Risk factors for equine glandular and squamous gastric disease in show jumping Warmbloods.

BACKGROUND: Prevalence of, and risk factors for, equine squamous gastric disease (ESGD) are well established. Limited data exists on risk factors for equine glandular gastric disease (EGGD). OBJECTIVES: To identify management factors associated with EGGD in show jumping Warmbloods in training. A secondary objective was to identify management factors associated with ESGD. STUDY DESIGN: Cross-sectional. METHODS: Gastroscopies were performed in horses following a 12-16 h fast. Management questionnaires were collected for each horse. Risk factors were determined using multivariable logistic regression modelling. RESULTS: Eighty-three horses were included in the final analysis. Exercising ≥6 days per week increased the odds of EGGD grade ≥1/4 (odds ratio [OR] = 3.5; 95% confidence interval [CI] 1.2-10.7) compared to less frequent exercise. Currently showing increased the risk of EGGD grade ≥2/4 (OR = 10.2; 95% CI, 1.04-100), while competing at the international level decreased the odds of EGGD grade ≥2/4 (OR = 0.11; 95% CI, 0.01-0.97). Exercise intensity increased the odds of grade ≥1/4 ESGD (OR = 2.8; 95% CI, 1.03-7.8) and feeding beet pulp decreased odds (OR = 0.22; 95% CI, 0.07-0.7). Exercise intensity (OR = 3.8; 95% CI, 1.1-12.8) increased the likelihood of grade ≥2/4 ESGD and feeding beet pulp decreased the odds of grade ≥2/4 ESGD (OR = 0.1; 0.02-0.64) respectively. MAIN LIMITATIONS: This study used a convenience sample of horses within a relatively small (approximately 200 km) geographic radius. The sample size was relatively small, particularly within the international competition level group. CONCLUSIONS: Training and feeding strategies and competition level appear to influence the occurrence of EGGD and ESGD. Prospective studies evaluating the impact of training frequency, duration, and intensity on gastric physiology may clarify the role of exercise in gastric disease.

Phenylbutazone induces equine glandular gastric disease without decreasing prostaglandin E2 concentrations.

In equids, phenylbutazone at high doses induces gastric disease, primarily in the glandular portion of the stomach. However, the mechanism of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric disease in horses has yet to be determined. While phenylbutazone-associated ulceration is often attributed to a decrease in basal gastric prostaglandins, this has not been demonstrated in the horse. Twelve horses were randomly assigned to treatment (n = 6; 4.4 mg/kg phenylbutazone PO in 20 ml molasses q 12 hr for 7 days) or placebo (n = 6; 20 ml molasses PO q 12 hr for 7 days) groups. Before treatment and 3 and 7 days after initiation of treatment, gastroscopy was performed and glandular gastric biopsies were collected and frozen at -80°C. Glandular disease was assessed on a scale of 0-4. Prostaglandin E2 concentrations in biopsies were measured using a commercially available enzyme-linked immunosorbent assay. All phenylbutazone-treated horses developed grade ≥2 glandular disease. Prostaglandin concentrations increased over time (p = .0017), but there was no effect of treatment (p = .49). These findings indicate that despite induction of glandular disease grade ≥2, phenylbutazone did not decrease basal glandular gastric prostaglandin E2 concentration.

Viral clearance by flow-through mode ion exchange columns and membrane adsorbers.

Anion exchange (AEX) is a common downstream purification operation for biotechnology products manufactured in cell culture such as therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins. We present a head-to-head comparison of the viral clearance efficiency of AEX adsorbers and column chromatography using the same process fluids and comparable run conditions. We also present overall trends from the CDER viral clearance database. In our comparison of multiple brands of resins and adsorbers, clearance of three model viruses (PPV, X-MuLV, and PR772) was largely comparable, with some exceptions which may reflect run conditions that had not been optimized on a resin/membrane specific basis.

Monoclonal antibody capture and viral clearance by cation exchange chromatography.

Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step.

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications.

Process analytical technology (PAT) has been gaining momentum in the biotech community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. In this two part series, we address PAT as it applies to processes that produce biotech therapeutic products. In the first part, we address evolution of the underlying concepts and applications in biopharmaceutical manufacturing. We also present a literature review of applications in the areas of upstream and downstream processing to illustrate how implementation of PAT can help realize advanced approaches to ensuring product quality in real time. In the second part, we will explore similar applications in the areas of drug product manufacturing, rapid microbiology, and chemometrics as well as evolution of PAT in biotech processing.

Process analytical technology (PAT) for biopharmaceutical products: Part II. Concepts and applications.

