Dry potato extracts improve water holding capacity, shelf life, and sensory characteristics of fresh and precooked beef patties.

The objective was to examine shelf stability, cooked product yield, and sensory characteristics of beef patties that had no binder (Control), incorporated soy flour (Textured Vegetable Protein; TVP) or one of three dry potato extracts: X-TRATOS™ (potato extract), X-TRATOS™ O (potato extract with mustard), or X-TRATOS™ W (potato extract with sodium acid pyrophosphate). In retail display patties, all binders decreased discoloration and lipid oxidation compared to Control, and X-TRATOS™ O was superior (P < 0.05) to all other treatments. Cooking yield was higher (P < 0.05) in patties containing potato extracts compared with patties containing TVP, which had higher yield than Control patties. Beef patties with potato extracts were juicier (P < 0.05) than Control and TVP patties and had higher (P < 0.05) overall acceptability than Control patties. We conclude that potato extracts are effective binders for use in fresh or precooked beef patties because they improve retail shelf life, cooked product yield, and sensory characteristics.

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis therapy.

A gene therapeutic approach to treat osteoarthritis (OA) appears to be on the horizon for millions of people who suffer from this disease. Previously we described optimization of a scAAVIL-1ra gene therapeutic vector and initially tested this in an equine model verifying long-term intrasynovial IL-1ra protein at therapeutic levels. Using this vector, we carried out a dosing trial in six horses to verify protein levels and establish a dose that would express relevant levels of therapeutic protein for extended periods of time (8 months). A novel arthroscopic procedure used to detect green fluorescence protein (GFP) fluorescence intrasynovially confirmed successful transduction of the scAAVGFP vector in both the synovial and cartilage tissues. No evidence of intra-articular toxicity was detected. Immune responses to vector revealed development of neutralizing antibodies (Nabs) within 2 weeks of administration, which persisted for the duration of the study but did not lower protein expression intra-articularly. Re-dosing with a different serotype to attain therapeutic levels of protein confirmed establishment of successful transduction. This is the first study in an equine model to establish a dosing/redosing protocol, as well as examine the Nab response to capsid and supports further clinical investigation to determine the clinical efficacy of scAAVIL-1ra to treat OA.

Four-month outbreak of invasive meningococcal disease caused by a rare serogroup B strain, identified through the use of molecular PorA subtyping, England, 2013.

Molecular PorA subtyping provides information that increasingly requires the adaptation of standard public health approaches to outbreak management. We report an outbreak of a rare subtype of meningococcal infection not previously identified in the United Kingdom (UK). The outbreak occurred in the Warwickshire area in England between February and June 2013. Molecular subtyping allowed the identification of additional cases, prompting an enhanced public health response that included efforts to identify potential social networks that might benefit from chemoprophylaxis. It also prompted swabbing to define nasopharyngeal carriage in the focal nursery and helped explain the unusual epidemiological pattern. Without subtyping to identify a link, the additional cases would have been managed as sporadic cases in accordance with current UK guidance.

Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates.

Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ∼2 × 10(12) vector genome (vg) per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases.

Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs.

Adeno-associated viral vector 9 (AAV9) has recently been shown to penetrate the blood-brain barrier via intravascular administration, making it a good candidate for diffuse gene delivery. However, the potential side effects of systemic delivery are unknown. Intrathecal viral vector administration may be more invasive than intravenous injections, but it requires far less vector and it can be performed on an outpatient basis, making it an ideal route of delivery for clinical translation. A total of 12 domestic farm pigs (<20 kg) underwent a single-level lumbar laminectomy with intrathecal catheter placement for AAV9 delivery. Animals were perfused and the tissue was harvested 30 days after treatment. Gene expression was assessed by anti-green fluorescent protein immunohistochemistry. Although a single lumbar injection resulted in gene expression limited to the lumbar segment of the spinal cord, three consecutive boluses via a temporary catheter resulted in diffuse transduction of motor neurons (MNs) throughout the cervical, thoracic and lumbar spinal cords. We now present the first successful robust transduction of MNs in the spinal cord of a large animal via intrathecal gene delivery using a self-complementary AAV9. These promising results can be translated to many MN diseases requiring diffuse gene delivery.

Usefulness of porA sequencing in distinguishing sporadic and linked cases of serogroup B invasive meningococcal disease in Suffolk, United Kingdom, December 2009 to January 2010.

