Prediction of clinically important acquired cardiac disease without an echocardiogram in large breed dogs using a combination of clinical, radiographic and electrocardiographic variables.

INTRODUCTION: Large breed (LB) dogs develop dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD). Echocardiography is required for a definitive diagnosis but is not always available. Our objective was to assess the clinical utility of thoracic radiographs alone and in combination with physical examination and electrocardiography findings for the prediction of clinically important DCM or MMVD in LB dogs. ANIMALS: Four hundred fifty-five client-owned dogs ≥20 kg with concurrent thoracic radiographs and echocardiogram. MATERIALS AND METHODS: Medical records were reviewed and stored thoracic radiographs and echocardiographic images were measured to classify dogs as normal heart size (NHS), preclinical DCM, clinical DCM, preclinical MMVD (with cardiomegaly), clinical MMVD, or equivocal. Dogs with preclinical MMVD, without cardiomegaly, were classified as NHS. Vertebral heart size (VHS) and vertebral left atrial size (VLAS) were measured. Receiver operating characteristic curves and prediction models were derived. RESULTS: Prevalence of MMVD (39.3%) was higher than the prevalence of DCM (24.8%), though most MMVD dogs (67.0%) lacked cardiomegaly and were classified as NHS for analysis. The area under the curve for VHS to discriminate between NHS and clinical DCM/MMVD or preclinical DCM/MMVD was 0.861 and 0.712, respectively, while for VLAS, it was 0.891 and 0.722, respectively. Predictive models incorporating physical examination and electrocardiography findings in addition to VHS/VLAS increased area under the curve to 0.978 (NHS vs. clinical DCM/MMVD) and 0.829 (NHS vs. preclinical DCM/MMVD). CONCLUSIONS: Thoracic radiographs were useful for predicting clinically important DCM or MMVD in LB dogs, with improved discriminatory ability when physical examination abnormalities and arrhythmias were accounted for.

T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses.

We have generated mice with a deficiency in T1/ST2 expression to clarify the roles of T1/ST2 in T helper cell type 2 (Th2) responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of T1/ST2-deficient mice with those generated by wild-type mice. Using a primary pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that granuloma formation, characterized by eosinophil infiltration, is abrogated in T1/ST2-deficient mice. Furthermore, we clearly demonstrate that in the absence of T1/ST2 expression, the levels of Th2 cytokine production are severely impaired after immunization. Thus, in a secondary pulmonary granuloma model, draining lymph node cells from the T1/ST2-deficient animals produced significantly reduced levels of IL-4 and IL-5, despite developing granulomas of a magnitude similar to those of wild-type mice and comparable antigen-specific immunoglobulin isotype production. These data clearly demonstrate that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence.

An assessment of the accuracy and usability of a novel optical wound measurement system.

AIMS: Measurement of wound size can predict healing and provide information to guide treatment. This study assesses a novel optical wound imaging system that creates a three-dimensional image of the ulcer. METHODS: Using a new camera-based digital system and traditional elliptical wound measurements, 36 foot ulcers from 31 patients (aged 44-94 years, median 70 years) were examined during a 12-week period at two centres. Median diabetes duration was 18 years (range 6-56 years). Seventeen percent had Type 1 diabetes, 93% had peripheral neuropathy and 57% had peripheral artery disease. Twenty-five were reviewed consecutively, resulting in 76 ulcer examinations. Median ulcer size was 94 mm(2), with size ranging from 3.1 to 2195 mm(2). RESULTS: Pearson, Spearman and Kendall rank coefficients showed a strong correlation (in all cases P < 0.001) between digital measurements of wounds against traditional hand-measured estimates. Intra-observer variation of wound length using digital elliptical measurement (DEM) gave a coefficient of variation of < 3.0%. Interobserver variation of wound length using DEM was < 6.5%. Variation from a standard known-size wound area was < 8.0% across 30 trials. CONCLUSIONS: This study shows a strong correlation between digital and traditional measurement techniques. The system can be easily deployed in routine clinical practice, providing an objective visual record, allowing remote in-depth analysis.

Substrate phage: selection of protease substrates by monovalent phage display.

A method is described here for identifying good protease substrates among approximately 10(7) possible sequences. A library of fusion proteins was constructed containing an amino-terminal domain used to bind to an affinity support, followed by a randomized protease substrate sequence and the carboxyl-terminal domain of M13 gene III. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). Phage were then bound to an affinity support and treated with the protease of interest. Phage with good protease substrates were released, whereas phage with substrates that resisted proteolysis remained bound. After several rounds of binding, proteolysis, and phagemid propagation, sensitive and resistant substrate sequences were identified for two different proteases, a variant of subtilisin and factor Xa. The technique may also be useful for studying the sequence specificity of a variety of posttranslational modifications.

