjuvant immunotherapy for patients with melanoma: are patients with melanoma of the head and neck candidates for this therapy?

BACKGROUND: Although a wealth of information is available on adjuvant immunotherapy for melanoma, little is known about adjuvant immunotherapy for head and neck melanoma. Interestingly, a few immunotherapy clinical trials report the observation of clinical responses in a subset of patients with head and neck melanoma. METHOD: An up-to-date literature search was performed to identify the current information on adjuvant immunotherapy for patients with melanoma, including head and neck melanoma. Moreover, a retrospective analysis of a subset of primary head and neck melanoma was performed using data from a phase III, randomized, double-blind, multi-institutinal, vaccinia melanoma oncolysate adjuvant immunotherapy trial that was performed in our laboratory for patients with stage III (AJCC) melanoma. RESULTS: In a passive immunotherapy trial with an antibody to melanoma ganglioside antigen GM2, a complete regression was observed in one patient with lesions of the right cheek. In three active specific immunotherapy trials, including our phase III trial, a subset of patients with head and neck primary melanoma showed a longer disease-free and overall survival with immunotherapy. Moreover, these clinical responses were correlated to the induction of immune response, delayed-type hypersensitivity response and melanoma-specific antibody response. CONCLUSIONS: The above results therefore suggest that patients with head and neck melanoma clinically respond to immunotherapy. However, these results need to be confirmed in a prospectively randomized trial for patients with head and neck melanoma.

Induction of CYP1A1, but not CYP1A2, in adrenals of 3, 3'-methylcholanthrene-treated guinea pigs.

To test the inducibility of CYP1A homologs in guinea pig adrenal, the effects of 3,3'-methylcholanthrene, an archetypal inducer of CYP1A, were compared in guinea pig adrenal and liver. Western blot analysis showed that levels of both CYP1A1 (53 kDa) and CYP1A2 (56 kDa) increasedin liver microsomes of 3,3'-methylcholanthrene-treated guinea pigs. In adrenals, an immunoreactive protein comigrating with liver CYP1A1 was detected only after 3,3'-methylcholanthrene treatment. Protein comigrating with CYP1A2 was never detected in adrenal microsomes. A third inducible immunoreactive protein (57 kDa) was seen in liver, but not adrenal, after 3, 3'-methylcholanthrene treatment. Another immunoreactive protein (52 kDa), present constitutively in liver and adrenal microsomes, was not induced in either tissue by 3,3'-methylcholanthrene. The precise identities of the inducible 57-kDa and the noninducible 52-kDa proteins remain to be determined. However, the identity of the 53-kDa protein in the adrenal as CYP1A1 was confirmed by RT-PCR, Northern blot, and sequence analysis. Similar analyses demonstrated that, despite the fact that the 56-kDa protein was not detectable in adrenal microsomes, CYP1A2 mRNA was present in adrenals of control animals. Strikingly, CYP1A2 mRNA decreased in adrenal, but increased in liver, following 3,3'-methylcholanthrene treatment, underscoring differences in the regulation of CYP1A expression in the two tissues. Levels of ethoxyresorufin and methyoxyresorufin metabolism correlated with levels of CYP1A1 and CYP1A2 protein, respectively.

Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes.

Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16alpha-position but was less active in estrone 16alpha-hydroxylation; the latter reaction and also estradiol 16alpha-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16alpha-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16alpha-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. alpha-Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, alpha-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis [Service, R. F. (1998) Science 279, 1631-1633].