A cluster of three fatal cases of invasive meningococcal disease due to Neisseria meningitidis serogroup Bin a town in Suffolk, United Kingdom, during December 2009 to January 2010 was reported to the local Health Protection Unit. This paper describes the investigation undertaken to identify any potential epidemiological links among the cases, to determine if this was an outbreak and to consider whether to implement community-wide interventions and control measures. Case epidemiological information in addition to serogroup and genosubtyping (porA gene sequencing) data of the infecting organism was gathered on all cases in this reported cluster. Genosubtyping was also retrospectively requested for all serogroup B cases confirmed in Suffolk during 2009. Extensive investigation failed to establish an epidemiological link among the cluster of fatal cases of serogroup B invasive meningococcal disease in Suffolk. By demonstrating a number of distinct strains, the genosubtyping of isolates proved to be useful in the public health management of this incident by serving to exclude a community outbreak and preventing unnecessary mass chemoprophylaxis.

Invasive meningococcal disease: completeness and timeliness of reporting of confirmed cases in Thames Valley, 2006-2007.

OBJECTIVES: Regular evaluation of disease surveillance systems is essential. This study assessed the completeness and timeliness of reporting of invasive meningococcal disease (IMD) in Thames Valley in 2006-2007. STUDY DESIGN: Retrospective review of two data sources used in disease surveillance: the list of notified cases to the Thames Valley Health Protection Unit (TVHPU) and the list of confirmed cases at the reference laboratory during 2006-2007. METHODS: The datasets were compared by checking patient name, date of birth, sample date and date of onset of illness. Completeness was estimated using Tilling's capture-recapture method. Timeliness was assessed by calculating the difference between the date of admission and the date of notification to the TVHPU. RESULTS: The estimated completeness of reporting of IMD cases was calculated as 90.5% (95% confidence interval 88.6-92.4). Thirty-six percent of cases were notified on the day of admission, 63% were notified within 1 day and 72% were notified within 2 days (range 0-36 days). CONCLUSIONS: Timeliness and completeness of reporting of IMD was clearly suboptimal. It is critical to educate clinicians on the need to notify all suspected cases of IMD to public health authorities in a timely manner.

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay.

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.

Risk of laboratory-acquired meningococcal disease.

Five probable secondary cases of meningococcal disease were identified in microbiology laboratory workers in England and Wales during a 15-year period. All cases had prepared suspensions of Neisseria meningitidis outside a safety cabinet upto seven days before onset of illness. Relative risk in laboratory workers compared with the background adult population was 184 (95% CI 60-431). In view of the potentially serious outcome from this infection, a safety cabinet should always be used when preparing or working with suspensions of meningococci. Vaccination policy for microbiology laboratory workers should be reviewed.

Novel erythropoiesis stimulating protein for treatment of anemia in chronic renal insufficiency.

BACKGROUND: Novel erythropoiesis stimulating protein (NESP) is a glycoprotein with a threefold longer terminal half-life than recombinant human erythropoietin (rHuEPO) in humans. The aim of this study was to determine whether NESP is effective for the treatment of anemia at a reduced dosing frequency relative to rHuEPO in patients with chronic renal failure not yet on dialysis [chronic renal insufficiency (CRI)]. METHODS: This was a multicenter, randomized, open-label study. A total of 166 rHuEPO-naive patients with CRI were randomized in a 3:1 ratio to receive NESP (0.45 microg/kg once weekly) or rHuEPO (50 U/kg twice weekly) administered subcutaneously for up to 24 weeks. Dose adjustments were made as necessary to achieve a hemoglobin response, defined as an increase > or =1.0 g/dL from baseline and a concentration > or = 11.0 g/dL. RESULTS: During the 24-week treatment period, 93% (95% CI, 87 to 97%) of patients receiving NESP and 92% (95% CI, 78 to 98%) of patients receiving rHuEPO achieved a hemoglobin response. The median time to response was seven weeks (range of 3 to 25 weeks) in both groups. After correction of anemia, mean hemoglobin concentrations were maintained within the target range of 11.0 to 13.0 g/dL for the remainder of the 24-week treatment period. The safety profiles of NESP and rHuEPO were similar, and no antibodies were detected to either drug. CONCLUSIONS: These results demonstrate that NESP safely and effectively corrects and maintains hemoglobin concentrations at a reduced dosing frequency relative to rHuEPO in patients with CRI, providing a potential benefit to patients and health care providers.

Meningococcal serology.