Five-year prospective clinical and radiological results of a new cannulated cemented polished Tri-Taper femoral stem.

We describe the results at five years of a prospective study of a new tri-tapered polished, cannulated, cemented femoral stem implanted in 51 patients (54 hips) with osteoarthritis. The mean age and body mass index of the patients was 74 years and 27.9, respectively. Using the anterolateral approach, half of the stems were implanted by a consultant orthopaedic surgeon and half by six different registrars. There were three withdrawals from the study because of psychiatric illness, a deep infection and a recurrent dislocation. Five deaths occurred prior to five-year follow-up and one patient withdrew from clinical review. In the remaining 51 hips the mean pre-operative Oxford hip score was 47 points which decreased to 19 points at five years (45 hips). Of the stems 49 (98%) were implanted within 1 degrees of neutral in the femoral canal. The mean migration of the stem at five years was 1.9 mm and the survivorship for aseptic loosening was 100%. There was no significant difference in outcome between the consultant and registrar groups. At five years, the results were comparable with those of other polished, tapered, cemented stems. Long-term surveillance continues.

Engineering an interfacial zinc site to increase hormone-receptor affinity.

BACKGROUND: Human growth hormone (hGH) binds to both the hGH and human prolactin (hPRL) receptors. Binding to the hPRL receptor, however, is approximately 50-fold tighter and requires a single Zn2+ cation, unlike binding of hGH to the hGH receptor. Previous mutational studies have identified putative ligands from hGH and the hPRL receptor responsible for coordinating the interfacial Zn2+. RESULTS: One of these ligands was introduced at a structurally analogous site in the extracellular domain of the hGH receptor by mutating Asn218 to His, and the resulting mutant protein showed a 20-fold increase in hGH binding in the presence of ZnCl2. Alanine-scanning mutagenesis showed that the binding site on hGH for the Asn218-->His hGH receptor in the presence of Zn2+ resembled that for the hPRL receptor. CONCLUSIONS: It is possible to introduce the metal-binding site from the hPRL receptor into the homologous hGH receptor. More generally, these studies indicate that affinity between two proteins may be enhanced by design of an interfacial metal-binding site.

Interfacial metal-binding site design.

In recent years, much attention has focused on the characterization of metal-binding sites in natural metalloproteins and the design of novel metal-binding motifs. As a result, it is now possible to harness the high specificity and potency of metal-ion binding to modulate intermolecular interactions. Some encouraging results have been obtained using designed metal-binding sites in such diverse applications as the stabilization of artificial peptide assembly, regulation of membrane channels, control of enzyme activity and enhancement of hormone-receptor interactions.

Proliferation of MIELIKI a novel t(7;9) early pre-B acute lymphoblastic leukemia cell line is inhibited concomitantly by IL-4 and IL-7.

The present study describes a novel cell line, MIELIKI, established from bone marrow of a pediatric patient with B lineage acute lymphoblastic leukemia (ALL) at diagnosis. The MIELIKI cell line displays an early pre-B cell phenotype (CD10+, CD19+, CD20+, CD34-, Cmu-, sIg-) with rearrangements on both Ig heavy chain and k light chain alleles, and carries an unfrequent t(7;9) chromosomal translocation identical to the freshly isolated leukemic blasts. The proliferation of MIELIKI cells was abrogated by IL-4 and by IL-7, as measured by DNA replication and viable cell recovery. The effects of IL-4 and IL-7 were mediated, respectively, through the CDw124 and CDw127 IL-4 and IL-7 receptor components. Growth inhibition by IL-4 was not mediated by soluble factors released by MIELIKI cells in response to IL-4, suggesting the existence of an intrinsic negative signaling pathway. Finally, neither IL-4 nor IL-7 were found to induce maturation of MIELIKI into cells expressing cytoplasmic or surface membrane mu chain. The present cell line should constitute a useful model of t(7;9) early pre-B ALL and allow investigation of the relationship between IL-4 and IL-7 negative signaling in leukemic B cell ontogeny.

Characterization of the receptor binding determinants of granulocyte colony stimulating factor.

We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

High-throughput structural biology in drug discovery: protein kinases.

Structural biology is an invaluable tool in modern drug discovery, providing key insights into the interactions of small-molecule drugs with their protein targets. As in many aspects of the drug discovery process, significant synergies can be realized in structural biology by the contemporaneous pursuit of many target proteins from a single structural and functional class. We will review some of those synergies here using the example of the protein kinases--an important class of drug targets that has recently been the subject of intensive study. We conclude by discussing some of the technical advances in X-ray crystallography that have enabled implementation of high-throughput structural biology as applied to drug lead optimization.

Inhibition of axoplasmic transport in the developing visual system of the rat-III. Electron microscopy and Golgi studies of retino-fugal synapses and post-synaptic neurons in the dorsal lateral geniculate nucleus.

