Inter-laboratory comparisons of assessment of the allergenic potential of proteins in mice.

Assessment of the potential allergenicity of novel proteins, including those expressed in genetically modified plants, is an important issue. In previous studies, we have shown that the IgE measurement induced by systemic exposure of BALB/c mice to a range of proteins correlates broadly with what is known of their allergenic potential in humans. The approach used a homologous passive cutaneous anaphylaxis (PCA) assay that reflects IgE-dependent biological activity and is of sufficient sensitivity to detect IgE production in the absence of adjuvant. In previous studies, the immunization phase was conducted independently in two separate facilities, and the subsequent analytical work (PCA) conducted in a single facility. The purpose here was to further evaluate the transferability of this approach. To this end, BALB/c mice were exposed to a range of doses of peanut agglutinin or ovalbumin, allergenic proteins of peanut and hen's egg, respectively, in two independent laboratories. Serial doubling dilutions of serum pooled for each treatment group were analyzed for specific IgE. At higher doses of allergen very similar, or identical, IgE titers were achieved in both laboratories, although at lower doses, responses were somewhat more variable. These data demonstrate that, although technically demanding, the measurement of protein allergen-induced IgE antibody production in mice using PCA is relatively robust and is transferable and reproducible between laboratories. This approach may provide a useful tool for the safety assessment of novel proteins and suggests that continued evaluation of the approach with a wider range of protein allergens and non-sensitising proteins is justified.

Preconditioning of skeletal muscle against contraction-induced damage: the role of adaptations to oxidants in mice.

Adaptations of skeletal muscle following exercise are accompanied by changes in gene expression, which can result in protection against subsequent potentially damaging exercise. One cellular signal activating these adaptations may be an increased production of reactive oxygen and nitrogen species (ROS). The aim of this study was to examine the effect of a short period of non-damaging contractions on the subsequent susceptibility of muscle to contraction-induced damage and to examine the changes in gene expression that occur following the initial contraction protocol. Comparisons with changes in gene expression in cultured myotubes following treatment with a non-damaging concentration of hydrogen peroxide (H(2)O(2)) were used to identify redox-sensitive genes whose expression may be modified by the increased ROS production during contractions. Hindlimb muscles of mice were subjected to a preconditioning, non-damaging isometric contraction protocol in vivo. After 4 or 12 h, extensor digitorum longus (EDL) and soleus muscles were removed and subjected to a (normally) damaging contraction protocol in vitro. Muscles were also analysed for changes in gene expression induced by the preconditioning protocol using cDNA expression techniques. In a parallel study, C(2)C(12) myotubes were treated with a non-damaging concentration (100 microM) of H(2)O(2) and, at 4 and 12 h following treatment, myotubes were treated with a damaging concentration of H(2)O(2) (2 mM). Myotubes were analysed for changes in gene expression at 4 h following treatment with 100 microM H(2)O(2) alone. Data demonstrate that a prior period of non-damaging contractile activity resulted in significant protection of EDL and soleus muscles against a normally damaging contraction protocol 4 h later. This protection was associated with significant changes in gene expression. Prior treatment of myotubes with a non-damaging concentration of H(2)O(2) also resulted in significant protection against a damaging treatment, 4 and 12 h later. Comparison of changes in gene expression in both studies identified haem oxygenase-1 as the sole gene showing increased expression during adaptation in both instances suggesting that activation of this gene results from the increased ROS production during contractile activity and that it may play a role in protection of muscle cells against subsequent exposure to damaging activity.

Is it justified to obtain routine peritoneal fluid cultures during appendectomy in children?

