The lipopolysaccharide model for the experimental induction of transient lameness and synovitis in Standardbred horses.

An established lipopolysaccharide (LPS) model previously described in Warmbloods, was inconsistent in Standardbred horses, where lameness was not detected despite the presence of synovitis. The present study aimed to determine the dose of LPS from E. coli O55:B5 required to induce mild to moderate lameness following middle carpal joint injection in Standardbred horses and to quantitate the induced lameness over time, with and without anti-inflammatory pre-treatment. In a baseline trial, eight healthy, clinically sound Standardbred horses were used in a rule-based dose-escalation design trial, starting at a dose of 10 endotoxin units (EU). Lameness at trot was evaluated visually and quantitatively (using an inertial-sensor system and pressure plate analysis). Synovial fluid aspirates were analysed for total nucleated cell counts, total protein and prostaglandin E2 (PGE2). Following 2 months wash-out, the effective LPS-dose determined in the baseline trial was used to evaluate the effect of anti-inflammatory treatment. A mixed model for repeated measures with horse as random effect was used for analysis. After injection of 10 EU LPS, the desired degree of lameness was observed in the baseline trial, with maximal lameness at post-injection hour (PIH) 4, followed by a rapid decline and return to baseline by PIH 48. No lameness was observed following pre-treatment with meloxicam. In synovial fluid, PGE2 was significantly higher at PIH 8 and PIH 24 in the baseline trial compared with following meloxicam pre-treatment. In conclusion, injection of the middle carpal joint with 10 EU LPS consistently induces a transient lameness and synovitis in Standardbred horses.

The use of equine chondrogenic-induced mesenchymal stem cells as a treatment for osteoarthritis: A randomised, double-blinded, placebo-controlled proof-of-concept study.

BACKGROUND: There is a need to improve therapies for osteoarthritis in horses. OBJECTIVES: To assess the efficacy of equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma as a novel therapy for osteoarthritis in horses. STUDY DESIGN: Randomised, double-blinded, placebo-controlled experiment. METHODS: In 12 healthy horses, osteoarthritis was induced in the metacarpophalangeal joint using an osteochondral fragment-groove model. Five weeks after surgery, horses were randomly assigned to either an intra-articular injection with chondrogenic-induced mesenchymal stem cells + equine allogeneic plasma (= intervention) or with 0.9% saline solution (= control). From surgery until the study end, horses underwent a weekly joint and lameness assessment. Synovial fluid was collected for cytology and biomarker analysis before surgery and at Weeks 5, 5 + 1d, 7, 9 and 11. At Week 11, horses were subjected to euthanasia, and the metacarpophalangeal joints were evaluated macroscopically and histologically. RESULTS: No serious adverse events or suspected adverse drug reactions occurred during the study. A significant improvement in visual and objective lameness was seen with the intervention compared with the control. Synovial fluid displayed a significantly higher viscosity and a significantly lower glycosaminoglycan concentration in the intervention group. Other biomarkers or cytology parameters were not significantly different between the treatment groups. Significantly less wear lines and synovial hyperaemia were present in the intervention group. The amount of cartilage oligomeric matrix protein, collagen type II and glycosaminoglycans were significantly higher in the articular cartilage of the intervention group. MAIN LIMITATIONS: This study assessed the short-term effect of the intervention on a limited number of horses, using an osteoarthritis model. This study also included multiple statistical tests, increasing the risk of type 1 error. CONCLUSIONS: Equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma may be a promising treatment for osteoarthritis in horses. The Summary is available in Spanish - see Supporting Information.

Can the hoof be shod without limiting the heel movement? A comparative study between barefoot, shoeing with conventional shoes and a split-toe shoe.

Conventional shoeing restricts heel movement, which may have a negative effect on the orthopaedic health of the horse. A randomised crossover experimental study using noninvasive techniques was performed to compare the mediolateral heel movement in barefoot horses, horses shod with a conventional toe clipped shoe and with a new type of shoe with a split toe. In eight horses, 16 forelimbs were tested barefoot, shod with a conventional shoe and with the split-toe (ST) shoe, in random order. A displacement sensor was secured on the heels and measurements were collected continuously at a frequency of 679Hz while horses were exercised on a treadmill at the walk (1.8m/s), trot (3.5m/s) and canter (8m/s). Differences in heel movement between the conditions were analysed using a generalised estimating equations approach. The conventional shoe was associated with significantly less heel expansion compared with the ST shoe and barefoot situation in all gaits (P≤0.001). Heel expansion with the ST shoe was not significantly different from the barefoot condition. For all gaits, shoeing was associated with a significant reduction in heel contraction compared with the barefoot situation (P≤0.038), except for the heel contraction at the canter using a conventional shoe. In conclusion, the heel expansion with the ST shoe did not differ significantly from when the horse was barefoot, in contrast with the significant restriction of the heel movement when a conventional shoe was used.

