bluuebunny/crossref_metadata_2025 huggingface dataset:
SHP-2 phosphatase is required for hematopoietic cell transformation by Bcr-Abl
Authors: Chen Jing , Yu Wen-Mei , Daino Hanako , Broxmeyer Hal E. , Druker Brian J. , Qu Cheng-Kui
Published: September 2006

SHP-2 phosphatase forms a stable protein complex with and is heavily tyrosine-phosphorylated by the oncogenic tyrosine kinase Bcr-Abl. However, the role of SHP-2 in Bcr-Abl–mediated leukemogenesis is unclear. In the present report, we provide evidence that SHP-2 is required for hematopoietic cell transformation by Bcr-Abl. In vitro biological effects of Bcr-Abl transduction were diminished in SHP-2Δ/Δ hematopoietic cells, and the leukemic potential of Bcr-Abl–transduced SHP-2Δ/Δ cells in recipient animals was compromised. Further analyses showed that Bcr-Abl protein (p210) was degraded, and its oncogenic signaling was greatly decreased in SHP-2Δ/Δ cells. Treatment with proteasome inhibitors or reintroduction of SHP-2 restored p210 level in Bcr-Abl–transduced SHP-2Δ/Δ cells. Subsequent investigation revealed that SHP-2 interacted with heat shock protein 90, an important chaperone protein protecting p210 from proteasome-mediated degradation. The role of SHP-2 in the stability of p210 is independent of its catalytic activity. Blockade of SHP-2 expression in p210-expressing cells by antisense or small-interfering RNA approaches decreased p210 level, causing cell death. Inhibition of SHP-2 enzymatic activity by overexpression of catalytically inactive SHP-2 mutant did not destabilize p210 but enhanced serum starvation-induced apoptosis, suggesting that SHP-2 also plays an important role in downstream signaling of p210 kinase. These studies identified a novel function of SHP-2 and suggest that SHP-2 might be a useful target for controlling Bcr-Abl–positive leukemias.
Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia
Authors: Byrd John C. , Lin Thomas S. , Dalton James T. , Wu Di , Phelps Mitch A. , Fischer Beth , Moran Mollie , Blum Kristie A. , Rovin Brad , Brooker-McEldowney Michelle , Broering Sarah , Schaaf Larry J. , Johnson Amy J. , Lucas David M. , Heerema Nyla A. , Lozanski Gerard , Young Donn C. , Suarez Jose-Ramon , Colevas A. Dimitrios , Grever Michael R.
Published: September 2006

Despite promising preclinical studies with the cyclin-dependent kinase inhibitor flavopiridol in chronic lymphocytic leukemia (CLL) and other diseases, previous clinical trials with this agent have been disappointing. The discovery of differential protein binding of flavopiridol in human and bovine serum contributed to an effective pharmacokinetic-derived schedule of administration of this agent. On the basis of pharmacokinetic modeling using our in vitro results and data from a previous trial, we initiated a phase 1 study using a 30-minute loading dose followed by 4 hours of infusion administered weekly for 4 of 6 weeks in patients with refractory CLL. A group of 42 patients were enrolled on 3 cohorts (cohort 1, 30 mg/m2 loading dose followed by 30 mg/m2 4-hour infusion; cohort 2, 40 mg/m2 loading dose followed by 40 mg/m2 4-hour infusion; and cohort 3, cohort 1 dose for treatments 1 to 4, then a 30 mg/m2 loading dose followed by a 50 mg/m2 4-hour infusion). The dose-limiting toxicity using this novel schedule was hyperacute tumor lysis syndrome. Aggressive prophylaxis and exclusion of patients with leukocyte counts greater than 200 × 109/L have made this drug safe to administer at the cohort 3 dose. Of the 42 patients treated, 19 (45%) achieved a partial response with a median response duration that exceeds 12 months. Responses were noted in patients with genetically high-risk disease, including 5 (42%) of 12 patients with del(17p13.1) and 13 (72%) of 18 patients with del(11q22.3). Flavopiridol administered using this novel schedule has significant clinical activity in refractory CLL. Patients with bulky disease and high-risk genetic features have achieved durable responses, thereby justifying further study of flavopiridol in CLL and other diseases.
Paucity of CD4+CCR5+ T cells is a typical feature of natural SIV hosts
Authors: Pandrea Ivona , Apetrei Cristian , Gordon Shari , Barbercheck Joseph , Dufour Jason , Bohm Rudolf , Sumpter Beth , Roques Pierre , Marx Preston A. , Hirsch Vanessa M. , Kaur Amitinder , Lackner Andrew A. , Veazey Ronald S. , Silvestri Guido
Published: September 2006