Meningococcal serology has been mainly used over the last 20 years in the field of vaccinology, to evaluate candidate vaccines and quantify individuals' immune responses. With the increasing usage of pre-admission antibiotic treatment (1), nonculture diagnostic methods such as polymerase chain reaction (PCR) ( Chapter 3 ), antigen detection ( Chapter 4 ), and serology have become important tools. Nonculture diagnosis of meningococcal disease is rapidly becoming of equal importance for the confirmation of meningococcal infection as the isolation of Neisseria meningitidis organisms (2). This has occurred at a time when accurate case ascertainment of meningococcal disease has become a crucial aspect of assessing effectiveness of the recently introduced serogroup C polysaccharide-protein conjugate vaccine in the UK (3). N. meningitidis polysaccharide vaccines have been available for over 20 years (4) and evaluation of candidate vaccines and assessment of levels of antibody requires accurate and reproducible assays.

Performance characteristics of the polymerase chain reaction assay to confirm clinical meningococcal disease.

BACKGROUND: Confirmation of clinical meningococcal disease (MCD) is essential for management of patients, contacts, and outbreaks. Blood and CSF cultures, the traditional gold standard diagnostic tests, have been adversely affected by preadmission parenteral penicillin and fewer lumbar punctures. Rapid, reliable serogroup determination without the need to grow isolates could improve laboratory confirmation of MCD. AIMS: To determine performance characteristics of the currently available meningococcal polymerase chain reaction (PCR) assays in a clinical setting. METHODS: Prospective study of 319 children presenting with a suspected diagnosis of MCD (fever and a rash, or suspected bacterial meningitis) over a 16 month period. RESULTS: A total of 166 (52% of all) children had clinical MCD: diagnosis was confirmed microbiologically in 119 (72%) of these. Performance characteristics (sensitivity, specificity, negative predictive value, positive predictive value) in confirmation of clinical MCD were respectively (95% confidence interval): blood culture 31% (24-38%), 100%, 57% (49-65%), 100%; blood PCR 47% (39-55%), 100%, 65% (58-73%), 100%; any test positive 72% (65-79%), 100%, 77% (70-84%), 100%. CONCLUSIONS: Meningococcal DNA detection in blood or CSF by PCR is a useful method of diagnosis of MCD. PCR of peripheral blood performs better than blood culture. In a child with clinically suspected MCD, PCR assays, bacterial antigen tests, and oropharyngeal swabbing for meningococcal carriage should be performed in addition to blood or CSF culture, to improve case confirmation.

Measurement of serum antigen concentration by ultrasound-enhanced immunoassay and correlation with clinical outcome in meningococcal disease.

The distribution of Neisseria meningitidis serogroup B and C polysaccharide antigen in blood and the prognostic significance of antigen concentration was examined by ultrasound-enhanced immunoagglutination of coated microparticles. Specimens (169 sera/plasma from 145 patients with confirmed meningococcal disease) were tested retrospectively. The ultrasonic immunoassay detected serum antigen in 136 samples from 112 patients. Titration of antigen-positive specimens allowed estimation of blood antigen concentration. The modal blood antigen titre was 1/16, corresponding to an estimated polysaccharide concentration of 0.85 microg/ml. The lowest mean blood antigen concentration found ultrasonically was 0.05 microg/ml; compared to the 1.98 microg/ml found by conventional latex agglutination, this represents an approximately 30-fold improvement in sensitivity. Three grades of outcome were correlated with the presenting antigen titre in 83 patients: (i) <2 weeks hospitalisation, (ii) > or =2 weeks hospitalisation and (iii) mortality. High polysaccharide concentrations correlated with mortality. Nine of 15 patients with a serum antigen titre of 1/64 or greater (> or =3.4 microg/ml polysaccharide) died, whereas no patient with titres equal to or less than 1/4 (< or = 0.21 microg/ml) died, including those patients in whom antigen was undetectable by ultrasonic immunoassay. Increasing antigen concentration significantly correlated with severity of outcome (P<0.001). Ultrasound-enhanced agglutination provides a rapid prognostic indicator by sensitive measurement of serum antigen level.

Prevalence of de-O-acetylated serogroup C meningococci before the introduction of meningococcal serogroup C conjugate vaccines in the United Kingdom.