Combinatorial Design of a Nanobody that Specifically Targets Structured RNAs.

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.

Equine allogeneic chondrogenic induced mesenchymal stem cells: A GCP target animal safety and biodistribution study.

The safety of the intra-articular use of mesenchymal stem cells (MSCs) is scarcely reported. Therefore, the goal of this study was to investigate the safety of a single intra-articular injection with allogeneic chondrogenic induced MSCs combined with equine plasma (=the investigational product: IVP) compared to a saline (0.9% NaCl) placebo control (=control product: CP). Sixteen healthy experimental horses were randomly assigned to receive a single intra-articular injection with either the IVP (n=8) or the CP (n=8) in the left metacarpophalangeal joint. All horses underwent a daily clinical assessment throughout the entire study period of 42days to assess adverse events. Additionally, a local joint assessment and a lameness examination were performed daily during the first two weeks, and weekly the following 4weeks. Blood samples were taken weekly for hematological and biochemical analysis. At the end of the study period, horses of the IVP group were euthanized for a thorough necropsy and to check for biodistribution. Tissue samples of the injected joint were collected for histological examination. In both CP and IVP treated horses a mild transient subjective increase in periarticular temperature and lameness was noted after the intra-articular injection with no significant differences between the treatment groups. No distribution of the cells was found using immunohistochemistry and no ectopic tissue formation or signs of inflammation were found on histology. A single intra-articular injection of allogeneic chondrogenic induced MSCs combined with allogeneic plasma in horses had the same clinical side effects as an intra-articular injection with saline solution.

Evaluation of direct Etest for antimicrobial susceptibility testing of bacteria isolated from synovial fluid of horses using enrichment bottles.

This study evaluated the Etest for direct antimicrobial susceptibility testing (AST) of bacteria from equine synovial specimens, incubated in BACTEC enrichment bottles. Ninety-four culture-positive broths were inoculated onto agar to directly determine the minimum inhibitory concentrations (MICs) of 13 antimicrobials, using the Etest (direct Etest). Results were compared with those obtained with the agar dilution reference method, the standard Etest, and the disc diffusion method, after subculture and standardisation of the inoculum. For categorical comparison of AST results, MICs were translated into susceptibility categories, using clinical breakpoints. The direct Etest predicted categorical susceptibility/resistance of bacteria from equine synovial fluid with acceptable accuracy (overall categorical agreement, 91%) and was more reliable than the disc diffusion test. The direct Etest was less accurate than the standard Etest for generating MICs ± 1 log dilution relative to the reference method (overall essential agreement, 69% vs. 89%). As the Etest generated a high percentage of inaccuracies with trimethoprim and sulfadiazine, these were less suitable antimicrobial agents for inclusion. In conclusion, the direct Etest reliably predicted the susceptibility of isolates from equine synovial fluid for the tested antimicrobials, except for trimethoprim and sulfadiazine. Since it did not require subculture and preparation of a standardised inoculum, direct Etest results were available at least 24 h earlier than with other methods, which could facilitate the diagnosis of synovial infections. However, when accuracy is prioritised over speed for MIC determination, the standard Etest is preferred over the direct Etest.

The preventive effects of two nutraceuticals on experimentally induced acute synovitis.

