Vacuolar myelopathy in transgenic mice expressing human immunodeficiency virus type 1 proteins under the regulation of the myelin basic protein gene promoter.

Vacuolar myelopathy is a common neurological complication in AIDS patients. The pathogenesis of this spinal cord white matter disease remains unclear and it is still debated whether infection of spinal cord with the human immunodeficiency virus type 1 (HIV-1) is causing the disease. We have generated transgenic mice expressing the entire HIV-1 genome under the regulation of an oligodendrocyte-specific promoter. These mice develop spinal cord vacuolar lesions similar to those found in AIDS patients. This animal model provides in vivo evidence linking the expression of HIV-1 proteins in oligodendrocytes to the spinal cord damage found in vacuolar myelopathy.

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice.

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

Involvement of Notch1 in the development of mouse mammary tumors.

The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5' truncated approximately 7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3' Notch1 transcripts (3.5 - 4.5 kb) and proteins (86 - 110 kDa) deleted of most of the extracellular sequences (Notch1intra). We found that 3' truncated Notch1intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 - 1821) are required, while a signal peptide, the two conserved cysteines (C1652 and C1685) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1intra protein behaves as an oncogene in this system.

Improving the cancer chemotherapy use process.

PURPOSE: Reports of the tragic consequences of erroneous cancer chemotherapy overdoses at a prominent cancer center and a university hospital prompted a review of our institution's practices and those of 123 other hospitals to ascertain for each the current in-house process to prevent chemotherapy errors. METHODS: A multidisciplinary committee of oncologists, nurses, and pharmacists reviewed the chemotherapy use process and identified opportunities for improvement. A 1-page facsimile survey was answered by 150 of 215 members of the American Society of Clinical Oncology (ASCO) who received it. RESULTS: We further restricted the writing of cytotoxic chemotherapy orders to physicians who were board-certified or -eligible in hematology or medical, pediatric, and gynecologic oncology and their approved fellows. Dispensation of drugs is limited to oncology-certified pharmacists, and administration to chemotherapy-certified nurses. Standard orders are used either on special oncology forms or designated order sets in the computer. Procedures to regulate the ordering of antineoplastic drugs for nonmalignant indications by nononcology specialists are outlined. A process to prevent chemotherapy errors is in place in 95% of hospitals. Dedicated medical oncology units are ubiquitous, and most cancer centers and university hospitals have dedicated gynecologic and pediatric oncology units. Chemotherapy orders are generally written by oncology fellows and countersigned by an attending oncologist in cancer centers and university hospitals, whereas private oncology attending physicians write them in most community hospitals. Drugs are administered by oncology-certified nurses in most institutions. CONCLUSIONS: These recommendations should improve the safety and effective use of chemotherapy and reduce the error rate to as close to zero as human fallibility will allow.

Health plan characteristics and consumers' assessments of quality.

Many purchasers and consumers of health care have become concerned about the quality of care being delivered in managed care plans. Little is known, however, about the health plan characteristics that are associated with better performance. We used survey responses from 82,583 Medicare beneficiaries from 182 health plans to study the association of consumers' assessments of care with health plan characteristics. For-profit and nationally affiliated health plans received much worse scores on the outcomes of interest, particularly for overall ratings of the health plan and composite measures of customer service and access to care. Health plans accredited by the National Committee for Quality Assurance did not receive higher scores.

High-performance liquid chromatographic methods for the determination of ranitidine and related substances in raw materials and tablets.

High-performance liquid chromatographic methods have been developed for the determination of ranitidine and related compounds in drug raw material and tablets. The method has been shown to resolve at least nine related compounds from the drug. The sensitivity of the method to related compounds is better than 0.01%. Eight raw material samples and 11 tablet samples were examined for related compounds. Total impurities found ranged from 0.31 to 0.79% in raw materials and from 0.40 to 1.75% in tablets. Drug raw materials and tablets were assayed by HPLC; results for raw materials were between 98.2 and 101.1%, and those for tablets were between 96.1 and 102.2%, with a relative standard deviation for the assay of less than 1%. Raw material assay results were confirmed by nonaqueous titration.

High-performance liquid chromatography method for assay of diltiazem hydrochloride and its related compounds in bulk drug and finished tablets.

The method provides for the resolution of trans-diltiazem and seven known and several unknown related compounds from diltiazem HCl. Minimum detectable amounts were less than 0.1%, except for an intermediate which originates early in the synthetic process, for which the sensitivity is approximately 2%. The relative standard deviation of the assay procedure is 0.15%. Total related compounds in four bulk drug and four tablet samples were less than 0.25%. The specific rotation of four samples of diltiazem HCl analyzed in duplicate was between +112 and +114 degrees. The UV absorption spectra of all compounds exhibited two maxima, one between 203 and 213 nm and the other between 230 and 244 nm.

Liquid chromatographic methods for the determination of albuterol (salbutamol), albuterol sulphate and related compounds in drug raw materials, tablets and inhalers.