In contrast to lentiviral infections of humans and macaques, simian immunodeficiency virus (SIV) infection of natural hosts is nonpathogenic despite high levels of viral replication. However, the mechanisms underlying this absence of disease are unknown. Here we report that natural hosts for SIV infection express remarkably low levels of CCR5 on CD4+ T cells isolated from blood, lymph nodes, and mucosal tissues. Given that this immunologic feature is found in 5 different species of natural SIV hosts (sooty mangabeys, African green monkeys, mandrills, sun-tailed monkeys, and chimpanzees) but is absent in 5 nonnatural/recent hosts (humans, rhesus, pigtail, cynomolgus macaques, and baboons), it may represent a key feature of the coevolution between the virus and its natural hosts that led to a nonpathogenic infection. Beneficial effects of low CCR5 expression on CD4+ T cells may include the reduction of target cells for viral replication and a decreased homing of activated CD4+ T cells to inflamed tissue.
In vitro–expanded donor alloantigen–specific CD4+CD25+ regulatory T cells promote experimental transplantation tolerance
Authors: Golshayan Dela , Jiang Shuiping , Tsang Julia , Garin Marina I. , Mottet Christian , Lechler Robert I.
Published: September 2006

CD4+CD25+ regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25− effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro–generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.
Characterization of a progenitor cell population in childhood T-cell acute lymphoblastic leukemia
Authors: Cox Charlotte V. , Martin Hannah M. , Kearns Pamela R. , Virgo Paul , Evely Roger S. , Blair Allison
Published: September 2006

A significant proportion of children with T-cell acute lymphoblastic leukemia (T-ALL) continue to fail therapy. Consequently, characterization of the cells that proliferate to maintain the disease should provide valuable information on the most relevant therapeutic targets. We have used in vitro suspension culture (SC) and nonobese diabetic–severe combined immune deficient (NOD/SCID) mouse assays to phenotypically characterize and purify T-ALL progenitor cells. Cells from 13 pediatric cases were maintained in vitro for at least 4 weeks and expanded in 8 cases. To characterize the progenitors, cells were sorted for expression of CD34 and CD4 or CD7 and the subfractions were evaluated in vitro and in vivo. The majority of cells capable of long-term proliferation in vitro were derived from the CD34+/CD4− and CD34+/CD7− subfractions. Moreover, the CD34+/CD4− or CD7− cells were the only subfractions capable of NOD/SCID engraftment. These T-ALL cells successfully repopulated secondary and tertiary recipients with equivalent levels of engraftment, demonstrating self-renewal ability. The immunophenotype and genotype of the original leukemia cells were preserved with serial passage in the NOD/SCID mice. These data demonstrate the long-term repopulating ability of the CD34+/CD4− and CD34+/CD7− subfractions in T-ALL and suggest that a cell with a more primitive phenotype was the target for leukemic transformation in these cases.
Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias
Authors: Jeeninga Rienk E , Jan Barbara , van den Berg Henk , Berkhout Ben
Published: September 2006

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3) and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.
Comparative gene expression by WC1+ γδ and CD4+ αβ T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ γδ T cells
Authors: Lahmers Kevin K , Hedges Jodi F , Jutila Mark A , Deng Mingqi , Abrahamsen Mitchell S , Brown Wendy C
Published: September 2006

The functions of γδ T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized αβ T cells. However, their conservation throughout evolution suggests that γδ T cells are biologically unique. In ruminants, γδ T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from αβ T cells. In fact, bovine WC1+ γδ T cells can act as APC for αβ T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1+ γδ and CD4+ αβ T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific αβ and γδ T cell clones express many of the same genes, γδ T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1+ γδ T cells when compared with CD4+ αβ T cells selected from peripheral blood.
CYP11B2 −344T/C Gene Polymorphism and Blood Pressure in Patients with Acromegaly
Authors: Mulatero Paolo , Veglio Franco , Maffei Pietro , Bondanelli Marta , Bovio Silvia , Daffara Fulvia , Leotta Giannina , Angeli Alberto , Calvo Chiara , Martini Chiara , degli Uberti Ettore C. , Terzolo Massimo
Published: September 2006

Context: The pathogenesis of increased blood pressure (BP) in acromegaly is unclear, and the role of IGF-I levels and the renin-angiotensin-aldosterone system (RAAS) in this disease remains controversial.

Objective and Design: The aim of this study was to investigate the role of gene polymorphisms of the RAAS and involved in sodium handling on BP in acromegaly.

Setting and Patients: We conducted a multicentric retrospective study that included 100 consecutive patients with acromegaly referred during the period 2000–2003.

Intervention: All patients were genotyped for ACE I/D, AGT M235T, CYP11B2 −344T/C, B2R −58T/C, and α-adducin G460W polymorphisms.

Main Outcome Measure: We assessed the prevalence of hypertension and BP according to the genotype.