BACKGROUND: Nutraceuticals are often used in the management of equine osteoarthritis, but scientific evidence of their efficacy is lacking. OBJECTIVES: To study the preventive effects of two new nutraceuticals after the experimental induction of synovitis in comparison with positive and negative control treatments. STUDY DESIGN: Blinded, controlled, randomised experiment. METHODS: Twenty-four healthy Standardbred horses were randomly allocated to supplement AT (multi-ingredient, 28 days), supplement HP (collagen hydrolysate, 60 days), meloxicam (4 days) or placebo (60 days). Synovitis was induced in the right intercarpal joint by intra-articular injection of 0.5 ng lipopolysaccharide (LPS) of Escherichia coli while treatments were continued. Blood and synovial fluid were sampled before treatment, immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Synovial fluid samples were analysed for total nucleated cell count (TNCC), total protein (TP) and selected biomarkers (prostaglandin E2 [PGE2 ], interleukin-6 [IL-6], glycosaminoglycans [GAGs], type II collagen synthesis [CPII], matrix metalloproteinase [MMP]). Lameness was scored by visual examination and pressure plate analysis immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Clinical examinations were performed before treatment, immediately prior to LPS injection, at 2, 4 and 6 h post-injection, and then twice per day during the test period. RESULTS: Before treatment and intra-articular challenge, there were no statistically significant differences among the treatment groups for any of the parameters. After intra-articular challenge, the placebo group showed significantly higher synovial fluid TP, TNCC and PGE2 compared with the meloxicam group, although the model did not induce a relevant amount of lameness. Both nutraceuticals resulted in significantly lower synovial fluid TP, TNCC and PGE2 compared with placebo. No statistical differences in IL-6, GAGs, CPII or MMPs were observed among treatment groups. No adverse effects were observed. MAIN LIMITATIONS: Despite evidence of synovitis, lameness was too mild to detect. CONCLUSIONS: The preventive administration of these nutraceuticals showed anti-inflammatory effects in this validated synovitis model. Therefore, further studies of their clinical applicability are warranted.

Cryptosporidium parvum infection and associated risk factors in dairy calves in western France.

This study was conducted to determine the prevalence and risk factors for Cryptosporidium infection in calf neonates on dairy farms in Normandy. Fecal samples were randomly collected between July 2010 and September 2011 from 968 calves (7-21 days old) on 97 farms. Up to 10 calves were selected and sampled per farm, and feces examined for oocysts by microscopy. C. parvum oocyst shedding was scored semi-quantitatively (0-5). A questionnaire about calf-level care and management was completed, and mortality rates were obtained from the French national registration database (BDNI). Bivariable and multivariable analyses of potential risk factors for C. parvum oocyst shedding were conducted using generalized estimating equation (GEE) models (family=Binomial).Overall, 402 out of 968 calves (41.5%) were positive for oocysts, and 25.1% of animals had a shedding score >2. Seven of the 97 farms (7%) were negative for oocysts in all fecal samples. At the time of collection, 375 calves (39%) had diarrhea, and its prevalence strongly correlated with the score for C. parvum oocyst shedding (p<0.0001). The mortality rate at 90 days was significantly greater for calves with high combined scores of diarrhea and shedding. Factors associated with the shedding of C. parvum were the Normande breed (odds ratio=1.49; 95% confidence interval (CI): 0.93-2.37), dispensing of colostrum using a bucket (odds ratio=1.37; 95% CI: 1.00-1.89), treatment with halofuginone (odds ratio=0.46; 95% CI: 0.19-1.15) and feeding with fermented milk (odds ratio=0.32; 95% CI: 0.17-0.63). C. parvum is widespread among calves under 21 days old in dairy herds of western France. Shedding of C. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. This study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of C. parvum.

[Statistical and epidemiological methods used in biomedical research: implications for initial medical education]. / Méthodes biostatistiques et épidémiologiques employées pour la recherche biomédicale : implications pour la formation médicale initiale.

BACKGROUND: The main source of key medical information consists in original articles published in peer-reviewed biomedical journals. Reported studies use increasingly sophisticated statistical and epidemiological approaches that first require a solid understanding of core methods. However, such understanding is not widely shared among physicians. Our aim was to assess whether the basic statistical and epidemiological methods used in original articles published in general biomedical journals are taught during the first years of the medical curriculum in France. METHODS: We selected original articles published in The New England Journal of Medicine, The Lancet, and The Journal of the American Medical Association, over a period of six months in 2007 and in 2008. A standardized statistical content checklist was used to extract the necessary information in the "Abstract", "Methods", "Results", footnotes of tables, and legends of figures. The methods used in the selected articles were compared to the national program and the public health program of biostatistics and epidemiology taught during the first six years of medical school. RESULTS: The 237 analyzed original articles all used at least one statistical or epidemiological method. Descriptive statistics, confidence interval and Chi(2) or Fisher tests, methods used in more than 50% of articles, were repeatedly taught throughout the medicine curriculum. Measures of association, sample size, fit and Kaplan-Meier method, used in 40 to 50% of articles, were specifically taught during training sessions on critical reading methods. Cox model (41% of articles) and logistic regression (24% of articles) were never taught. The most widely used illustrations, contingency tables (92%) and flowcharts (48%), were not included in the national program. CONCLUSION: More teaching of the core methods underlying the understanding of sophisticated methods and illustrations should be included in the early medical curriculum so that physicians can read the scientific literature critically for their medical education.