Implementing real-time product quality control meets one or both of the key goals outlined in FDA's PAT guidance: "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." The first part of the paper presented an overview of PAT concepts and applications in the areas of upstream and downstream processing. In this second part, we present principles and case studies to illustrate implementation of PAT for drug product manufacturing, rapid microbiology, and chemometrics. We further present our thoughts on how PAT will be applied to biotech processes going forward. The role of PAT as an enabling component of the Quality by Design framework is highlighted. Integration of PAT with the principles stated in the ICH Q8, Q9, and Q10 guidance documents is also discussed.

Ovarian and placental production of progesterone and oestradiol during pregnancy in reindeer.

We obtained uterine and peripheral venous plasma, and samples of luteal and placental tissues from 2- to 7-year-old, Eurasian mountain reindeer (Rangifer tarandus tarandus) from a free-living, semi-domesticated herd in northern Norway in November 1995, and February and March 1996. In November, ovarian venous blood was also collected from four animals. Plasma samples were assayed for progesterone and oestradiol. The tissue samples were examined by light and electron microscopy, steroid dehydrogenase histochemistry, and northern blot analysis for RNAs for 3beta-hydroxy-steroid dehydrogenase (3beta-HSD) and P450 (side chain cleavage (scc)). Peripheral blood was taken from non-pregnant females in the same herd on the same dates. Peripheral progesterone concentrations in pregnant reindeer (3.4 +/- 0.5 ng/ml, n = 8) clearly exceeded those in non-pregnant animals (0.40 +/- 0.14 ng/ml; P < 0.0004 , n = 10) but oestradiol levels were only marginally higher in pregnant (6.0 +/- 0.7 pg/ml) than in non-pregnant (4.8 +/- 0.5 pg/ml; P = 0.35) reindeer at the stages examined. In pregnant animals, peripheral progesterone and oestradiol concentrations rose slightly between November and March but the differences did not reach significance (progesterone, P = 0.083; oestradiol, P = 0.061). In November, progesterone concentrations in the ovarian vein (79 +/- 15 ng/ml) greatly exceeded (P < 0.03) those in the uterine vein ( 10 +/- 4 ng/ml) which in turn exceeded the levels in the peripheral blood (2.8 +/- 0.4 ng/ml; P < 0.29). Oestradiol concentrations were slightly but significantly (P < 0.05) higher in the ovarian (20 +/- 3 pg/ml) than the uterine vein (13 +/- 1 pg/ml) and, in turn, greater (P < 0.03) than in peripheral blood (4.6 +/- 0.4 pg/ml). All samples of luteal tissue consisted exclusively of normal fully-differentiated cells and stained intensely for 3beta-HSD. Isolated groups of placental cells also stained strongly for 3beta-HSD. RNA for P450 (scc) and 3beta-HSD was abundant in all corpora lutea and lower concentrations of P450 (scc) were present in the placenta. 3beta-HSD RNA in the placenta was below the limit of detection. We conclude that the corpus luteum remains an important source of progesterone throughout pregnancy in reindeer but that the placenta is also steroidogenic.

Specific recognition of DNA by integration host factor. Glutamic acid 44 of the beta-subunit specifies the discrimination of a T:A from an A:T base pair without directly contacting the DNA.

Integration host factor (IHF) is a protein that binds to the H' site of bacteriophage lambda with sequence specificity. Genetic experiments implicated amino acid residue Glu(44) of the beta-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E. C., Hales, L. M., Gumport, R. I., Gardner, J. F. (1992) EMBO J., 11, 305-313). We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and 45 of the H' site. IHF failed to bind these H' site substitution mutants in vivo. The K(d)(app) value for each H' site substitution, except for H'45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid beta-Glu(44) with alanine prevented IHF from discriminating against the H'44A variant but not the other H' site substitution mutants. Further analysis with other substitutions at position beta44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the beta-Glu(44) residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.

Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered specificity and enhanced affinity.

To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes. We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.

Spatial and temporal quantitation of plane thallium myocardial images.

A computer method has been developed to determine the relative initial uptake and segmental washout rates of thallium-201 from sequential myocardial images. Initial images in multiple projections are obtained at 10 min after thallium-201 injection, and delayed images 2-3 hr after injection. A modified interpolative method was used to construct a background reference plane, and net myocardial counts was used to construct a background reference plane, and net myocardial counts above this reference plane were determined from multiple count profiles. Washout rates were determined by linear regression of time-activity curves constructed from the sequential images. In this approach, both relative temporal as well as relative spatial quantitation is performed. Data from 25 normal subjects were used to establish numerical criteria and normal ranges for relative focal defects and abnormal segmental washout slopes. Normal ranges were set to include the 90-percentile limits of the distribution of values obtained from the normal population. From these values we derived a quantitative criterion for thallium scan interpretation that can be used for analysis and interpretation of scintigrams in clinical situations.