Results: Patients with the CYP11B2 −344CC genotype displayed a significant increase in the risk of hypertension compared with patients with CT/TT genotypes (odds ratio = 4.0; 95% confidence interval = 1.4–11.6; P = 0.01). Consistently, a significant proportion of patients with the CYP11B2 −344CC genotypes were under antihypertensive treatment (73.1%) compared with patients with the TT/TC genotypes (38.2%; P = 0.003). Patients with the −344CC genotype displayed a significant increase in systolic BP (10.2 ± 4.3 mm Hg; P = 0.02) but not a significant increase in diastolic BP (2.6 ± 2.6 mm Hg; P = 0.32) compared with patients with the CT/TT genotype.

Conclusions: We have shown an association of the −344T/C CYP11B2 gene polymorphism with BP in patients affected by acromegaly. These findings suggest that the RAAS is implicated in the pathogenesis of hypertension in acromegaly.
Sleep-Related Breathing Disorders in Prepubertal Children with Prader-Willi Syndrome and Effects of Growth Hormone Treatment
Authors: Festen D. A. M. , de Weerd A. W. , van den Bossche R. A. S. , Joosten K. , Hoeve H. , Hokken-Koelega A. C. S.
Published: September 2006

Context: Recently, several cases of sudden death in GH-treated and non-GH-treated, mainly young Prader-Willi syndrome (PWS), patients were reported. GH treatment in PWS results in a remarkable growth response and an improvement of body composition and muscle strength. Data concerning effects on respiratory parameters, are however, limited.

Objective: The objective of the study was to evaluate effects of GH on respiratory parameters in prepubertal PWS children.

Design: Polysomnography was performed before GH in 53 children and repeated after 6 months of GH treatment in 35 of them.

Patients: Fifty-three prepubertal PWS children (30 boys), with median (interquartile range) age of 5.4 (2.1–7.2) yr and body mass index of +1.0 sd score (−0.1–1.7).

Intervention: Intervention included treatment with GH 1 mg/m2·d.

Results: Apnea hypopnea index (AHI) was 5.1 per hour (2.8–8.7) (normal 0–1 per hour). Of these, 2.8 per hour (1.5–5.4) were central apneas and the rest mainly hypopneas. Duration of apneas was 15.0 sec (13.0–28.0). AHI did not correlate with age and body mass index, but central apneas decreased with age (r = −0.34, P = 0.01). During 6 months of GH treatment, AHI did not significantly change from 4.8 (2.6–7.9) at baseline to 4.0 (2.7–6.2; P = 0.36). One patient died unexpectedly during a mild upper respiratory tract infection, although he had a nearly normal polysomnography.

Conclusions: PWS children have a high AHI, mainly due to central apneas. Six months of GH treatment does not aggravate the sleep-related breathing disorders in young PWS children. Our study also shows that monitoring during upper respiratory tract infection in PWS children should be considered.
Insulinomas May Present with Normoglycemia after Prolonged Fasting but Glucose-Stimulated Hypoglycemia
Authors: Kar Partha , Price Paul , Sawers Stewart , Bhattacharya Satya , Reznek Rodney H. , Grossman Ashley B.
Published: September 2006

Background: Insulinomas are rare but are the most common cause of hyperinsulinemic hypoglycemia in the adult population. Diagnosis of this pathology relies on clinical features along with laboratory tests and imaging investigations to aid in localization. One of the most robust standard tests used for establishing a biochemical diagnosis is the prolonged (72 h) fast. Currently, it is recommended that a prolonged supervised fast be performed, at least for 48 h if not for 72 h, and many would take the absence of hypoglycemia after a 72-h fast as evidence excluding the diagnosis.

Methods: We employed prolonged fasts and standard glucose tolerance tests, plus imaging studies and surgical pathology, in two patients with suspected insulinomas.

Results: The prolonged 72-h fast was normal in both the patients, whereas in both cases a prolonged oral glucose tolerance test clearly demonstrated the induction of severe hyperinsulinemia followed by significant hypoglycemia. Surgical removal confirmed the presence of insulinomas in each case.

Conclusions: Although the sensitivity of the 72-h fast is high and still plays an important role in the diagnosis of an insulinoma, we suggest that a “normal” test result should be interpreted in the light of clinical symptoms.
Induction of Surfactant Protein A Expression by Cortisol Facilitates Prostaglandin Synthesis in Human Chorionic Trophoblasts
Authors: Sun Kang , Brockman Diane , Campos Begona , Pitzer Brad , Myatt Leslie
Published: September 2006

Context: Surfactant protein A (SP-A) may be an important link between the maturation of fetal organs and the initiation of parturition. However, the local expression of SP-A and the effect of SP-A on prostaglandin synthesis in human fetal membranes have not been resolved.

Objective: Our objective was to examine SP-A expression and the effect of SP-A on prostaglandin synthesis in human fetal membranes.

Design: SP-A expression was examined with immunohistochemistry and PCR. The effect of SP-A on prostaglandin synthesis was investigated in cultured human chorionic trophoblasts.

Patients: Patients were normal-term pregnant women undergoing elective cesarean sections.

Results: Both SP-A protein and mRNA were present in amnion epithelial cells, fibroblasts, and chorionic trophoblasts. Cortisol (10−7 and 10−6m, 24 h) induced SP-A expression in cultured chorionic trophoblasts, which could be blocked by the glucocorticoid receptor antagonist RU486. Treatment of chorionic trophoblasts with SP-A (10–100 μg/ml, 24 h) caused a dose-dependent increase of prostaglandin E2 release and an induction of cyclooxygenase type 2 but not cytosolic phospholipase A2 and microsomal prostaglandin E synthase expression.

Conclusions: SP-A can be synthesized locally in human fetal membranes, which can be induced by glucocorticoids. SP-A appeared to induce prostaglandin E2 synthesis in chorionic trophoblasts via induction of cyclooxygenase type 2 expression.
PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells
Authors: Afonso Cristina , Henrique Domingos
Published: September 2006

Neural progenitors in the vertebrate nervous system are fully polarized epithelial cells, with intercellular junctions at the apical region. These progenitor cells remain within the neuroepithelium throughout neurogenesis, and will ultimately give rise to all the neurons in the mature nervous system. We have addressed the role of the PAR polarity complex in vertebrate neuroepithelial polarity and show that PAR3 functions as the initial scaffold to assemble and organize the PAR complex at the apical region of neuroepithelial cells, coordinating also the recruitment of additional polarity complexes and junction-associated proteins to the same region, while restricting other polarity proteins to the basolateral membrane. We propose that PAR3 acts as a molecular organizer to connect the acquisition of apico-basal polarity with the positioning and formation of junctional structures in neuroepithelial cells, a function of upmost importance for the morphogenesis of embryonic neural tissue and the process of neurogenesis.
Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis
Authors: Karamboulas Christina , Dakubo Gabriel D. , Liu Jun , De Repentigny Yves , Yutzey Katherine , Wallace Valerie A. , Kothary Rashmi , Skerjanc Ilona S.
Published: September 2006

Myocyte enhancer factors (MEF2s) bind to muscle-specific promoters and activate transcription. Drosophila Mef2 is essential for Drosophila heart development, however, neither MEF2C nor MEF2B are essential for the early stages of murine cardiomyogenesis. Although Mef2c-null mice were defective in the later stages of heart morphogenesis, differentiation of cardiomyocytes still occurred. Since there are four isoforms of MEF2 factors (MEF2A, MEF2B, MEF2C and MEF2D), the ability of cells to differentiate may have been confounded by genetic redundancy. To eliminate this variable, the effect of a dominant-negative MEF2 mutant (MEF2C/EnR) during cardiomyogenesis was examined in transgenic mice and P19 cells. Targeting the expression of MEF2C/EnR to cardiomyoblasts using an Nkx2-5 enhancer in the P19 system resulted in the loss of both cardiomyocyte development and the expression of GATA4, BMP4, Nkx2-5 and MEF2C. In transiently transgenic mice, MEF2C/EnR expression resulted in embryos that lacked heart structures and exhibited defective differentiation. Our results show that MEF2C, or genes containing MEF2 DNA-binding sites, is required for the efficient differentiation of cardiomyoblasts into cardiomyocytes, suggesting conservation in the role of MEF2 from Drosophila to mammals.
CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage
Authors: Schmitt Estelle , Boutros Rose , Froment Carine , Monsarrat Bernard , Ducommun Bernard , Dozier Christine
Published: September 2006

CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1–cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1–cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome.
Thermal degradation of polyethylene film materials due to successive recycling
Authors: Marsh R , Griffiths A J , Williams K P , Evans S L
Published: September 2006

Mechanical recycling of plastic film involves subjecting plastic materials to a series of heat cycles that can potentially degrade the material, causing brittleness and increased melt viscosity. Plastic film recycling in the UK is in its infancy, in need of an increased understanding of how the physical properties of polymeric materials change before and during the process. Reliable data are required to estimate the behaviour of such film products when recycled. Measurements were made as to the changes in physical properties of four different varieties of polyethylene (PE) film products when subjected to a series of successive simulated heat cycles and evaluated after each step. Results showed that although changes in tensile properties were fairly small, changes in processing properties such as melt-flow index for highly branched or low-density PE are substantial and could be a concern during recycling operations.
Creep failure behaviour of a circumferential P91 pipe weldment with an anisotropic weld metal subjected to internal pressure and end load
Authors: Hyde T H , Sun W
Published: September 2006