[Salvage radiotherapy outcomes for biochemical recurrence following radical prostatectomy among the African-Caribbean population of Guadeloupe]. / Résultats de la radiothérapie de rattrapage pour récidive biologique après prostatectomie radicale dans la population africaine-caribéene de la Guadeloupe.

OBJECTIVES: To make the first analysis of the salvage radiotherapy outcomes for biochemical recurrence of prostate cancer after radical prostatectomy in African descendant people, witch has no healthcare access difficulties: the French West-Indies African-Caribbean people of Guadeloupe. PATIENTS AND METHOD: Ninety successive patients, with biological failure after radical prostatectomies were treated in the University Hospital of Pointe-à-Pitre with salvage radiotherapy between April 2003 and December 2008. The retrospective study analysed the biochemical disease free survival (bDFs) after irradiation with Kaplan-Meier method, and the independent predictors of bDFs using the Cox model, with P<0.05. The treatment failure was defined for PSA superior or equal to nadir +0.1 ng/mL. RESULTS: The median dose was 64 Gy. At the median follow-up of 24.63 months, 35 (38.9%) patients had biochemical recurrence. The median bDFs was 55.3 months, the bDFs probabilities at months 12, 24, 36 and 48 were 8.1%, 70.3%, 61.9% and 56.1%. Multivariate analysis determined that the independents predictors of treatment failure were the seminals vesicles invasion (P=0.0094, Hazard ratio=2.63 CI 95%: [1.28-5.55]), the PSA velocity superior or equal to 0.75 ng/mL per year (P=0.0002, Hazard ratio=3.88 CI95%: [1.86-7.75]) and the pre-irradiation PSA level superior or equal to 1.5 ng/mL (P=0.0093, Hazard ratio=2.89 CI95%: [1.30-6.45]). CONCLUSION: Salvage radiotherapy for African descendent people with no healthcare difficulty access was an efficient treatment for the biochemical recurrence and had the same outcomes than others Caucasian people.

Final cerebral infarct volume is predictable by MR imaging at 1 week.

BACKGROUND AND PURPOSE: Stroke volume, an increasingly used end point in phase II trials, is considered stationary at least 30 days after the ictus. We investigated whether information conveyed by MR imaging measurements of the "final" infarct volume could be assessed as early as the subacute stage (days 3-6), rather than waiting for the chronic stage (days 30-45). MATERIALS AND METHODS: Ninety-five patients with middle cerebral artery stroke prospectively included in a multicenter study underwent MR imaging during the first 12 hours (MR imaging-1), between days 3 and 6 (MR imaging-2), and between days 30 and 45 (MR imaging-3). We first investigated the relationship between subacute (FLAIR-2) and chronic volumes (FLAIR-3), by using a linear regression model. We then tested the relationship between FLAIR volumes (either FLAIR-2 or FLAIR-3) and functional disability, measured by the mRS at the time of MR imaging-3, by using logistic regression. The performances of the models were assessed by using the AUC in ROC. RESULTS: A linear association between log FLAIR-2 and log FLAIR-3 volumes was observed. The proportion of FLAIR-3 variation, explained by FLAIR-2, was high (R(2) = 81%), without a covariate that improved this percentage. Both FLAIR-2 and FLAIR-3 were independent predictors of mRS (OR, 0.79 and 0.73; 95% CI, 0.64-0.97 and 0.56-0.96; P = .026 and .023). The performances of the models for the association between either FLAIR volume and mRS did not differ (AUC = 0.897 for FLAIR-2 and 0.888 for FLAIR-3). CONCLUSIONS: Stroke damage may be assessed by a subacute volume because subacute volume predicts the "true" final volume and provides the same clinical prognosis.

Use of blood culture medium enrichment for synovial fluid culture in horses: a comparison of different culture methods.