Meningococcal serogroup C conjugate (MCC) vaccines have been introduced in the UK to combat the rise in serogroup C meningococcal disease. Serogroup C meningococci may occur naturally expressing either O-acetylated (Oac(+)) or de-O-acetylated (Oac(-)) polysaccharide capsules. In a small study in the USA in the 1970s 15% of serogroup C meningococcal case isolates were reported to be Oac(-) though the prevalence of these Oac(-) isolates has not been recorded in the UK. This is of interest as the first MCC vaccines to be introduced are Oac(+) and the potential impact of this on Oac(-) serogroup C isolates is unclear. Serogroup C isolates submitted to the Public Health Laboratory Service Meningococcal Reference Unit in January 1998 (n=113) and January 1999 (n=162) were investigated by dot blotting using monoclonals specific for Oac(+) and Oac(-) serogroup C polysaccharides. This revealed 12% Oac(-) isolates for both January 1998 and January 1999. The proportion of fatal cases was found to similar for both Oac(-) and Oac(+), 14 and 9% for 1998 and 5 and 3% for 1999, indicating that the pathogenic potential of these Oac(-) isolates is similar to Oac(+). The acetylation status of serogroup C isolates needs to be monitored throughout and after the introduction of MCC vaccines.

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak.

Multilocus sequence typing and antigen gene sequencing were used to investigate an outbreak of meningococcal disease in a university in the United Kingdom. The data obtained showed that five distinct Neisseria meningitidis strains belonging to the ET-37 complex were present in the student population during the outbreak. Three of these strains were not associated with invasive disease, and two distinct strains caused invasive disease, including several fatalities. The initial case of the disease cluster was caused by a strain distinct from that responsible for at least two subsequent cases and two cases remote from the university, which were epidemiologically linked to the outbreak. These observations were consistent with pulsed-field gel electrophoresis data, but the sequence data alone were sufficient to resolve the strains involved in the disease cluster. Interpretation of the nucleotide sequence data was more straightforward than interpretation of the fingerprint patterns, and the sequence data provided information on the genetic differences among the isolates.

Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients.

Novel erythropoiesis stimulating protein (NESP) is a hyperglycosylated analogue of recombinant human erythropoietin (Epoetin) which has an increased terminal half-life in animal models. The aim of this study was to extend these observations to humans. Using a double-blind, randomized, cross-over design, the single-dose pharmacokinetics of Epoetin alfa (100 U/kg) and an equivalent peptide mass of NESP were compared following intravenous bolus in 11 stable peritoneal dialysis patients. This was followed by an open-label study to determine the single-dose pharmacokinetics of an equivalent peptide mass of NESP by subcutaneous injection in six of these patients. The mean terminal half-life for intravenous NESP was threefold longer than for intravenous Epoetin (25.3 versus 8.5 h), a difference of 16.8 h (95% confidence interval, 9.4 to 24.2 h, P = 0.0008). The area under the serum concentration-time curve was significantly greater for NESP (291.0 +/- 7.6 ng x h per ml versus 131.9 +/- 8.3 ng x h per ml; mean +/- SEM; P < 0.0005), and clearance was significantly lower (1.6 +/- 0.3 ml/h per kg versus 4.0 +/- 0.3 ml/h per kg; mean +/- SEM; P < 0.0005). The volume of distribution was similar for NESP and Epoetin (52.4 +/- 2.0 ml/kg versus 48.7 +/-2.1 ml/kg; mean +/-SEM). The mean terminal half-life for subcutaneous NESP was 48.8 h. The peak concentration of subcutaneous NESP was approximately 10% of that following intravenous administration, and bioavailability was approximately 37% by the subcutaneous route. The longer half-life of NESP is likely to confer a clinical advantage over Epoetin by allowing less frequent dosing in patients treated for anemia.

The nutrient-toxin dosage continuum in human evolution and modern health.

Recent findings support the long-recognized principle that nutritive and toxic effects of an ingested material depend not only on its nature but very much on its quantity. The well known observation that essential nutrients can be toxic at high dosages suggests that the same reversal of effect may be true of many substances that could be beneficial but not essential at low dosages (the phenomenon of hormesis). This has been demonstrated for many well known toxins. We suggest a mathematical model that describes these dosage effects as an expected result of the evolution of human metabolic and dietary adaptations for maximizing benefits and minimizing costs of the ingestion or other intake of any substance. Evolved mechanisms for achieving benefits may be unrelated to those for reducing costs. These evolutionary considerations suggest important consequences demonstrable by experimental or epidemiological studies. They also suggest ways in which our evolved dietary adaptations may be currently maladaptive, and individual development of taste preferences poorly calibrated by early experience in modern environments. The apparent reality of hormesis raises the possibility of counterproductive effects of current dosage recommendations and limits for nutrients and pollutants. We propose that some conceptual and factual problems are urgently in need of resolution. Fundamental to evolutionary biology is the tendency for organisms to become increasingly adapted to those environments to which they are most commonly exposed (Parsons 1990).