Quantitative light and electron microscopy, together with Golgi methodology, were used to study alterations in retino-fugal terminals and postsynaptic neurons within the dorsal lateral geniculate nucleus of the rat at various intervals following inhibition of axoplasmic transport in the optic nerve induced by intraocular injections of colchicine from 1-20 days postnatal. Colchicine concentrations used in this study ranged from 10-5 M-10-2 M. These were selected on the basis of our measurements of axon transport suppression described in the preceding article. 61. The volume of the nucleus was determined by section planimetry and reconstruction. Growth of the contralateral dorsal lateral geniculate nucleus was significantly retarded by intraocular colchicine at 1 and 5 days of age, only achieving a volume of between 61-75% of the normal or the control (ipsilateral) nucleus depending upon dosage. Application of colchicine at 10 days of age resulted in minimal stunting of nuclear growth, (79-93% of normal). Mean numbers of neurons in the contralateral and ipsilateral nucleus remained stable throughout all postnatal ages examined suggesting that nuclear volume loss was caused principally by a reduction in the amount of neuropil. Golgi impregnation displayed dendritic stunting in relay neurons characterized by narrowing of the portion of the shaft between 40-80 micrometer from the soma and a reduced incidence of spinous protrusions, particularly those shown by other studies to engage the retino-fungal terminal. 27,69 A concomitant Sholl76 analysis of dendritic branching in relay neurons demonstrated no significant differences in the number of intersections between normal and experimental nuclei. No alterations were observed in intrinsic neurons. Electron microscopy of postsynaptic neurons following concentrations ranging from 10-4 M at birth revealed altered patterns of granular endoplasmic reticulum in many cells characterized by reduced numbers of cisternae and scattered instances of cisternal dilation, together with enhanced infolding of the nuclear membrane at 20 days postnatal. Those animals which were given 5 X 10-3 M-10-2 M colchicine demonstrated an increased incidence of cisternal dilation, loss of ribosomes, disruption of the nuclear membrane and occasionally, complete degeneration. A similar array of alterations took place following intraocular injection at 5 days of age; however, animals receiving colchicine at 10 days postnatal displayed minimal alterations in relay neurons. Synaptic glomeruli, which contain the retinofugal terminal, displayed dose and age-dependent reduction in the size of the presynaptic element of the complex following intraocular colchicine, together with fewer post-synaptic spinous protrusions. Synaptic vesicles remained normal in appearance and distribution and our quantitative analysis demonstrated no loss of such terminals in accordance with colchicine concentrations which were previously found not to be lethal to retinal ganglion cells and optic axons.

Motor point blocks in children. A technique to relieve spasticity using phenol injections.

The careful attention that the day-surgery staff provides to the child who undergoes a motor point block procedure can reduce the anxiety of the parents and child and significantly contribute to the overall interdisciplinary care of the patient. The experience should be a positive one for the family and for the surgical staff.

Using model-system genetics for drug-based target discovery.

The combination of medicinal chemistry and model-organism genetics is emerging as a powerful tool for the discovery and validation of drug targets. Model systems can be used to identify the cognate target for compounds that demonstrate in vivo efficacy but have unknown mechanisms of action. Alternatively, drugs with known cognate targets can be used to probe biochemical pathways in model organisms, revealing new targets and mechanisms within these pathways. In both cases, the availability of human genomic sequence data is opening up new opportunities for accelerating target discovery.

Ethical issues encountered in pediatric rehabilitation.

Ethical issues in pediatric rehabilitation should be viewed within the framework of the understanding of the terms of autonomy, nonmaleficence, beneficence and justice. In dealing with the pediatric patient, health professionals are most frequently treating the parents who have the decision-making authority. Normalization which underlies the concept of mainstreaming has become an important issue in pediatric rehabilitation. Normalization is based on the provision of opportunities for choosing, for independent decision making, and for autonomy. The right of the disabled child to have sexual information and to be acknowledged as a sexual individual is essential to the child's autonomy. More children are surviving catastrophic injuries and living with severe disabilities. Birth defects such as myelodysplasias point to the issues of treatment selection involving ethical, moral, philosophical, religious, financial and social values. The phrase 'quality of life' is often applied in these situations and is frequently understood as the 'best interests of the child' which should result in child-centered decisions. In this way, a decision to begin or to withhold treatment can be made in a more ethically considered manner. Resource allocation is of special concern in disabled children since they may consume significant resources in medical and rehabilitation costs spent throughout a lifetime. However, these costs may be justifiable when a disabled child matures into a productive adult. If maximum benefit of limited resources is to be achieved, these children should receive their care from rehabilitation professionals in established centers of rehabilitation expertise. However, we may soon need to accept the responsibility for rationing rehabilitative care as the number of disabled children grows beyond the dollars available to be spent.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-4 and IL-13 receptors: are they one and the same?

