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.

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.

Handedness in boys with gender identity disorder.

Handedness preference was assessed in 205 boys with gender identity disorder and 205 clinical control boys referred for other reasons. Boys with gender identity disorder were significantly more likely to be left-handed than the clinical control boys (19.5% vs. 8.3%, respectively). The boys with gender identity disorder, but not the clinical control boys, also had a significantly higher rate of left-handedness compared to three independent, general population studies of nonreferred boys (11.8%; N = 14,253) by Hardyck, Goldman, and Petrinovich (1975), Calnan and Richardson (1976), and Eaton, Chipperfield, Ritchot, and Kostiuk (1996). Left-handedness appears to be a behavioral marker of an underlying neurobiological process associated with gender identity disorder in boys.

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.

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.

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.

Dimensions of consumer-assessed quality of Medicare managed-care health plans.

OBJECTIVES: We investigated relationships at the health-plan level among member ratings of and reports on plans in the Consumer Assessment of Health Plans Survey (CAHPS). We sought a more parsimonious description of the reports that can be used in analyses of the distribution and correlates of consumer-assessed quality. SUBJECTS: There were 89,419 Medicare beneficiaries enrolled in 212 Medicare managed-care health plans who responded to CAHPS in 1998. MEASURES: There were 39 survey items measuring consumer ratings of and reports on care. METHODS: We adjusted correlations for sampling variability in the plan means and performed a principal factor analysis of the report items with oblique rotation. We grouped items that loaded heavily on the different factors, formed composites, and regressed rating items on the report composites. RESULTS: Four factors explained 75% of the variance in the reports. The corresponding groups of items were concerned with the following subjects: (1) interactions around delivery of care in the doctor's office; (2) customer service from the plan; (3) access to medical services provided by the plan, such as specialist care, equipment, therapy, or drugs; and (4) advice on health-promoting activities. Corrected Cronbach alpha for composites were 0.97, 0.93, 0.86, and 0.60. The "delivery" composite was strongly predictive of overall ratings of care, doctor, and specialist; the "customer" composite was strongly predictive of overall ratings of the plan. CONCLUSIONS: CAHPS distinguishes among dimensions of between-plan variability of consumer-assessed quality. Different global ratings are related to distinct groups of consumer reports on their experiences.

Characterization of the human DNA methyltransferase splice variant Dnmt1b.

Tissue- and gene-specific patterns of cytosine-DNA methylation are characteristic features of vertebrate genomes. The generation and proper maintenance of DNA methylation patterns are essential for embryonic development, as demonstrated by the lethal phenotypes of mice with either a targeted disruption of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generation of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, resulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estimated by semiquantitative reverse transcription polymerase chain reaction and was suggested to encode a major C-5 DNA methyltransferase isoform. Here we report characterization of this novel DNA methyltransferase transcript, Dnmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcript, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisense inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumulation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a baculovirus expression system was demonstrated to be a functional DNA methyltransferase, and to have Michaelis constants for both DNA and S-adenosyl-L-methionine similar to baculovirus-expressed Dnmt1. However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at approximately 2-5% of the level of Dnmt1 and therefore represents only a minor DNA methyltransferase isoform in human cells.

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.

Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms.

A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5' CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzyme (DNA MeTase) are also prevalent features of human neoplasia. However, direct evidence that elevated DNA MeTase levels alter gene expression and influence oncogenesis has been difficult to obtain, in part due to the lack of specific DNA MeTase inhibitors. Here we show that specific reduction of cellular DNA MeTase levels in human cancer cells with potent antisense inhibitors: 1) causes demethylation of the p16(ink4A) gene promoter; 2) causes re-expression of the p16(ink4A) protein; 3) leads to accumulation of the hypophosphorylated form of the retinoblastoma protein (pRb); and 4) inhibits cell proliferation. Stepwise reduction of cellular DNA MeTase protein levels also induced a corresponding rapid increase in the cell cycle regulator p21(WAF/Cip1) protein demonstrating a regulatory link between DNA MeTase and the growth regulator p21(WAF/Cip1) that is independent of methylation of DNA. These results suggest that the elevated levels of DNA MeTase seen in cancer cells can inhibit tumor suppressors by distinct mechanisms involving either transcriptional inactivation through DNA methylation or by a methylation independent regulation.

The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl.

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells.

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.

Frequent provirus insertional mutagenesis of Notch1 in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notch1 for oncogenesis.

The MMTVD/myc transgenic mice spontaneously develop oligoclonal CD4+CD8+ T-cell tumors. We used provirus insertional mutagenesis in these mice to identify putative collaborators of c-myc. We found that Notch1 was mutated in a high proportion (52%) of these tumors. Proviruses were inserted upstream of the exon coding for the transmembrane domain and in both transcriptional orientations. These mutations led to high expression of truncated Notch1 RNAs and proteins (86-110 kD). In addition, many Notch1-rearranged tumors showed elevated levels of full-length Notch1 transcripts, whereas nearly all showed increased levels of full-length (330-kD) or close to full-length (280-kD) Notch1 proteins. The 5' end of the truncated RNAs were determined for some tumors by use of RT-PCR and 5' RACE techniques. Depending on the orientation of the proviruses, viral LTR or cryptic promoters appeared to be utilized, and coding potential began in most cases in the transmembrane domain. Pulse-chase experiments revealed that the 330-kD Notch1 proteins were processed into 110- and 280-kD cleavage products. These results suggest that Notch1 can be a frequent collaborator of c-myc for oncogenesis. Furthermore, our data indicate that Notch1 alleles mutated by provirus insertion can lead to increased expression of truncated and full-length (330/280-kD) Notch1 proteins, both being produced in a cleaved and uncleaved form.

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.

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%.

Radioimmunoassay for albuterol using a monoclonal antibody: application for direct quantification in horse urine.

A monoclonal antibody was synthesized in mouse against the O-(3-carboxypropionyl) derivative of albuterol linked to bovine serum albumin. Isotyping of this material revealed the IgG1 class characterized by an affinity constant of 1.03 nM-1 and a density of sites of 0.55 nM. This antibody was found specific as its cross-reactivity to structurally related molecules was less than 1% except for clenbuterol (75%). A radioimmunoassay was set up with culture supernatant (final dilution 1/1000) and [3H] albuterol. The calibration curve was characterized by a maximum binding of 28%, an ED50 of 1.15 pmol per tube, the detection limit was 28.8 fmol/tube and the linearity of the response was up to 39.8 pmol/tube. This RIA method has been used for direct quantitation of albuterol in horse urine without any clean-up or extraction step.

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.

Analysis of albuterol in human plasma based on immunoaffinity chromatographic clean-up combined with high-performance liquid chromatography with fluorimetric detection.

A method combining immunoaffinity chromatography with high-performance liquid chromatography was developed for the determination of albuterol in human plasma. The immunoaffinity chromatography, based on the specific interaction of albuterol with the immobilized antibody raised against it, was used as a clean-up step. Albuterol eluted from this immunochemical solid-phase clean-up step was analysed by reversed-phase high-performance liquid chromatography with fluorimetric detection. The performance of the assay was validated on six normal volunteers after a 4-mg oral dose of albuterol, which gave a peak plasma concentration in the range 6.67-15.31 ng/ml at 3-4 h after the dose. Plasma levels (0.79-1.56 ng/ml) of albuterol could be detected up to 24 h after the dose.