The creep failure behaviour of a P91 pipe weldment that has an ‘anisotropic’ weld metal (WM), under internal pressure and end load, was investigated using the finite-element (FE) method. It is assumed that the WM consists of a columnar region and a weaker equiaxed (fine-grained and coarse-grained) region. A practical pipe weld and bead dimensions were used for the FE model. Creep damage analyses were conducted using a single-variable, Kachanov type damage law, for which the material properties used were determined on the basis of the experimental data. Life prediction was also performed using a steady-state approach. Three representative cases, i.e. ‘matched’, ‘under-matched’, and ‘over-matched’, with respect to WM strength, were considered. Failure lives and failure positions of the weldment, for various cases, were investigated. The results were compared with those obtained from the corresponding weldment with an isotropic WM, from which the effects of the WM anisotropy on the failure life and failure location can be evaluated.
Synthesis and Application of Fluorous-tagged Oligonucleotides
Authors: Mishra Roli , Mishra Satyendra , Misra Krishna
Published: September 2006

Nucleoside phosphoramidites bearing a adsorbent tert-butylphenyl-1H,1H,2H,2H-heptafluorodecyloxysilyl (BPFOS) group were used to synthesize fluorous-tagged oligonucleotides, viz. 5-mer, 10-mer, 13-mer, 17-mer, and 19-mer, which were subjected to liquid-phase extraction. The labeled oligomers were found to be highly hydrophobic in nature, become precipitated in water, therefore allowing easy purification from the failure sequences. The silyl fluorous group was deprotected by TBAF treatment and oligomers were easily purified by CH3CN/FC-72 liquid–liquid extraction. Fluorous-tagged oligonucleotides have high selectivity for the removal of failure sequences, high recoveries (typically 70–100%).
Urine free cortisol analysis by automated immunoassay and high-performance liquid chromatography for the investigation of Cushing's syndrome
Authors: Ching Simon Y L , Lim Ee Mun , Beilby John , Bhagat Chotoo , Rossi Enrico , Walsh John P , Pullan Peter
Published: September 2006

Background: We compared a widely used immunoassay method for urine free corticoids with a high-performance liquid chromatography (HPLC) method for the investigation of Cushing's syndrome.

Methods: Urine free corticoid analysis using an Immulite:2000 automated immunoassay analyser and urine free cortisol analysis by a specific HPLC method on 39 patients with an elevated baseline immunoassay 24 h urine free corticoid result who were referred from 2000 to 2004.

Results: Reference ranges for urine free corticoids by immunoassay and for urine free cortisol by HPLC were <900 and <170 nmol/day, respectively. Of the 39 patients with an elevated immunoassay urine free corticoid result, Cushing's syndrome was surgically proven in 22 patients, giving a positive predictive value of 56%. Cushing's syndrome was excluded in 12 of the remaining 17 patients by applying the specific HPLC method and at least one of the following: a clinical evaluation which was not suggestive or adequate suppression of plasma cortisol following dexamethasone. Urine free cortisol analysis by HPLC gave a positive predictive value of 81% for Cushing's syndrome.

Conclusions: Although immunoassay has a low specificity for urine cortisol, it is a widely available test which serves as a valid initial investigation of Cushing's syndrome.
Evaluation of four portable self-monitoring blood glucose meters
Authors: Lippi Giuseppe , Salvagno Gian Luca , Guidi Gian Cesare , Negri Mauro , Rizzotti Paolo
Published: September 2006

Self-monitoring of blood glucose (SMBG) is a cornerstone of diabetes care. However, the effectiveness of any glucose-monitoring programme depends on the ability to integrate SMBG into a program of self-care and therapeutic decision-making. Because the accuracy of SMBG is instrument dependent, we analysed 45 heparinized whole blood specimens using four marketed portable glucose meters to evaluate whether their precision and accuracy would be efficient and safe for clinical use. All measurements were standardized and performed by a single expert health-care professional at the same clinical chemistry laboratory. Results were compared with those obtained on the same plasma samples by the hexokinase method on a secondary reference analyser and further analysed according to the error tolerance criteria and the current American Diabetes Association (ADA), Clinical Laboratory Improvement Amendments (CLIA), and National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The within-run imprecision ranged from 2.2% to 3.2%. Passing and Bablok regression analysis yielded slope values from 0.93 to 1.07 and correlation coefficients between 0.994 and 0.998. When compared with the secondary reference analyser, mean variations were between -4.9% and 14.1%, fulfilling in three out of four cases the 5.5% current desirable analytical quality specifications for total error. Nevertheless, when considering the two standard deviations level of this bias, several results exceeded this limit. Although three out of four devices tested achieved or came closer to the NCCLS C30-A2, CLIA and error tolerance targets, none of them met the current analytical ADA thresholds. Despite the acceptable analytical performances, we demonstrated that standardization and harmonization of results in SMBG have not been fully achieved.
Reflective testing - what do our service users think?
Authors: Darby Denise , Kelly Anne-Marie
Published: September 2006

Background: Reflective testing incorporates the clinical judgement, knowledge and experience of an individual biochemist to request additional tests appropriate to the clinical scenario. Despite being vigorously debated within the biochemistry profession, little is known about how the clinicians directly involved in patient care feel about it. We have conducted a survey to elicit our service users' opinion of reflective testing.