Liquid chromatographic methods for the determination of albuterol (salbutamol), albuterol sulphate and related compounds in drug raw materials, tablets and inhalers are described. The methods resolve five known related compounds from the drug and, in the case of inhalers, several compounds not related to the drug. Two of these were identified as 2,6-di-t-butyl-4-methylphenol, a common antioxidant, and 2,2'-methylene bis(6-t-butyl-4-methylphenol). Related compounds are detectable at levels of about 0.03%. Eleven albuterol and 12 albuterol sulphate raw materials and eight tablet formulations were found to contain related compounds ranging from 0.03 to 0.54%, 0.09 to 0.50% and 0.32 to 0.95%, respectively. Non-drug compounds in three inhaler samples ranged from 4.6 to 12% of the drug delivered through the valve. Some of the non-drug compounds may be excipients.

Validation of a method for the assay of related compounds in famotidine raw materials and formulations.

A high-performance liquid chromatographic (HPLC) method has been developed for the determination of famotidine and related compounds in drug raw materials and formulations. The minimum detectable amount of the available related compounds is less than 0.02% and the minimum quantifiable amount is less than 0.1%. Famotidine impurity levels were between 0.5 and 2.5% in raw materials. 0.44% in one tablet sample and about 3% in an IV solution, allowing for stabilizers.

How consumer assessments of managed care vary within and among markets.

This study investigated the extent to which the Consumer Assessments of Health Plans Survey (CAHPS) distinguishes performance of Medicare managed care (MMC) health plans. Results indicate that CAHPS ratings and report composites distinguish among plans both nationally and within markets. Global ratings of a health plan and reports on customer services varied strongly at the individual plan level, with smaller effects seen at regional and Metropolitan Statistical Area (MSA) levels. Ratings of doctors and health care, and reports on experiences in the doctor's office varied more among regions and among MSAs than among plans within the same MSA. These patterns are consonant with our hypotheses about the determinants of these ratings: customer service is a distinct plan function, but medical services are provided by networks that often overlap for plans in the same area. We conclude that the CAHPS-MMC survey can inform consumers choosing among plans as well as policymakers and researchers.

Healthcare costs of GERD and acid-related conditions in pediatric patients, with comparison between histamine-2 receptor antagonists and proton pump inhibitors.

BACKGROUND: Gastroesophageal reflux disease and acid-related conditions (GERD/ARC) are common in pediatric practice but their costs have not been well characterized. AIM: To compare healthcare costs (HCC) and healthcare utilization (HCU) of pediatric GERD/ARC between groups of GERD/ARC patients initiated on histamine-2 receptor antagonists (H(2)RAs) or proton pump inhibitors (PPIs) and matched controls. PATIENTS AND METHODS: Children (age < 18 years) diagnosed with GERD or ARC (exploratory category) were identified from a large US claims database (1999-2005) using ICD-9 codes. Costs of pediatric GERD/ARC were estimated by comparing 6-month post-diagnosis HCC between cases and matched controls. GERD/ARC-related HCC and HCU for the year 2005 were further compared between GERD/ARC patients initiated with PPIs vs. H(2)RAs in terms of the cost differences relative to pre-initiation (difference-in-difference) and using multivariate regression to adjust for demographics, pre-treatment health status and pre-treatment costs. RESULTS: A total of 27 865 matched pairs were identified. GERD/ARC patients incurred on average more 6-month total HCC than controls ($2386). In 2005, 1010 pediatric patients were initiated on H(2)RAs or PPIs. About 61% were initiated on PPIs and incurred 1.8 times higher 6-month post-initiation GERD/ARC-related HCC than H(2)RA-initiated patients ($661 vs. $372, p < 0.001). Although total 6-month GERD/ARC-related HCC increased for both PPI- and H(2)RA-treated patients, the increase was 30% less for PPI-treated patients ($173 vs. $246, p = 0.521) in the difference-in-difference analysis and 69% less in the multivariate analysis ($109 vs. $347, p = 0.040). LIMITATIONS: The use of an exploratory definition for GERD/ARC, administrative claims data and potential coding errors in diagnosis codes used in selection process may limit the generalizability of the results. CONCLUSION: Pediatric GERD/ARC patients incurred significantly higher healthcare costs compared to similar children without GERD/ARC. Compared to patients initiated with H(2)RAs, patients initiated with PPIs had more baseline comorbidities, and lower GERD/ARC-related HCC after beginning treatment.

Pediatric gastroesophageal reflux disease and acid-related conditions: trends in incidence of diagnosis and acid suppression therapy.

OBJECTIVE: To describe the incidence of diagnosis of gastroesophageal reflux disease and acid-related conditions (GERD/ARC) throughout childhood and characterize patterns of diagnosis and treatment with proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H(2)RAs). METHODS: Cohorts of GERD/ARC children (age 0-18 years) were identified from a large US administrative claims database covering 1999-2005 using ICD-9 codes. Incidence, healthcare utilization (HCU), costs, therapy discontinuation and switching rates were compared between various age and patient groups. RESULTS: Between 2000 and 2005 annual incidence of GERD/ARC diagnosis among infants (age ≤1 year) more than tripled (from 3.4 to 12.3%) and increased by 30% to 50% in other age groups. Patients diagnosed by GI specialists (9.2%) were more likely to be treated with PPIs compared to patients diagnosed by primary care physician (PCP). PPI-initiated patients doubled (from 31.5% in 1999 to 62.6% in 2005) and, when compared with H(2)RA-initiated patients, were associated with 30% less discontinuation and 90% less therapy switching in the first month, and with higher comorbidity burden and pre-treatment total HCU and costs when diagnosed by GI specialists. LIMITATIONS: The use of an exploratory definition for GERD/ARC, administrative claims data and potential coding errors in diagnosis codes used in selection process may limit the generalizability of the results. CONCLUSIONS: GERD/ARC incidence increased for children of all ages between 2000 and 2005. PCPs made the majority of diagnoses. PPI initiations have now surpassed H(2)RA initiations.

