Genomics and outbreaks: foot and mouth disease.

Foot and mouth disease virus (FMDV) is an animal pathogen of global economic significance. Identifying the sources of outbreaks plays an important role in disease control; however, this can be confounded by the ease with which FMDV can spread via movement of infected livestock and animal products, aerosols or fomites, e.g. contaminated persons and objects. As sequencing technologies have advanced, this review highlights the uses of viral genomic data in helping to understand the global distribution and transboundary movements of FMDV, and the role that these approaches have played in control and surveillance programmes. The recent application of next-generation sequencing platforms to address important epidemiological and evolutionary challenges is discussed with particular reference to the advent of 'omics' technologies.

Le virus de la fièvre aphteuse est un agent pathogène affectant les animaux d'élevage, avec des conséquences économiques considérables à l'échelle mondiale. La détection des sources des foyers est un aspect important de la lutte contre cette maladie ; l'efficacité de cette stratégie est toutefois compromise par la facilité avec laquelle le virus de la fièvre aphteuse se propage à la faveur des mouvements d'animaux ou de produits d'origine animale infectés, d'aérosols ou de personnes ou matières contaminées. Les auteurs décrivent, au fur et à mesure des avancées des technologies du séquençage, les données de la génomique virale qui ont permis de mieux comprendre la distribution mondiale et la propagation transfrontalière du virus de la fièvre aphteuse et le rôle que ces approches ont commencé à jouer dans les programmes de contrôle et de surveillance. Les auteurs examinent également les applications récentes des plates-formes de séquençage de nouvelle génération pour résoudre des problèmes épidémiologiques et évolutifs importants, en se référant particulièrement à l'avènement des technologies dites «­omiques ¼.

El virus de la fiebre aftosa es un patógeno animal que reviste importancia planetaria. A la hora de combatir la enfermedad es útil poder determinar el origen de los brotes, tarea que sin embargo puede verse frustrada por la facilidad con que el virus es capaz de diseminarse siguiendo los desplazamientos de animales o derivados animales infectados o por aerosoles o fómites (por ejemplo personas u objetos contaminados). Los autores hacen hincapié en la utilización de datos de genómica vírica para ayudar a aprehender la distribución mundial y los movimientos transfronterizos del virus de la fiebre aftosa, lo cual es posible gracias a los avances que han conocido las técnicas de secuenciación, así como en la función que pueden cumplir estos métodos dentro de los programas de control y vigilancia. También examinan la reciente aplicación de dispositivos de secuenciación de próxima generación para abordar importantes problemas epidemiológicos y evolutivos, refiriéndose especialmente al advenimiento de las técnicas «ómicas¼.

Data on the annealing of NbTiVZr at 1200 °C with slow cooling rate.

The data presented here is complementary to the publication entitled "High temperature, low neutron cross-section high-entropy alloys in the Nb-Ti-V-Zr system" [1]. A homogenization methodology with slower cooling rate (∼2 °C/min) was performed. X-ray diffraction and scanning electron microscopy (backscattered electron and energy dispersive spectroscopy) data pertaining to annealed high-entropy alloy composition NbTiVZr is presented.

A systematic review of cross-sectional differences and longitudinal changes to the morphometry of the brain following paediatric traumatic brain injury.

Paediatric traumatic brain injury (pTBI) is a leading cause of disability for children and young adults. Children are a uniquely vulnerable group with the disease process that occurs following a pTBI interacting with the trajectory of normal brain development. Quantitative MRI post-injury has suggested a long-term, neurodegenerative effect of TBI on the morphometry of the brain, in both adult and childhood TBI. Changes to the brain beyond that of anticipated, age-dependant differences may allow us to estimate the state of the brain post-injury and produce clinically relevant predictions for long-term outcome. The current review synthesises the existing literature to assess whether, following pTBI, the morphology of the brain exhibits either i) longitudinal change and/or ii) differences compared to healthy controls and outcomes. The current literature suggests that morphometric differences from controls are apparent cross-sectionally at both acute and late-chronic timepoints post-injury, thus suggesting a non-transient effect of injury. Developmental trajectories of morphometry are altered in TBI groups compared to patients, and it is unlikely that typical maturation overcomes damage post-injury, or even 'catches up' with that of typically-developing peers. However, there is limited evidence for diverted developmental trajectories being associated with cognitive impairment post-injury. The current review also highlights the apparent challenges to the existing literature and potential methods by which these can be addressed.

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge.

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.

Therapeutic antibody fragments with prolonged in vivo half-lives.

Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.

Implications for anaesthesia in a patient established on clozapine treatment.

Clozapine is an atypical antipsychotic agent with a novel pharmacological profile and multiple clinical properties. Because of its side effects, it is recommended in treatment of severe resistant schizophrenia for which purpose it is remarkably effective. Little is known about the safety profile of clozapine during pregnancy and labour and because it is now used more commonly to manage schizophrenia, it is important that we as anaesthetists are aware of its many interactions and potential side effects. We present a case of a successful emergency caesarean section in a schizophrenic patient on clozapine treatment.

Improved tumor targeting with chemically cross-linked recombinant antibody fragments.

The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.

Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells.

Radiolabeled antibodies have produced encouraging remissions in patients with chemotherapy-resistant hematological malignancies; however, the selection of therapeutic radionuclides for clinical trials remains controversial. In this study, we compared the internalization, lysosomal targeting, metabolism, and cellular retention of radiolabeled murine and humanized monoclonal antibodies targeting the CD33 antigen (monoclonal antibodies mP67 and hP67, respectively) on myeloid leukemia cell lines (HEL and HL-60) and of anti-carcinoma antibodies (monoclonal antibodies hCTM01 and hA33) targeting breast cancer and colorectal carcinoma cell lines (MCF7 and Colo 205, respectively). Each antibody was labeled with 125I (by the IodoGen method) and with 111In and 90Y using macrocyclic chelation technology. Targeted tumor cells were analyzed for retention and metabolism of radioimmunoconjugates using cellular-radioimmunoassays, Percoll gradient fractionation of cell organelles, SDS-PAGE, and TLC of cell lysates and culture supernatants. Our results suggest that antibodies are routed to lysosomes after endocytosis, where they are proteolytically degraded. [125I]monoiodotyrosine is rapidly excreted from cells after lysosomal catabolism of antibodies radioiodinated by conventional methods, whereas small molecular weight 111In and 90Y catabolites remain trapped in lysosomes. As a consequence of the differential disposition of small molecular weight catabolites, 111In and 90Y conjugates displayed superior retention of radioactivity compared with 125I conjugates when tumor cells were targeted using rapidly internalizing antibody-antigen systems (e.g., hP67 with HEL cells and hCTM01 with MCF7 cells). When tumor cells were targeted using antibody-antigen systems exhibiting slow rates of endocytosis (e.g., hP67 on HL-60 cells and hA33 on Colo 205 cells), little differences in cellular retention of radioactivity was observed, regardless of whether 125I, 111In, or 90Y was used.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells.

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.

A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody.

Human immunoglobulin G4 (IgG4) exists in two molecular forms due to the heterogeneity of the inter-heavy chain disulphide bridges in the hinge region in a proportion of secreted human IgG4. This heterogeneity is only revealed under denaturing, non-reducing conditions in which an HL "half antibody" is detected, a phenomenon not seen in other human IgG isotypes. In native conditions noncovalent interactions hold the antibody together as the H2L2 tetramer. Analysis of the hinge sequences of human IgG heavy chains suggested that the presence of serine at residue 241 might be the cause of this heterogeneity. We therefore changed the serine at 241 to proline (found at that position in IgG1 and IgG2) in a mouse/human chimeric heavy chain. This single residue substitution leads to the production of a homogeneous antibody. Further, the variant IgG4 has significantly extended serum half-life and shows an improved tissue distribution compared to the original chimeric IgG4.

Psychomotor performance and counterregulatory responses during mild hypoglycemia in healthy volunteers.

The effect of mild hypoglycemia on psychomotor performance and counterregulatory responses was studied among 12 healthy volunteers. Each volunteer received two modified hyperinsulinemic glucose clamps. One morning, plasma glucose was held constant at euglycemic levels (4.9 mM) for 95 min, and another morning, it was lowered over 35 min and then held constant at hypoglycemic levels (3.4 mM) for 60 min. A battery of psychomotor tests and a questionnaire assessing hypoglycemic symptoms were administered before and repeated during the last 30 min of each clamp. The questionnaire and three selected psychomotor tests were also administered repetitively during the 1st h of each clamp. During the hypoglycemic studies, a rise was seen in plasma epinephrine and pancreatic polypeptide at 45 min. An increase in symptom scores was first recorded at 50 min during the hypoglycemic studies [median 4 (range 0-13) vs. 2 (5-6), P less than .05]. Performance was impaired on two psychomotor tests included in the battery. One was the trail making test on fine motor performance (-19.3 +/- 4.2 targets/min, mean +/- SE vs. 1.2 +/- 4.8 targets/min, P less than .05), and the other was the digit-symbol substitution (DSS) test on information processing and memory (18 +/- 3 vs. 29 +/- 4 symbols/min, P less than .03). Of the tests administered during the 1st h, performance was impaired on the DSS. This impairment became significant at 45 min (14 +/- 4 vs. 22 +/- 4 symbols/min, P less than .005). In conclusion, mild hypoglycemia selectively impairs psychomotor performance in healthy volunteers but not before the onset of glucose counterregulation and warning symptoms.

The effects of chlorpromazine and lorazepam on abnormal antisaccade and no-saccade distractibility.

BACKGROUND: Abnormally high levels of saccadic distractibility have been demonstrated to occur in patients with schizophrenia. Converging evidence implicates frontal cortical dysfunction as a mechanism; however, much of the neuropharmacology of saccadic distractibility has not yet been established. METHODS: We measured antisaccade, no-saccade, and visually guided saccade components in healthy subjects following single doses of lorazepam 2 mg, chlorpromazine 50-100 mg, and placebo. Visual analogue rating scales (VARS) provided a subjective measure of sedation. RESULTS: Lorazepam, but not chlorpromazine, was shown to cause an increase in saccadic distractibility in both the antisaccade and no-saccade tasks. Peak visually guided saccade velocity was decreased by lorazepam and chlorpromazine in a dose-dependent manner, with corresponding changes seen in VARS. Lorazepam, unexpectedly, did not affect peak antisaccade velocity. The background level of antisaccade directional errors was 6.43%, which is relatively low compared to control groups in patient studies. CONCLUSIONS: These results support the view that abnormal saccadic distractibility in patients with schizophrenia is not due to an acute effect of antipsychotic medication. The use of benzodiazepines and the level of task practice are highlighted as possible confounding variables in patient studies. The implications of these results for the current neuropathological theories of abnormal saccadic distractibility are discussed.

trans Fatty acids in human milk are inversely associated with concentrations of essential all-cis n-6 and n-3 fatty acids and determine trans, but not n-6 and n-3, fatty acids in plasma lipids of breast-fed infants.

BACKGROUND: Human milk fatty acids vary with maternal dietary fat composition. Hydrogenated dietary oils with trans fatty acids may displace cis n-6 and n-3 unsaturated fatty acids or have adverse effects on their metabolism. The effects of milk trans, n-6, and n-3 fatty acids in breast-fed infants are unclear, although n-6 and n-3 fatty acids are important in infant growth and development. OBJECTIVE: We sought to determine the relations between trans and cis unsaturated fatty acids in milk and plasma phospholipids and triacylglycerols of breast-fed infants, and to identify the major maternal dietary sources of trans fatty acids. DESIGN: We collected milk from 103 mothers with exclusively breast-fed 2-mo-old infants, blood from 62 infants, and 3-d dietary records from 21 mothers. RESULTS: Mean (+/-SEM) percentages of trans fatty acids were as follows: milk, 7.1 +/- 0.32%; infants' triacylglycerols, 6.5 +/- 0. 33%; and infants' phospholipids, 3.7 +/- 0.16%. Milk trans fatty acids, alpha-linolenic acid (18:3n-3), arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3) (P < 0.001), and linoleic acid (18:2n-6) (P = 0.007) were each related to the same fatty acid in infant plasma phospholipids. Milk trans fatty acids were inversely related to milk 18:2n-6 and 18:3n-3, but not to milk or infant plasma 20:4n-6 or 22:6n-3. trans Fatty acids represented 7.7% of maternal total fat intake (2.5% of total energy); the major dietary sources were bakery products and breads (32%), snacks (14%), fast foods (11%), and margarines and shortenings (11%). CONCLUSIONS: There were comparable concentrations of trans fatty acids in the maternal diet, breast milk, and plasma triacylglycerols of breast-fed infants. Prepared foods were the major dietary source of trans fatty acids.

Development of visual acuity in relation to plasma and erythrocyte omega-6 and omega-3 fatty acids in healthy term gestation infants.

The development of preferential looking acuity was studied prospectively to 3 mo of age in exclusively breast-fed and formula-fed term gestation infants. The formula contained (% of total fatty acids) 17.9% linoleic acid (18:2 omega-6) and 2.1% alpha-linolenic acid (18:3 omega-3) but no docosahexaenoic acid (22:6 omega-3) or arachidonic acid (20:4 omega-6). The breast milk contained (mean +/- SEM) 13.4 +/- 0.8% 18:2 omega-6, 1.5 +/- 0.1% 18:3 omega-3, 0.51 +/- 0.03% 20:4 omega-6, and 0.22 +/- 0.02% 22:6 omega-3. Preferential looking acuity, assessed by the acuity-card procedure, and plasma phospholipid and erythrocyte phosphatidylcholine and phosphatidylethanolamine fatty acids were determined at 14 d and 3 mo of age. There were no significant differences in acuity at 14 d or 3 mo, despite substantial differences in erythrocyte and plasma lipid 22:6 omega-3. Visual acuity was [mean (cycles/degree) +/- SD (octaves)] 3.93 +/- 0.54 and 4.77 +/- 0.48 and erythrocyte phosphatidylethanolamine %22:6 omega-3 was (mean +/- SE) 7.6 +/- 0.5 and 4.0 +/- 0.2 in the 3-mo-old breast-fed and formula-fed infants, respectively. These studies show that feeding formula containing 2.1% 18:3 omega-3 (approximately 1.0% energy) results in development of visual acuity similar to breast-feeding in term infants to > or = 3 mo of age.

Plasma and red blood cell fatty acids of low-birth-weight infants fed their mother's expressed breast milk or preterm-infant formula.

The fatty acid composition of plasma phospholipids, red blood cell (RBC) phosphatidylcholine (PC), and phosphatidylethanolamine (PE) was determined for low-birth-weight (LBW) infants when full oral feeding commenced (day 0) and after a further 28 d (day 28). They were fed their mother's expressed breast milk (PTM, n = 9), formula (SCF, n = 16) with 2% 18:3n-3 fatty acids, 20% 18:2n-6 fatty acids, or a combination of SCF and PTM (n = 11). Concentrations of all 20- and 22-carbon n-6 and n-3 fatty acids were similar among the infant groups on days 0 and 28 (mean postnatal age 42 +/- 1.3 d). The results suggest that formula with greater than or equal to 2% 18:3n-3 and a ratio of 18:2n-6 to 18:3n-3 similar to that of human milk may permit incorporation of n-3 fatty acids in LBW infant tissues equivalent to that from human milk.

F(ab')2 molecules made from Escherichia coli produced Fab' with hinge sequences conferring increased serum survival in an animal model.

Fab's with hinges based on the human gamma1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab')2. We find that the F(ab')2 made with hinges containing 2 or 4 cysteines have a high level (approximately 70%) of multiple disulphide bonds. These F(ab')2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab')2 formed by chemical cross-linking. F(ab')2 made from the Fab' with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols. These hinge sequences provide a simple method for producing robust F(ab')2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates.

Association of renal failure with Lewis incompatibility after allogeneic bone marrow transplantation.

The cause of the renal failure that occurs in approximately 20 percent of patients following allogeneic bone marrow transplantation is poorly understood. A patient is described in whom acute renal failure occurred one week after allogeneic bone marrow transplantation. The onset of the renal failure was associated with the demonstration of anti-Lewis antibodies in the patient's serum, which could only have been derived from donor lymphocytes. Recovery of renal function coincided with the disappearance of the Lewis antibody. It is postulated that Lewis incompatibility between graft and host tissue may have contributed to the renal failure in this patient and that incompatibility associated with determinants present on renal cells may account for other instances of acute renal failure following allogeneic bone marrow transplantation.

Recombinant antibodies for the diagnosis and therapy of human diseases.

It is nearly 25 years since the first description of monoclonal antibodies. During that time, monoclonal antibodies and derivatives have become a major group of pharmaceuticals in development. Technical problems have, however, hindered their development and initial optimism led to disappointment at the rate of progress in generating products. Many of the technical problems have now been overcome and in recent years a significant number of licensed products have emerged. Many more are in late-stage development and are expected to gain product approval in the near future to establish antibodies, and in particular, recombinant engineered antibodies as a major therapeutic class. This review highlights recent progress in the development of recombinant antibodies for the diagnosis and therapy of human disease.