Methods: Ten clinical scenarios, each involving the possible addition of a specific test, were circulated to 520 hospital doctors and 152 general practitioners. The four response options were to add further tests, phone and discuss the case, add a comment to the original results or do nothing.

Results: A total of 216 (32%) responses were received. Overall, the majority were in favour of 'adding on' free triiodothyronine (86%), γ-glutamyltransferase (78%), lipid profile (59%), thyroid peroxidase antibodies (63%), pituitary hormones (58%), troponin (55%) and serum electrophoresis (68%) in the given scenarios. However, only 30% would like a pregnancy test added and only 45% a prostate-specific antigen added without prior consultation. The response differed according to grade and specialty.

Conclusions: Reflective testing is generally welcomed by our service users, provided the nature and implications of the specific test is considered in addition to the full clinical scenario.
The evaluation of a novel conductometric device for the diagnosis of cystic fibrosis
Authors: Losty Helen C , Wheatley Heather , Doull Iolo
Published: September 2006

Background: We evaluated the diagnostic discrimination of a new micro-flow cell device (Nanoduct®) which measures sweat conductivity in situ at a regional referral centre for cystic fibrosis (CF).

Methods: The device was evaluated in comparison to the measurement of sweat chloride with the established quantitative pilocarpine iontophoresis test (QPIT) and extended in a number of patients to conductivity measurements in liquid sweat collected with the Macroduct® system. Sweat testing was conducted simultaneously on patients referred for diagnostic sweat testing, on patients known to have CF and on adult volunteers. The intra-individual variability, the failure rate and diagnostic accuracy were determined.

Results: A total of 110 tests were performed on 100 individuals, 36 of whom had classical CF and six of whom had non-classical CF. The Nanoduct system produced a false negative result in one quarter of the patients with classical CF. Moreover, conductivity was negatively biased compared with chloride in this group. Repeat testing of the false negatives using a new batch of sensors and/or measuring conductivity in liquid sweat collected with the Macroduct device gave accurate diagnostic discrimination indicating that the original sensors were faulty. Photographic examination confirmed that a batch of sensors were defective.

Conclusions: Our experience suggests that the prototype microflow cell conductometric device cannot be used for the diagnosis of CF due to the high false negative rate. As a consequence of this study, the manufacturers have implemented a pre-testing system to quality control the sensors prior to issue.
Gilbert's syndrome: an overview for clinical biochemists
Authors: Hirschfield G M , Alexander G J
Published: September 2006

Gilbert's syndrome (GS) is a benign and inherited state characterized by mild, lifelong, unconjugated hyperbilirubinaemia in the absence of haemolysis or evidence of liver disease. Its molecular basis, mutations in the TATA box upstream of the uridine diphosphoglucose glucuronyltransferase gene, leads to impaired bilirubin glucuronidation. This synopsis outlines the pathophysiology and investigation appropriate for this innocent anomaly.
Acute intermittent porphyria associated with hyperthyroidism
Authors: Vithian Karvnakaran , Samat Ashish , Jones M Keston
Published: September 2006

The differential diagnosis of abdominal pain with associated hyponatraemia should include acute intermittent porphyria. Development of hyperthyroidism in a patient with latent porphyria may precipitate an acute attack and increase disease severity. Treatment of hyperthyroidism may prevent recurrent episodes.
Determining urinary calcium/creatinine cut-offs for the paediatric population using published data
Authors: Metz Michael P
Published: September 2006

Background: The purpose of this study was to determine a cut-off above which a result is considered abnormal for 'spot' calcium-to-creatinine ratio (Ca/Cr) in urine in the paediatric age group. While the cut-offs are not well established, there are many published reports of urine Ca/Cr excretion ratio in the literature. Rather than undertaking a difficult, time-consuming, expensive, and ultimately redundant study, an analysis of these published works was undertaken to determine urine Ca/Cr cut-offs for the laboratory.

Methods: Fourteen studies reporting urine Ca/Cr ratio available in the literature were reviewed. Each study was abstracted for several characteristics. The data were plotted and the line of best fit describing the data was determined. Using the function describing the line, cut-offs by pertinent age were recalculated. These cut-offs were then compared to the original data and to in-house data.

Results: The function describing the line of best fit was y = -0.3175 ln( x) +1.46 ( y = Ca/Cr in mmol/mmol and x = age in years), r2 = 0.85. After consultation with the paediatric nephrologists and comparison with our in-house data, cut-offs for age groups of <1, 1-<2, 2-<5, 5-<10, and 10-18 years in mmol/mmol of 1.50, 1.25, 1.00, 0.70, and 0.60, respectively, were derived.