Liquid chromatographic method for perphenazine and its sulfoxide in pharmaceutical dosage forms for determination of stability.

A liquid chromatographic method is described for determination of perphenazine and perphenazine sulfoxide in representative dosage forms. Sulfoxide levels were nondetectable or less than 1% in tablets and in an injectable product. Sulfoxide levels increase with time in some syrup formulations and may be as high as 11% in syrup formulations before their expiration date.

cDNA sequence and tissue-specific expression of an anionic flax peroxidase.

A flax (Linum usitatissimum L.) lambda gt10 cDNA library was screened with a probe coding for the amino terminus of a flax peroxidase. The probe was generated by PCR amplification of the library with one of the lambda gt10 sequencing primers and a mixed oligonucleotide derived from a well-conserved amino acid region (distal heme ligand) found in all plant peroxidases. A positive clone (FLXPER2) was isolated and characterized. The cDNA contains 1153 nucleotides, excluding the poly(A) tail, and encodes a mature protein of 297 amino acids with a molecular mass of 31.9 kDa. The mature protein's amino acid sequence contains three highly conserved regions, two of which contain histidine ligands for the heme group. The deduced amino acid sequence has nine cysteine residues. Eight are identically located to those of horseradish C peroxidase, which participate in four disulfide bridges; these cysteines are highly conserved in all plant peroxidases. One potential N-glycosylation site (Asn-X-Thr/Ser) is present. The predicted pI value of 4.5 identifies FLXPER2 as an anionic peroxidase. Northern blot analysis shows that its mRNA expression is unique to stem tissue. Amino acid sequence comparisons show high similarity between FLXPER2 and peanut, rice, and tobacco peroxidases.

Efficient production of human immunodeficiency virus proteins in transgenic mice.

Transgenic mice containing the complete human immunodeficiency virus (HIV) coding sequences fused to the mouse mammary tumor virus long terminal repeat were generated. They were found to produce high levels of authentic gag and env HIV proteins in several tissues known to support mouse mammary tumor virus-driven transcription. HIV proteins were also detected in serum and in body fluids (milk and epididymal secretions) known to be natural sites of retrovirus, and specifically of HIV, production. These results indicate that primary mouse cells from different tissues have the capacity to produce HIV proteins. These mice represent a novel animal model for HIV infection.

Determination of diclofenac sodium and related compounds in raw materials and formulations.

A liquid chromatographic method has been developed for determination of drug and related compounds in diclofenac sodium raw material, slow-release, and enteric coated tablets. The method specifies a 5 microns octadecylsilane bonded phase column, a mobile phase of tetrahydrofuran-acetonitrile-buffer, pH 5 (1 + 4 + 8.3), and detection at 229 nm. The method resolves 10 known related compounds with limits of quantitation of 0.2% or less. Seventeen drug raw material samples were evaluated. Total impurity levels ranged from 0.1 to 0.9%. The method has also been used for determination of drug content in raw materials and formulations. Mean assay levels in drug raw materials ranged between 98.3% and 101.8%.

Liquid chromatographic determination of identity, content, and content uniformity of desipramine, fluphenazine, and promazine.

A liquid chromatographic procedure has been developed for the assay, content uniformity, and identification of single active ingredient formulations of desipramine, fluphenazine, and promazine. The drugs are extracted from formulations with methanol or dilute hydrochloric acid and quantitated against an internal standard (norephedrine). The drugs are identified by comparison of retention times with those of the reference standards.

A specific radio-immunoassay (RIA) for salbutamol (albuterol) in human plasma.

A specific RIA method for Salbutamol (S) employing a selective extraction procedure was developed to enable the quantitation of 0.5 ng/ml of (S) in plasma from human volunteers. The specificity of the assay was confirmed by GC-MS method.

Liquid chromatographic method for identity, assay, and content uniformity of five tricyclic drugs.

A liquid chromatographic (LC) procedure has been developed for the assay, content uniformity, and identification of single active ingredient solid and liquid formulations of amitriptyline, chlorpromazine, imipramine, thioridazine, and trifluoperazine. The drugs are extracted from their formulations with methanol or dilute hydrochloric acid, and identified by comparison of retention times with those of known standards; drugs are quantitated against these standards with dl-norephedrine hydrochloride as the internal standard. The precision of replicate injections is better than 2.5% for peak area and better than 1% for peak height. The precision of triplicate determinations of tablet composites is better than 2.2%.