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Figure 1 , panel B shows some examples among the 92 analyzed; all other samples were consistent with those in the figure data not shown. Results are the average of water soluble extracts obtained from four producers. Error bars, indicating variability among four assays, are reported above each allergen result. Several peptides were traced and identified in all compounds.

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Detailed information about the nature of additional IgE positive allergens detected by ISAC microarray system can be obtained from www. The quantitative trends observed were quite clear: proteolytic compounds deriving from CAS fragmentation were found to be the most abundant in the less aged samples 6 months and then continuously decreasing with the increasing of the maturation time, whereas non-proteolytic compounds always increased or maintained a constant amount by increasing the maturation time data not shown.

These results on the composition of peptide fraction were in perfect agreement with previous works on PR WSE [12] , [13] and on recent report detailing the evolution along time of the peptide fraction [Sforza S, Cavatorta V, Lambertini F, Galaverna G, Dossena A et al. In agreement with the molecular composition reported above, the main results of SPHIAa experiments performed on the 92 PR WSE are shown in Figure 2 and can be summarized as follows: the tested PR WSE samples showed to contain all proteins known as allergens, including milk whey proteins, as all preparation were equally able to exert IgE inhibition on allergens immobilized on the ISAC microarray Figure 2.


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No major differences were observed depending on the producer. PR extracts older than 20 months behaved on whey proteins similarly to those of 20 months or younger, whilst for CAS a decreasing IgE inhibition ability was found as a function of PR maturation Figure 2 , panels D to F.

Statistical analysis is reported in the Figure 3 legend. Enzymes without any PR preparation and PR WSE at the same maturation stages without enzyme preparations acted as negative and positive controls, respectively. Recorded results of no IgE inhibition for the former and full IgE binding inhibitions, in an almost overlapping way as in Figure 2 , for the latter were obtained data not shown.

Aggregated data are reported in Figure 4. A very partial effect was also recorded during pepsin digestion. All PR tolerant subjects were in the follow up phase. Parents were instructed to continue using the 36 months maturated PR at home by using the commercially available preparations as certified by the producer on the marketed packages.

No specific weekly schedule of PR intake was given assuming its use as normal in Italians daily life. Overall follow up period lasted from December up to December , with the shortest observation time of six months just for one child. None of the PR tolerant subjects reported any minor or major complaint in the follow up phase, all using PR as in the usual family habit.

Testing allergenic molecules for IgE using the microarray approach allowed us to detect sensitizations also to other important allergens from other sources as shown in Figure 5.

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Allergenic molecules were considered either as markers of allergen group sensitization Figure 5 , Panel A or as markers of sensitization to a given allergenic source Figure 5 , Panel B—C; i. Additional details on allergens are given in Figure 5 legend. Such trend was not recorded for allergens causing sensitization by inhalation like those from pollen, mites, mammal epithelia, and moulds. