Vaccination of poultry workers: delivery and uptake of seasonal influenza immunization.

Avian influenza is a highly infectious disease in poultry and although the risk of human infection is low, concerns exist that it could evolve into a new human strain of pandemic potential if reassortment with a human influenza virus occurs. In January 2007, the UK government introduced a programme to vaccinate poultry workers to reduce the potential of such an event. This study evaluates the delivery, uptake and costs of the programme in three counties of England. A questionnaire survey was completed by consultants in public health in all the Primary Care Trusts in Norfolk, Suffolk and Cambridgeshire in May 2007. The delivery of the programme varied between Primary Care Trusts, including being delivered in some cases by clinics in primary care, by general practitioners and occupational health services in others. The uptake of vaccination was low ranging from 7% to 29% at a cost of £29 to £132 per person vaccinated. Vaccination of poultry workers as a public health measure to prevent an influenza pandemic is likely to be ineffective with the level of coverage found in this evaluation in our region.

Systematic review of multidisciplinary interventions in heart failure.

OBJECTIVE: To determine the impact of multidisciplinary interventions on hospital admission and mortality in heart failure. DESIGN: Systematic review. Thirteen databases were searched and reference lists from included trials and related reviews were checked. Trial authors were contacted if further information was required. SETTING: Randomised controlled trials conducted in both hospital and community settings. PATIENTS: Trials were included if all, or a defined subgroup of patients, had a diagnosis of heart failure. INTERVENTIONS: Multidisciplinary interventions were defined as those in which heart failure management was the responsibility of a multidisciplinary team including medical input plus one or more of the following: specialist nurse, pharmacist, dietician, or social worker. Interventions were separated into four mutually exclusive groups: provision of home visits; home physiological monitoring or televideo link; telephone follow up but no home visits; and hospital or clinic interventions alone. Pharmaceutical and exercise based interventions were excluded. MAIN OUTCOME MEASURES: All cause hospital admission, all cause mortality, and heart failure hospital admission. RESULTS: 74 trials were identified, of which 30 contained relevant data for inclusion in meta-analyses. Multidisciplinary interventions reduced all cause admission (relative risk (RR) 0.87, 95% confidence interval (CI) 0.79 to 0.95, p = 0.002), although significant heterogeneity was found (p = 0.002). All cause mortality was also reduced (RR 0.79, 95% CI 0.69 to 0.92, p = 0.002) as was heart failure admission (RR 0.70, 95% CI 0.61 to 0.81, p < 0.001). These results varied little with sensitivity analyses. CONCLUSION: Multidisciplinary interventions for heart failure reduce both hospital admission and all cause mortality. The most effective interventions were delivered at least partly in the home.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Purification and quantitation of tumor necrosis factor receptor immunoadhesin using a combination of immunoaffinity and reversed-phase chromatography.

The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5-15 microg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml(-1). Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented.

Novel assays based on human growth hormone receptor as alternatives to the rat weight gain bioassay for recombinant human growth hormone.

Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

Multiple sclerosis: Etiology, incidence and prevalence.

The article examines the medical, psychological, and social aspects of multiple sclerosis (MS), one of the most common neurological disorders in the Western hemisphere. It presents current information concerning the symptomology, diagnosis, course, progression, and treatment of the illness.

Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function.

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.

Affinity purification and microcharacterization of recombinant DNA-derived human growth hormone isolated from an in vivo model.

A procedure has been developed for the isolation and purification of trace amounts of unlabeled proteins from biological solutions. Using a combination of affinity chromatography and reversed-phase HPLC, microgram amounts of recombinant DNA-derived human growth hormone (rhGH) were purified from an in vivo rat model. Microcharacterization techniques were developed, and picomole amounts of the recovered protein were digested with trypsin and characterized using capillary HPLC peptide mapping. The described procedures were used to study the chemical changes that occur in rhGH following intravenous administration. The study demonstrated that both deamidation and oxidation can occur in vivo, although the former would occur to a significant extent only in proteins with an extended half-life.

Application of capillary high-performance liquid chromatography to biotechnology, with reference to the analysis of recombinant DNA-derived human growth hormone.

Using capillary HPLC, femtomole amounts of recombinant DNA-derived human growth hormone (rhGH) have been successfully detected from solutions at nanomolar concentrations. The separation used capillaries of 15 cm x 320 microns I.D. and detection was with a UV absorbance detector containing a capillary Z-shaped flow-cell. A sample of rhGH that was recovered from rat serum was analyzed by capillary reversed-phase HPLC, using both acidic- and neutral-pH mobile phases, as well as by capillary ion-exchange chromatography. When compared to HPLC separations performed at flow-rates of 1 ml/min, the sensitivity of the detection was increased 200 times, without any loss in resolution. Sub-microgram amounts of rhGH were also analyzed by tryptic mapping using capillary HPLC and peptides were identified by capillary LC-MS.

Diketopiperazine formation and N-terminal degradation in recombinant human growth hormone.

A new degradation process has been identified that occurs in recombinant DNA-derived human growth hormone. Non-enzymatic cyclization of the first two amino acids from the N-terminus and subsequent cleavage results in the formation of a diketopiperazine and a truncated variant of rhGH. The truncated protein was separated using hydrophobic interaction chromatography and identified as desPhe1Pro2-rhGH using N-terminal sequence analysis, tryptic mapping, and mass spectrometry.

Signet-ring cell carcinoma of the breast: a clinicopathologic study of 24 cases.

Twenty-four cases of signet-ring cell carcinoma of the breast are presented. These represented 4.5% of 535 cases of surgically treated breast cancer at Indiana University Medical Center. Twelve cases were associated with ductal carcinoma, nine with lobular carcinoma, one with colloid carcinoma, and four were pure signet-ring cell carcinoma. The mortality rate, incidence of axillary lymph nodal metastases, and number of involved lymph nodes were greater in cases of signet-ring cell carcinoma than with other forms of mammary carcinomas without signet-ring cells. It is proposed that signet-ring cell carcinoma can manifest as a pure lesion or as a variant of ductal, lobular, or colloid carcinoma, and that it is an aggressive histologic variant of mammary carcinoma.