A single oral sensitization to peanut without adjuvant leads to anaphylaxis in mice.

BACKGROUND: A model of peanut food allergy has been developed in mice using a simple sensitization protocol leading to a quantitatively measurable allergic response. METHODS: C3H/HeJ mice received a single intragastric administration of whole peanut (80 mg) without adjuvant. Two weeks later, intraperitoneal challenge with peanut extract led to a severe anaphylaxis. RESULTS: Anaphylactic reaction was evidenced by vascular leakage, severe clinical symptoms, a drop in body temperature, a decrease in breathing rate and also by increased concentrations of serum mouse mast cell protease-1. Sensitization to peanut was demonstrated by positive skin tests (ear swelling test and intradermal skin testing) and increased peanut-specific IgE levels. CONCLUSIONS: Thus, we obtained a model of severe peanut hypersensitivity within 2 weeks following single oral exposure without adjuvant. This model may be useful for further basic and applied studies on peanut allergy.

Differential localization of autolyzed calpains 1 and 2 in slow and fast skeletal muscles in the early phase of atrophy.

Calpains have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. However, limited data are available about the specific involvement of each calpain in the early stages of muscle atrophy. The aims of this study were to determine whether calpains 1 and 2 are autolyzed after a short period of muscle disuse, and, if so, where in the myofibers the autolyzed products are localized. In the rat soleus muscle, 5 days of immobilization increased autolyzed calpain 1 in the particulate and not the soluble fraction. Conversely, autolyzed calpain 2 was not found in the particulate fraction, whereas it was increased in the soluble fraction after immobilization. In the less atrophied plantaris muscle, no difference was noted between the control and immobilized groups whatever the fraction or calpain. Other proteolytic pathways were also investigated. The ubiquitin-proteasome pathway was activated in both skeletal muscles, and caspase 3 was activated only in the soleus muscle. Taken together, our data suggest that calpains 1 and 2 are involved in atrophy development in slow type muscle exclusively and that they have different regulation and protein targets. Moreover, the activation of proteolytic pathways appears to differ in slow and fast muscles, and the proteolytic mechanisms involved in fast-type muscle atrophy remain unclear.

Predictive value of skin prick tests using recombinant allergens for diagnosis of peanut allergy.

BACKGROUND: Current diagnosis of peanut allergy relies on natural extracts that lack standardization. Recombinant DNA technology allows production of pure biochemically characterized proteins. Their usefulness for peanut allergy diagnosis is not established. OBJECTIVE: This study aimed to evaluate the diagnostic value of the 3 major recombinant peanut allergens. METHODS: Recombinant (r) Ara h 1, rAra h 2, and rAra h 3 were produced according to the recommendations of good manufacturing practice for recombinant allergens. Skin prick tests (SPTs) and IgE ELISA assays were performed in 30 patients with peanut allergy and 30 control subjects without food allergy: 15 nonatopic and 15 sensitized to birch pollen. Disease severity was graded by clinical scoring. RESULTS: All patients with peanut allergy showed positive SPT results to rAra h 2; 40% reacted with rAra h 1 and 27% with rAra h 3. No control subjects reacted with any of the recombinant allergens. Monosensitization to rAra h 2 was observed in 53% of patients. Neither SPT size nor levels of specific IgE were correlated with the disease severity. However, patients with monosensitization to rAra h 2 had a significantly lower severity score than polysensitized subjects and a lower level of specific IgE against peanut extract and rAra h 2. CONCLUSION: Skin prick tests to individual recombinant peanut allergens appear to be a safe and effective diagnostic tool. Cosensitization to rAra h 2 and rArah 1 and/or rAra h 3 is predictive of more severe reactions. CLINICAL IMPLICATIONS: Recombinant peanut allergens can be used by SPTs for diagnosis and evaluation of allergy severity.

Regulation of the actin-myosin interaction by titin.

Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin-myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca(2+)-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex.

Evidence for a direct but sequential binding of titin to tropomyosin and actin filaments.

Titin is a giant molecule that spans half a sarcomere, establishing several specific bindings with both structural and contractile myofibrillar elements. It has been demonstrated that this giant protein plays a major role in striated muscle cell passive tension and contractile filament alignment. The in vitro interaction of titin with a new partner (tropomyosin) reported here is reinforced by our recent in vitro motility study using reconstituted Ca-regulated thin filaments, myosin and a native 800-kDa titin fragment. In the presence of the tropomyosin-troponin complex, the actin filament movement onto coated S1 is improved by the titin fragment. Here, we found that two purified native titin fragments of 150 and 800 kDa, covering respectively the N1-line and the N2-line/PEVK region in the I-band and known to contain actin-binding sites, directly bind tropomyosin in the absence of actin. We have also shown that binding of the 800-kDa fragment with filamentous actin inhibited the subsequent interaction of tropomyosin with actin, as judged by cosedimentation. However, this was not the case if the complex of actin and tropomyosin was formed before the addition of the 800-kDa fragment. We thus conclude that a sequential arrangement of contacts exists between parts of the titin I-band region, tropomyosin and actin in the thin filament.