Inguinal hernia repair: results using an open preperitoneal approach.

BACKGROUND: Laparoscopic surgical approaches to the repair of inguinal hernias have shown the advantages of placing mesh in the preperitoneal space. Despite those advantages, laparoscopic hernia repairs have been associated with increased cost, longer operating times, and advanced laparoscopic skills. An open preperitoneal approach has the benefit of mesh in the preperitoneal position without the disadvantages of a laparoscopic procedure. METHODS: We present our experience with the use of an open preperitoneal mesh repair (KugelMesh, Bard, Inc.). The study was conducted in a prospective fashion from January 1998 through October 2001. 1072 hernias were repaired in two community hospitals by three general surgeons. Patients with recurrent hernias were excluded if the initial repair was from a preperitoneal approach. Operative time, cost, post-operative pain, and complications were analyzed. RESULTS: Recurrences occurred in five patients (0.47%) during a mean follow-up time of 23 months (range: 2-47). The average operating time was 32.4 min (range: 16-62). Post-operative narcotic pain medication usage averaged 5.8 pills (range: 0-26) per repair. Average surgical charges were less for the open preperitoneal approach ($2253) than for laparoscopic repairs ($4826). CONCLUSIONS: The open preperitoneal hernia repair using the Kugel mesh offers many advantages. It is inexpensive, has a low recurrence rate, and allows the surgeon to cover all potential defects with one piece of mesh. Postoperative recovery is short and postoperative pain is minimal.

Nursing interventions in the Kingston Paediatric and Perinatal HIV/AIDS Programme in Jamaica

BACKGROUND: Nursing care has been the [quot]grass roots[quot] of healthcare management even before nursing became a profession. Literature on the nursing experience with HIV is minimal and so it is challenging to comment on, or to compare experiences. PURPOSE: This paper highlights the nursing interventions as a key feature in the ongoing development and success of a prevention of mother-to-child HIV transmission (pMTCT) programme in a resource-limited setting. METHOD: In the Kingston Paediatric and Perinatal HIV/AIDS Programme, the nurses and midwives were carefully selected and then trained in the management of preventing mother-to-child transmission (pMTCT) of HIV/AIDS, voluntary counselling and testing and the identification and nursing management of paediatric and perinatal HIV/AIDS. The sites of the programme included three large maternity centres and four paediatric centres, with several feeder clinics for pregnant women. A nurse coordinator supervised the interventions at each site. A multidisciplinary team followed protocol-driven management for the care of pregnant HIV-positive women and children. There was strong collaboration with the Jamaican government and other agencies. RESULTS: The nursing interventions served to: sensitize and encourage other healthcare workers in the care of persons living with HIV/AIDS; sensitize persons in the community about the disease; improve the comfort level of women and families with accessing healthcare; enable prospective data collection for programme assessment and research purposes and to enhance multidisciplinary collaboration to widen the scope of patient care and prevent duplication of healthcare services. CONCLUSION: Nursing intervention is a vital part of a pMTCT HIV programme; however, ongoing education and training of the entire healthcare team needs to be continued in order to strengthen the programme. It is hoped that much of what is done in the Kingston Paediatric and Perinatal HIV/AIDS Programme will become integrated in the nursing management of maternal and child health nationally

Nursing interventions in the Kingston Paediatric and Perinatal HIV/AIDS Programme in Jamaica.

BACKGROUND: Nursing care has been the "grass roots" of healthcare management even before nursing became a profession. Literature on the nursing experience with HIV is minimal and so it is challenging to comment on, or to compare experiences. PURPOSE: This paper highlights the nursing interventions as a key feature in the ongoing development and success of a prevention of mother-to-child HIV transmission (pMTCT) programme in a resource-limited setting. METHOD: In the Kingston Paediatric and Perinatal HIV/AIDS Programme, the nurses and midwives were carefully selected and then trained in the management of preventing mother-to-child transmission (pMTCT) of HIV/AIDS, voluntary counselling and testing and the identification and nursing management of paediatric and perinatal HIV/AIDS. The sites of the programme included three large maternity centres and four paediatric centres, with several feeder clinics for pregnant women. A nurse coordinator supervised the interventions at each site. A multidisciplinary team followed protocol-driven management for the care of pregnant HIV-positive women and children. There was strong collaboration with the Jamaican government and other agencies. RESULTS: The nursing interventions served to: sensitize and encourage other healthcare workers in the care of persons living with HIV/AIDS; sensitize persons in the community about the disease; improve the comfort level of women and families with accessing healthcare; enable prospective data collection for programme assessment and research purposes and to enhance multidisciplinary collaboration to widen the scope of patient care and prevent duplication of healthcare services. CONCLUSION: Nursing intervention is a vital part of a pMTCT HIV programme; however, ongoing education and training of the entire healthcare team needs to be continued in order to strengthen the programme. It is hoped that much of what is done in the Kingston Paediatric and Perinatal HIV/AIDS Programme will become integrated in the nursing management of maternal and child health nationally.

Volvulus of the gallbladder: laparoscopic detorsion and removal.

A 73-year-old woman who presented with symptoms of acute cholecystitis was found to have a gangrenous gallbladder wrapped in three complete rotations around its pedicle. Detorsion and removal of the gallbladder were accomplished laparoscopically. Our review of the literature found no other case in which this degree of torsion was successfully treated laparoscopically.

Novel use of an established agent: Topotecan is anti-angiogenic in experimental Wilms tumor.

BACKGROUND/PURPOSE: Antiangiogenic agents offer a new approach to the treatment of aggressive neoplasms, yet very few agents are available for current use. The authors have shown previously the efficacy of antiangiogenic therapy in experimental Wilms tumor, using an investigative antibody. They hypothesized that topotecan, administered in a regimen targeting endothelial cells, would suppress tumor growth and angiogenesis in experimental Wilms tumor. METHODS: Experimental tumors were induced in the left kidneys of athymic mice by injection of cultured Wilms tumor cells. Topotecan (0.36, 0.6, 1.0, 2.0, and 3.0 mg/kg) or vehicle was injected intraperitoneally in 2 cycles over a 6-week period. Fluorescein angiograms and platelet endothelial cell adhesion molecule-1 staining of primary tumors were performed to ascertain vascular architecture. Endothelial apoptosis was assessed by TdT-mediated dUTP nick end labeling assay. RESULTS: Tumor weights were reduced significantly in treated versus control animals, even in the lowest-dose group. Endothelial cell staining and angiography results showed relatively sparse vascularity in treated xenografts. Endothelial apoptosis was observed in treated but not control tumors. CONCLUSIONS: Topotecan, delivered in an "antiangiogenic" regimen, even at very low doses, significantly inhibited growth of experimental Wilms tumors. No adverse effects were noted at low doses. Thus, the established chemotherapy agent topotecan may be useful in a novel role: as antiangiogenic therapy. J Pediatr Surg 36:1781-1784.

Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPAR alpha (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPAR alpha and histidine in PPAR gamma, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism.

Chemical genomics is the name we have given to the analysis of gene function through use of small molecule chemical tools. Orphan nuclear receptors are ideally suited to this technique of functional analysis, since their activity as transcription factors is regulated by small hydrophobic ligands. GW4064 is a potent and selective nonsteroidal ligand for the nuclear bile acid receptor FXR (NR1H4). Using GW4064 as a chemical tool, we have identified genes regulated by FXR in the liver, including those involved in bile acid synthesis and transport. We have also discovered that PXR (NR1I2) is a lithocholic acid receptor that controls the biosynthesis and metabolism of bile acids. Together FXR and PXR cooperate to control biliary and urinary bile acid excretion. These functions suggest that potent PXR and FXR ligands may offer a new approach to the treatment of cholestatic liver disease.

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes.

BACKGROUND: The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily. RESULTS: Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 gamma-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily. CONCLUSIONS: This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.

Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening.

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1). Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.

Combination antiangiogenic therapy: increased efficacy in a murine model of Wilms tumor.

BACKGROUND/PURPOSE: Antibody to vascular endothelial growth factor (anti-VEGF) suppresses tumor growth and metastasis in experimental Wilms tumor. However, tumor growth accelerates if antibody is withdrawn. As recently shown, low-dose, frequently administered topotecan, a topoisomerase-1 inhibitor, has anti-angiogenic activity. The authors hypothesized that combined topotecan/anti-VEGF therapy would suppress tumor growth and metastasis more durably than either agent alone. METHODS: Xenografts were induced by intrarenal injection of human Wilms tumor cells in athymic mice (n = 59). Mice were divided into control (n = 10), anti-VEGF (n = 16), topotecan (n = 17), and topotecan plus anti-VEGF (n = 16) groups. All control and half the treated mice were killed at week 6. Remaining ("rebound") mice were maintained without treatment until week 8. Tumor vasculature was mapped by fluorescein angiography/PECAM immunostaining. Endothelial apoptosis was assessed by TUNEL assay. RESULTS: 6 weeks: Tumor weights were reduced significantly in treated mice (P <.003 v control). Seven of ten control and 1 of 25 treated mice displayed lung metastases (P <.003). Rebound tumors were largest in topotecan-only, intermediate in antibody-treated, and smallest in combination-treated mice. Immunostaining and angiography results showed sparse vascularity in treated xenografts. Endothelial apoptosis was observed only in treated tumors. CONCLUSION: Combination low-dose topotecan and anti-VEGF antibody therapy is antiangiogenic and suppresses tumor growth and metastasis in experimental Wilms tumor more durably than either agent alone.

Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

Use of the nuclear receptor PXR to predict drug interactions.

We recently cloned the human, rabbit, rat, and mouse orthologs of a novel member of the steroid/retinoid/thyroid hormone receptor family, which we have named the Pregnane X Receptor (PXRs). The discovery and characterization of PXR has led to an increased understanding of the molecular basis of many drug-drug interactions as well as a better understanding of xenobiotic metabolism in general. The key insights into PXR action was the finding that this nuclear receptor is linked to regulation of the cytochrome P450 3A monooxygenase (CYP3A) genes. Several lines of evidence indicate that PXR mediates the induction of CYP3A gene transcription. First, PXR is selectively expressed in the liver and intestine, the same tissues in which CYP3A gene expression is induced. Second, PXR binds as a heterodimer with the retinoid X receptor (RXR) to xenobiotic response elements that have been identified in CYP3A gene promoters. Third, PXR is activated by the remarkable array of compounds that are known to induce CYP3A gene transcription. And finally, PXRs from different species are differentially activated by certain compounds such as rifampicin and pregnenolone 16alpha-carbonitrile (PCN) in a manner that correlates with species-specific induction of CYP3A gene expression. We are now employing high throughput PXR activation and binding assays to identify drug candidates that induce CYP3A gene expression so that these compounds can be removed from the drug development process.

Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail.

BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.

Identification of the human Mnk2 gene (MKNK2) through protein interaction with estrogen receptor beta.

We have identified and characterized the human Mnk2 gene (HGMW-approved gene symbol MKNK2) through a yeast two-hybrid screen in which the Mnk2 protein interacted with the ligand-binding domain of estrogen receptor beta (ERbeta). Human Mnk2 is homologous to murine Mnk2 ( approximately 94% identical) and human Mnk1 (71% identical), both of which encode MAP kinase interacting kinases that are phosphorylated and activated by ERK1 and 2. This report presents a thorough genomic sequence analysis revealing that the human Mnk2 gene has two C-terminal splice variants, designated here as Mnk2a and Mnk2b. These two isoforms are identical over the first 385 amino acids of the coding sequence and differ only in the final exon which encodes an additional 80 residues for Mnk2a and 29 residues for Mnk2b. A more detailed biological analysis in yeast showed that the Mnk2 interaction was selective for ERbeta as opposed to ERalpha and that the interaction was specific to Mnk2b as opposed to Mnk2a or Mnk1. This pattern was reproduced in a mammalian two-hybrid system using a completely different set of fusion partners; and in both yeast and mammalian systems, the addition of estradiol decreased the interaction. While it remains unknown whether ERbeta is a substrate of Mnk2, the interaction of these two proteins is reminiscent of ERalpha and ribosomal S6 kinase (p90-RSK), another MAP kinase-regulated kinase homologous to Mnk2 that is known to phosphorylate ERalpha.

The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution.

Transcription of genes encoding cytochrome P450 3A (CYP3A) monooxygenases is induced by a variety of xenobiotics and natural steroids. There are marked differences in the compounds that induce CYP3A gene expression between species. Recently, the mouse and human pregnane X receptor (PXR) were shown to be activated by compounds that induce CYP3A expression. However, most studies of CYP3A regulation have been performed using rabbit and rat hepatocytes. Here, we report the cloning and characterization of PXR from these two species. PXR is remarkably divergent between species, with the rabbit, rat, and human receptors sharing only approximately 80% amino acid identity in their ligand-binding domains. This sequence divergence is reflected by marked pharmacological differences in PXR activation profiles. For example, the macrolide antibiotic rifampicin, the antidiabetic drug troglitazone, and the hypocholesterolemic drug SR12813 are efficacious activators of the human and rabbit PXR but have little activity on the rat and mouse PXR. Conversely, pregnane 16alpha-carbonitrile is a more potent activator of the rat and mouse PXR than the human and rabbit receptor. The activities of xenobiotics in PXR activation assays correlate well with their ability to induce CYP3A expression in primary hepatocytes. Through the use of a novel scintillation proximity binding assay, we demonstrate that many of the compounds that induce CYP3A expression bind directly to human PXR. These data establish PXR as a promiscuous xenobiotic receptor that has diverged during evolution.

Production and characterization of an estrogen receptor beta subtype-specific mouse monoclonal antibody.

An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expression pattern of each of these receptors. We report the generation and characterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is ERbeta subtype-specific and capable of recognizing full-length human ERbeta as well as all of its known protein isoforms. ER15.64A, raised against a ERbeta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to the immunizing peptide and the full-length E. coli expressed ERbeta in ELISA and BIAcore assays. It also immunostained nuclei of Sf9 insect cells that were infected with an ERbeta-baculovirus. In Western analysis, ER15.64A recognized ERbeta1 and ERbeta2 proteins from a reticulocyte in vitro transcription/translation preparation. This antibody did not cross-react with recombinant ERalpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis. The specificity of ER15.64A should make this antibody a useful tool for monitoring expression of ERbeta and its isoforms at the protein level and should aid in distinguishing the pattern of ERbeta receptor expression from that of ERalpha.

Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer.

It has been shown in previous studies that a variety of estrogen receptor (ER) beta mRNA transcripts are expressed in human breast cancer cell lines and tumors. To complement the RNA expression studies, we have developed ER-beta-specific antibodies to characterize ER-beta protein expression in breast cancer cell lines and tumors. Monoclonal antibodies were made against a peptide representing the first 18 amino acids of the longest ER-beta open reading frame reported to date, and polyclonal antibodies were made against a peptide within the ER-beta B domain. By Western blot analysis, we show that ER-beta protein is expressed in all cancer cell lines tested and in three of five breast tumor samples. The breast cancer cell lines showed variation in the size of the expressed ER-beta protein. The longest form detected was consistent with the 530-amino acid, full-length ER-beta sequence. Shorter ER-beta isoforms were detected in the ER-alpha-negative MDA-MB-231 and MDA-MB-435 breast cancer cell lines, likely corresponding to previously described COOH-terminal RNA variant isoforms.

Human influenza: viral mutations and altered tropisms.

Influenza is a virus that is capable of causing a pandemic of the human race. Influenza has the ability to infect humans by mutating and altering its pathogenic characteristics. Efforts must be made worldwide to educate people about the possibilities of a potential outbreak. Awareness of optimal conditions which could lead to viral mutation and human to human transmission of a neogenetic strain of influenza appears to be a key deterrent against future cases.

The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.

The cytochrome P-450 monooxygenase 3A4 (CYP3A4) is responsible for the oxidative metabolism of a wide variety of xenobiotics including an estimated 60% of all clinically used drugs. Although expression of the CYP3A4 gene is known to be induced in response to a variety of compounds, the mechanism underlying this induction, which represents a basis for drug interactions in patients, has remained unclear. We report the identification of a human (h) orphan nuclear receptor, termed the pregnane X receptor (PXR), that binds to a response element in the CYP3A4 promoter and is activated by a range of drugs known to induce CYP3A4 expression. Comparison of hPXR with the recently cloned mouse PXR reveals marked differences in their activation by certain drugs, which may account in part for the species-specific effects of compounds on CYP3A gene expression. These findings provide a molecular explanation for the ability of disparate chemicals to induce CYP3A4 levels and, furthermore, provide a basis for developing in vitro assays to aid in predicting whether drugs will interact in humans.

Cloning and characterization of human estrogen receptor beta isoforms.

Multiple transcripts which arise from the human estrogen receptor beta (ER beta) gene have been characterized. Three full length isoforms of the hER beta gene, designated hER beta 1-3, were identified in a testis cDNA library. An additional two isoforms, designated hER beta 4 and hER beta 5, were identified by PCR amplification from testis cDNA and from the MDA-MB 435 cell line. hER beta 1 corresponds to the previously described hER beta. All five isoforms diverge at a common position within the predicted helix 10 of the ligand binding domain of hER beta, with nucleotide sequences consistent with differential exon usage. The hER beta isoform mRNAs displayed a differential pattern of expression in human tissues and in tumor cell lines when analyzed by RT-PCR. Further characterization of the three full length isoforms, hER beta 1-3, by in vitro band shift studies indicated that the isoforms were able to form DNA-binding homodimers and heterodimers with each other and with the ER alpha subtype.