Controversy exists regarding obtaining routine peritoneal cultures during appendectomy. The aim of the study was to determine the impact of obtaining routine peritoneal fluid cultures during appendectomy on the treatment and clinical outcome in children. The charts of 269 children who were operated with the diagnosis of appendicitis between January 1996 and January 2001 were reviewed. The microorganisms in peritoneal cultures, selection of antibiotics and clinical outcome were evaluated. Average age was 10.1+/-3.3 (range, 1 to 17 years) years with a male to female ratio of 1.7 (170/99). There were two groups of patients; Group 1: uncomplicated appendicitis (201/269=75%), and Group 2: complicated (perforated) appendicitis (49/269=18%). In the series, 19 patients were found to have a normal appendix in histopathological examination (7%). Cultures were obtained from 95 (35.3%) patients (group 1: 59/95, group 2: 36/95). In patients with uncomplicated appendicitis, 6.7% of the cultures (4/59) were positive while in group 2, the rate was 47.2% (17/36) ( p<0.05). Only in four patients who were in group 2, antibiotics were re-adjusted according to the cultures. Escherichia coli and Klebsiella pneumoniae were the most common microorganisms. There were no complications in group 1, while wound infection (18.3%) and intra-abdominal abscess (2%) were the two most common complications in group 2. Intra-operative peritoneal cultures during appendectomy do not add much to the treatment of children. Therefore, it is not necessary to get peritoneal swab cultures during the procedures, and empiric use of wide spectrum antibiotics when necessary is generally sufficient in the management of this group of children.

A comparison of the immediate effects of moderate exercise in the late morning and late afternoon on core temperature and cutaneous thermoregulatory mechanisms.

Twelve healthy male subjects each undertook two bouts of moderate exercise (70% VO2max for 30 minutes) in the morning (08:00) and late afternoon (18:00) at least 4 days apart. Measurements were made of heart rate, core (rectal) temperature, sternum skin temperature, and forearm skin blood flow during baseline conditions, during the bout of exercise, and throughout a 30-minute recovery period. Comparisons were made of the changes of heart rate, temperature, and skin blood flow produced by the exercise at the two times of day. Student t tests indicated that baseline values for core temperature (37.15 degrees C +/- 0.06 degrees C vs. 36.77 degrees C +/- 0.06 degrees C) and sternum temperature (33.60 degrees C +/- 0.29 degrees C vs. 32.70 degrees C + 0.38 degrees C) were significantly (p < .05) higher in the late afternoon than the early morning. Two-way analysis of variance (ANOVA) indicated that the increases in core and sternum temperatures during exercise were significantly less (p = .0039 and .0421, respectively) during the afternoon bout of exercise compared with the morning, even though the work loads, as determined by changes in heart rate, were not significantly different (p = .798) at the two times of testing. There were also tendencies for resting forearm skin blood flow to be higher in the afternoon than in the morning and for exercise to produce a more rapid rise in this variable in the afternoon. The possible mechanisms producing these responses to exercise are discussed in terms of those that are responsible for the normal circadian rhythm of core temperature. It is concluded that the body's ability to remove a heat load is less in the early morning, when the circadian system is in a "heat gain" mode, than in the late afternoon, when heat gain and "heat loss" modes are balanced more evenly.

Fluorometric determination of acid proteinase activity in vulvovaginal candidosis.

Vulvovaginal candidosis is one of the most frequent disorders in obstetrics and gynaecology. Candida albicans is commonly considered to be the true vaginopathic agent. The secreted acid proteinase might be especially relevant in the pathogenesis of vulvovaginal candidosis. A fluorometric determination of acid proteinase activity of clinical C. albicans isolates was carried out during the present work using fluorescamine. L-Leucyl-L-alanine was included as an internal standard and the results were expressed as nmoles of leucylalanine equivalents h-1 per 2 x 10(4) cells. The 13 isolates were taken from non-diabetic, non-pregnant women aged 22-35 years with vulvovaginal candidosis. Candida albicans ATCC 44858 was used as a control. The proteinase activity in culture supernatants was detectable starting from the mid- to late- exponential phase of growth, peaked between 30 and 46 h, and then declined. The control had an activity of 2.72 nmol h-1 per 2 x 10(4) cells, whereas eight of the samples had a lower activity (1.05 nmol h-1 per 2 x 10(4) cells on average) and five of the samples had a higher activity (6.53 nmol h-1 per 2 x 10(4) cells on average). The fluorometric determination of acid proteinase activity was found to be more reproducible and sensitive than the previously used spectrophotometric determinations.