Interleukin 4 (IL-4) and IL-13 share several biological properties, suggesting that they also share a common receptor or receptor component. Indeed, as discussed here by Robin Callard and colleagues, the IL-13 receptor appears to be a functional receptor for IL-4.

Biological activity of IL-4 and IL-13 on human endothelial cells: functional evidence that both cytokines act through the same receptor.

The cytokine IL-4 has unique effects on human endothelial cells. It specifically increases expression of vascular cell adhesion molecule (VCAM)-1 promoting adhesion of lymphocytes but not neutrophils, and causes profound effects on the morphology of endothelial monolayers characterized by formation of cell clusters and the appearance of holes in the cultured monolayer. In this study we show that the effects of IL-13 on human umbilical vein endothelial cells (HUVEC) are indistinguishable from those of IL-4. Both cytokines induce the same morphological changes in cultured HUVEC monolayers which are distinct from any other cytokine. In addition, IL-13 and IL-4 stimulate comparable levels of VCAM-1 expression with similar time kinetics, but at doses 10-fold less than those required for B cell activation and proliferation. Using a combination of mutant IL-4 antagonists and mAb to the IL-4R alpha chain (CD124), we show that expression of IL-4R alpha is essential for HUVEC responses to both IL-4 and IL-13, consistent with this receptor subunit being a component of the receptors for both cytokines. In contrast, the common gamma chain (gamma c), which is a component of the classical IL-4 receptor, was not detected on endothelial cells by flow cytometry or immunogold histochemistry. In addition, RT-PCR showed extremely low or absent gamma c mRNA, consistent with the absence of detectable surface protein. These results strongly suggest that the cytokines IL-4 and IL-13 are both important in modulating endothelial cell function, and may act through a single receptor complex on human endothelial cells that includes the IL-4R alpha chain but not the gamma c chain.

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor alpha chain (p140) but not the gamma c chain.

This study investigates the effect of interleukin (IL)-4 mutant proteins and a monoclonal antibody to the IL-4 receptor alpha chain on IL-4 and IL-13 response by B cells from X-linked severe combined immunodeficiency (X-SCID) patients in which the common gamma chain (gamma c chain) gene mutations have been fully characterized and no gamma c chain expression was detected. In this gamma c chain gene knockout model, it was confirmed that the gamma c chain is essential for B cell responses to IL-2 but not for IL-4 or IL-13. Dose-response curves for X-SCID and normal B cell responses to IL-4 were indistinguishable, showing that the loss of the gamma c chain did not diminish the sensitivity of B cells to IL-4. The mutant protein IL-4(Y124D) and an antibody to the IL-4R alpha chain both inhibited responses of X-SCID B cells to IL-4 and IL-13, showing that X-SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL-4R alpha chain but not the gamma c chain. Another mutant protein, IL-4(R88D), which has greatly reduced affinity for IL-4R alpha, was found to inhibit responses by normal B cells to IL-4 but not to IL-13. IL-4(R88D), did not, however, inhibit X-SCID B cell responses to IL-4. This result is consistent with IL-4(R88D) inhibition of responses mediated by receptor complexes that include the gamma c chain. We propose that X-SCID B cells responses to IL-4 are mediated by an IL-13 receptor complex comprised of the IL-4R alpha chain associated with the recently cloned IL-13R binding protein. This model has major implications for understanding normal B cell responses to IL-4.

Transferrin and its receptor in the development of genetically determined neural tube defects in the mouse embryo.

The iron-binding growth factor transferrin is taken up and localised in the hindgut of midgestation mouse embryos. We investigated whether the distribution of transferrin may be disturbed in mutant curly tail embryos, a proportion of which exhibit a cell proliferation defect affecting the hindgut endoderm, as part of the pathogenetic sequence leading to development of neural tube defects. Immunostaining revealed a reduction in the binding and/or uptake of transferrin by hindgut epithelial cells in affected curly tail embryos compared with their unaffected littermates. There was no apparent difference between the two embryo types, however, in the distribution or level of expression of the transferrin receptor. The receptor is expressed specifically in the hindgut endoderm of the 10.5-day embryo, although its mRNA is present in all tissues of the posterior neuropore region, suggesting posttranscriptional control of gene expression. These findings may indicate a role for transferrin binding and/or uptake in the regulation of cell proliferation in the hindgut endoderm, with a defect in this process in the curly tail mutant. However, an alternative explanation is suggested by our finding that transferrin immunostaining is more intense in the hindgut of unaffected curly tail embryos than in nonmutant CBA/Ca and CD-1 embryos. Thus, mutant embryos may increase their uptake of transferrin in an attempt to compensate for defective cell proliferation in the hindgut resulting from a defect in another pathway. Only a proportion of embryos are able to mount this compensatory response leading to the observed partial penetrance of developmental defects in the curly tail mutant mouse.