REASONS FOR PERFORMING STUDY: Standard methods for culturing equine synovial fluid (SF) are often unrewarding. Evidence-based information on the relative efficiency of different systems used for optimisation of isolation of microorganisms from equine SF is lacking. OBJECTIVES: To compare the results of different culture systems performed in parallel on SF samples from horses clinically diagnosed with synovial sepsis. METHODS: Synovial fluid specimens were collected between February 2007 and October 2008 from all horses admitted to a referral hospital that were clinically diagnosed with synovial sepsis and from control horses. Synovial fluid samples were cultured in parallel by: 1) direct agar culture (DA); agar culture after: 2) lysis-centrifugation pretreatment (LC); 3) conventional enrichment (CE); 4) combined LC/CE; or 5) blood culture medium enrichment using an automated system (BACTEC 9050). RESULTS: Ninety SF samples from 82 horses were included, together with 40 control samples. Seventy-one of 90 samples (79%) were culture-positive by using blood culture medium enrichment (BACTEC), which was significantly higher compared to all other methods. BACTEC enrichment was never negative while any of the other methods was positive. Although agar culture following LC and/or CE resulted in a slightly higher number of positive samples compared to DA, this difference was not significant. All control samples were culture negative by the 5 different techniques. Although the majority of samples containing isolates recovered without enrichment, culture results after BACTEC enrichment were available on the same day as for agar culture with or without LC (19/23 samples), while CE postponed recovery by at least one day in 20/23 samples. CONCLUSION: Blood culture medium enrichment is superior to other techniques for isolation of bacteria from SF of horses. The use of an automated system allows enrichment without substantially postponing recovery of microorganisms. POTENTIAL RELEVANCE: The efficient and fast isolation of microorganisms from infected SF by the BACTEC system allows for rapid susceptibility testing and a more appropriate antibiotic treatment.

[Practical questions in case of maternal death]. / Questions pratiques en cas de survenue d'un décès maternel.

Every year, in France, about 70 women die during their pregnancy or the delivery. Any maternal death during labour is a traumatic event for the medical team and the family. The medical team has to face many "new" problems. We try to identify all the problems which the medical team has to face in front of a maternal death and try to solve them by a medical literature and French laws review. The medical team often feels powerless when a maternal death occurs. This work was made to be a guideline.

Metabolic syndrome and Framingham risk score for prediction of cardiovascular events in Caribbean Indian patients with blood glucose abnormalities.

OBJECTIVE: To evaluate the metabolic syndrome (MS) and Framingham risk score (FRS) as predictors of cardiovascular (CV) events in Caribbean Indian patients who have type 2 diabetes (T2D) or impaired glucose tolerance (IGT). METHOD: A longitudinal and retrospective study was conducted involving patients classified as T2D or IGT in a first study in 1997 who responded for a second examination in 2006. Nonparametric tests and Cox's proportional hazards model were used. Hazard ratios (HRs) and their confidence intervals (95% CI) for risk of a first CV event, according to the presence of MS or a high FRS, were estimated. For MS, the models were adjusted for age, gender and smoking status. RESULTS: A total of 148 patients were included in the present study. The mean time without a CV event was 7.5 years (range 0.38-8.45 years). We noted 31 (25 nonfatal) first hospitalizations, for stroke (n=15), angina pectoris (n=8), acute coronary heart disease (n=7) and acute peripheral vascular disease (n=1). Ten (6.8%) patients died and six deaths were related to CV events. The HRs of CV events associated with metabolic syndrome, defined by the National Cholesterol Education Program's Adult Treatment Program III, were not significant. Conversely, HRs of CV events associated with the FRS were 4.78 (95% CI 1.65-13.5) and 2.94 (95% CI 1.42-6.06) for a risk score superior or equal to 10% and superior or equal to 20%, respectively. For coronary heart disease alone, the HRs associated with the FRS were 9.92 (95% CI 1.31-75.2) and 2.88 (95% CI 1.05-7.93), respectively. In these Caribbean Indian patients with blood glucose abnormalities, unlike the FRS, MS failed to identify subgroups at high cardiovascular risk in the short term (8.5 years). Nevertheless, the long-term risk-predictive value of these tools needs to be evaluated.

Upward fixation of the patella in the horse. A retrospective study.

The objective of the present study was to assess the incidence of upward fixation of the patella (UFP) in different types of patients and to evaluate the outcome of conservative and surgical treatment for correcting this condition. A particular attempt was made to find out whether corrective trimming and/or shoeing (CTS) is efficient in the conservative treatment of UFP, and whether a longer postoperative resting period reduces the risk of complications after medial patellar desmotomy (MPD). Medical records of 78 horses with intermittent or permanent UFP were analyzed retrospectively. Young animals and ponies were mostly affected, mainly during winter (P < 0.05). Seventy-six horses with UFP were treated conservatively, with follow-up being possible in 64 of them. This treatment, in which CTS seemed the most important aspect, was fully successful in 51.6% of these patients; 20.3% of them improved partially. In case of no response to conservative treatment, or in case of a permanent fixation, MPD was performed in 20 horses, which corrected UFP completely in 17 of the 18 followed-up patients. However, gait abnormalities were seen in seven of those 17 horses postsurgically, but with the incidence being lower in horses that had rested for at least three months (25%) compared to horses that had only rested for less than one month (66.6%). Results indicate that conservative treatment, with special attention for CTS, is worth trying before performing more radical procedures to correct UFP, and that a longer convalescence period after MPD is desirable.

In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8.

BACKGROUND: The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins. OBJECTIVE: This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins. METHODS: Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA. RESULTS: Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity. CONCLUSION: The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.

Stability of recombinant 2 S albumin allergens in vitro.

Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.

Omapatrilat: a new tool for understanding metabolism of bradykinin at the endothelium level.

As a kallikrein-kinin system has been evidenced at the endothelium level, bradykinin released locally could be responsible, at least in part, for the vasodilatory effect of omapatrilat. To objectivate this potential role of bradykinin, it is important to understand the respective role of angiotensin-converting enzyme and neutral endopeptidase in the metabolism of this vasodilatory peptide, and also to define the influence of different pathophysiologic factors on the respective role of these metallopeptidases in this metabolism.

The metabolism of bradykinin: a cornerstone for the understanding of the cardiovascular effects of metallopeptidase inhibitors.

The development of a sensitive and specific analytical approach for the quantification of bradykinin (BK) has enabled the identification of five metallopeptidases primarily responsible for the metabolism of BK, including angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11. Several metabolic studies have shown that the metallopeptidases that participate in BK metabolism vary by location (eg, plasma, endothelium or cardiomyocytes) and by pathology (eg, diabetes, myocardial infarction or left ventricular hypertrophy). These studies advance understanding of the role of endogenous BK in the cardiovascular effects of ACE inhibitors and other metallopeptidase inhibitors.

Comparison of a vasopeptidase inhibitor with neutral endopeptidase and angiotensin-converting enzyme inhibitors on bradykinin metabolism in the rat coronary bed.

The in vitro effects of omapatrilat, a dual vasopeptidase inhibitor that simultaneously inhibits neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), on exogenous bradykinin metabolism after a single passage through the coronary bed were compared with that of a NEP inhibitor (retrothiorphan, 25 nM), an ACE inhibitor (enalaprilat, 130 nM), and omapatrilat (25 nM). Bradykinin and inhibitors were infused into isolated Langendorff rat hearts perfused at 1 ml/min followed by reperfusion at 10 ml/min. Residual bradykinin was quantified in the coronary effluent by enzyme-linked immunosorbent assay to calculate bradykinin recovery and its kinetic parameters (Vmax/Km). Bradykinin degradation rate at 1 ml/min was 4.56 +/- 0.39 1/min per gram without inhibitors and was significantly reduced to 2.57 +/- 0.19 1/min per gram in the presence of enalaprilat, to 2.97 +/- 0.38 1/min per gram with retrothiorphan, to 1.82 +/- 0.17 1/min per gram with both enalaprilat and retrothiorphan, and to 1.14 +/- 0.35 1/min per gram with omapatrilat. In a second set of experiments, the effect of a 14-day treatment of rats with either ACE inhibitors (enalapril, quinapril, and ramipril), a NEP inhibitor (candoxatril), or omapatrilat on exogenous bradykinin metabolism was studied in Langendorff perfused hearts isolated from these long-term treated rats. In untreated rats, bradykinin degradation at a coronary perfusion of 1 ml/min was 4.35 +/- 0.41 1/min per gram. This value was reduced by 30% for the NEP inhibitor, by 50% for all ACE inhibitors, and by 75% for omapatrilat. All inhibitors administered either short term or long term significantly reduced bradykinin degradation during a single passage through the coronary bed. However, omapatrilat administration resulted in the greatest protection from bradykinin breakdown than ACE or NEP inhibitors alone.