Conclusions Using data from 14 published studies, suitable spot urine Ca/Cr cut-offs from birth to 18 years of age were determined.
Calculated free testosterone in men: comparison of four equations and with free androgen index
Authors: Ho Clement KM , Stoddart Mary , Walton Melanie , Anderson Richard A , Beckett Geoffrey J
Published: September 2006

Background: Serum testosterone remains the most important investigation in the diagnosis of androgen deficiency in men. Most of the circulating testosterone is bound to albumin and sex hormone-binding globulin (SHBG), whereas free testosterone accounts for approximately 2% of total testosterone. Because direct measurement of free testosterone is impractical in routine practice, several equations are used to provide clinically useful estimates of free testosterone concentration. This study aimed to (1) obtain locally derived reference limits for total testosterone and calculated free testosterone (CFT) concentrations, and (2) critically evaluate the equations commonly used to estimate free testosterone.

Methods: Serum total testosterone, SHBG and albumin were assayed in morning blood samples obtained from 126 healthy men (aged 20-45 years) known to have normal semen analysis. CFT concentrations calculated using four published methods (i.e. the Sodergard, Nanjee-Wheeler, Vermeulen and Ly-Handelsman equations) were compared with one another and the free androgen index.

Results: Reference intervals for total testosterone and CFT by the Vermeulen equation were 9.4-31.0 nmol/L and 0.245-0.785 nmol/L (2.5-97.5 percentile), respectively. CFT values varied considerably with the four equations examined. Mean biases ranged from 5.8 to 56.0%; the Nanjee-Wheeler and Ly-Handelsman equations yielded positive and negative biases, respectively, against the other equations. Free androgen index was shown to correlate poorly with CFT ( r2 = 0.21-0.46) and over-estimate the CFT at low SHBG concentrations.

Conclusions: We have used various equations to derive reference ranges for CFT in healthy men aged 20-45 years. We suggest that CFT be incorporated into the investigation regimen for suspected hypogonadism when total testosterone results are equivocal.
The measurement of whole blood pre-treatment cyclosporine A: metabolite ratios predicts the onset of renal dysfunction in recipients of allogeneic stem cell transplantation
Authors: Stephens E , Bolderson I , Clark B , Kinsey S , Gooi HC , Cook G
Published: September 2006

Background: In the context of post-transplant immunosuppression, cyclosporine A (CSA) is dose adjusted in accordance with whole blood drug monitoring. While currently available immunoassay systems primarily target the parent drug, cross-reactivity results in the detection of the major circulating CSA metabolites, though their contribution to both immunosuppression and toxicity remain unclear. This study examines the relationship of CSA metabolites to hepatic and renal dysfunction and the incidence of graft-versus-host disease (GvHD) through parallel assaying of parent drug and drug/metabolites expressed as a metabolite ratio (Cp:mR).

Method: Sequential pre-treatment (trough) whole blood samples ( n = 527) were collected from 31 allo-stem cell transplantation (SCT) recipients. Both parent drug and drug/metabolite levels were determined using the Abbott fluorescence polarization immunoassay.

Results: The average mean Cp:mR was significantly higher in patients with hepatic ( P = 0.004) and renal dysfunction ( P = 0.004) than in those without. Significantly higher Cp:mR were also found in patients with grades II-IV GvHD ( P = 0.001) than were observed in patients who did not experience significant GvHD. When measured prospectively, an increasing Cp:mR predated the rise in serum creatinine concentration by a median of two weeks.

Conclusions: This study demonstrates a clinically useful CSA metabolite ratio that shows association with hepatic and renal dysfunction and with GvHD. The measure can be used to predict those patients on CSA therapy who are likely to develop renal dysfunction.
Suppuration of the petrous pyramid
Authors: Bricker Sacks
Published: September 2006

This case must be considered one of suppuration of the petrous pyramid because of the following facts: 1. Following an acute mastoiditis, the development of intense deepseated pain behind the eye, severe pain over the right eye‐brow and the parietal region; 2. The sudden profuse discharge of pus from the mastoid wound and to some degree from the external auditory canal; 3. The period of low grade sepsis following the mastoidectomy, until the flare‐up of the temperature from the seventh to the tenth day after the first operation, when the inflammation in the petrous pyramid was at its height; 4. The extensively pneumatized development of the mastoid; 5. The roentgenologic findings showing positive evidence of bone destruction at the medial end of the right petron, and further evidence seven months later that there was some regeneration of the bone in this region; and 6. The abatement of the pain in the eye and the sudden improvement of the patient following the operation on his petrous pyramid.
Treatment of laryngeal carcinoma
Authors: Kramer Rudolph
Published: September 2006

This study of carcinoma of the larynx is an attempt to analyze for myself the results of treatment and to determine, if possible, any indications for the selection of the type of treatment most likely to be successful in any particular case. It is true that the number of cases in this series is relatively small and in some instances the time elapsed after treatment has been too short for clear‐cut conclusions. Nevertheless, some of the patients have been observed sufficiently long to evaluate the results of treatment. I am of the opinion that the general viewpoint of time elapsed after treatment of laryngeal cancer in relationship to cure is of little help in studying the results. The usual periods selected are three or five years after therapy. As a matter of fact, most recurrences, local or glandular, take place in much less than one year. In this series of cancer cases there has been no local or glandular recurrence after the first year, with one exception, which occurred 12 years postoperative. I do not for a moment deny that recurrences or metastases take place two, three or five years after operation, but they are comparatively infrequent. I am of the opinion that if patients are observed routinely by follow‐up every two or four weeks, as I do with all my patients, recurrences will be noted early; long before they fall into the two‐ or three‐year recurrence group. On the other hand, the three‐ or five‐year period is much too short because patients do occasionally get late local or distant metastases seven, 10 or 20 years after treatment. Therefore, reliable definitive statistics on the treatment of laryngeal carcinoma can only be obtained after many years of observation, a longer period of time than any one man is likely to cover in his active medical years. There are exceptions, such as Chevalier Jackson and St. Clair Thomson, who are able to report lengthy follow‐ups on their thyrotomy cases. It will require relays of laryngologists following the courses of therapeutic procedures for many years to estimate the value of each procedure if in the meantime the specific etiology and treatment of cancer are not discovered. For we must remember that we have not been treating carcinoma itself, but a local manifestation of a general disease or tendency. In the interim tentative reports such as this are in order so that a mass of material may be available for the study of various procedures and their indications in the treatment of laryngeal cancer.
The art of good tone‐production, with some helpful suggestions
Authors: Schatz Harry A.
Published: September 2006

Physicians in general, and especially otolaryngologists, should all be very much interested in voice‐production. The writer refers to correct voice‐production; i.e., good tone‐production. A good natural voice may be regarded as a “gift of God”; but the poor voice can be improved in the presence of a larynx that is normal in its anatomy and innervation, and therefore need not remain a lifelong affliction.
Tetanus associated with acute otitis media; probable atrium tetanus. Report of two cases
Authors: Stout Philip S.
Published: September 2006

Reporting two cases of tetanus occurring about the same time; coming to St. Luke's and Children's Hospital, Philadelphia, from northeastern Philadelphia. Both cases were boys under 12 years of age; one recovered, the other died about six hours after entering the hospital. Both were given the same amount of antitoxin. Both had acute otitis media a short time before the onset of the symptoms of tetanus. Neither of them had any scar, inflammation or history of any injury that might have caused this infection, except the acute otitis media. In one case, D. L., we were able to get a history of the enlarging of a cesspool in the yard of his home and that he played around the working men. He developed acute otitis media at this time.
Myxofibroma of the larynx. Report of two cases
Authors: Weinstein Samuel
Published: September 2006

Myxofibroma of the larynx occurs rarely enough to be reportable. Of the two cases reported in this paper, one is a private case of my own, and the other is from the service of Dr. Philip Leibowitz, Attending Otolaryngologist, Jewish Hospital, Brooklyn.
Placing the future in safe hands: review and future directions of legacy fundraising at the College
Authors: Louise Jary
Published: September 2006

Charitable bequests currently provide approximately a fifth of total voluntary income to the College and as such are a vitally important source of funding supporting a varied programme including education and research projects. These figures are in line with the UK charities average as estimated by the Charities Aid Foundation. Over the ten-year period from 1995 to 2005 the College has received approximately £14.4 million in legacy income, with an average annual income of £1.6 million.
The dying art of plastering: a survey of orthopaedic trainees' plastering experience
Authors: Sinha I , Barr L , Lee M
Published: September 2006

It is possible that some persons are more naturally equipped for acquiring manipulation skills than others, but it is unlikely that there is any special gift which cannot be conscientiously acquired by everyone.
The FGDP(UK)'s new dean sets out ambitions for the future
Authors: Hayward Richard
Published: September 2006

The Faculty of General Dental Practice (UK) is delighted to welcome Richard Hayward as its new dean. Having qualified in 1974, Richard spent 20 years in practice in the Navy, where he took advantage of postgraduate education opportunities. He was a founder member of the FGDP(UK) and is a long-standing Board member. Previously vice-dean in 1999 and 2005–2006, he has recently chaired the Faculty's examinations committee. In this article Richard reflects upon the achievements of the Faculty and sets out his visions for the future.
Recent progress in the synthesis of polymers based on carbon dioxide
Authors: Sugimoto Hiroshi , Inoue Shohei
Published: September 2006

The alternating copolymerization of carbon dioxide and epoxide to give polycarbonate has attracted the attention of many chemists, because it is one of the most promising methodologies for the utilization of carbon dioxide as a safe, clean, and abundant raw material in the synthetic chemistry. Recent developments of the catalysts for the alternating copolymerization are based on the rational design of metal complexes, especially complexes of transition metal with well-defined structures.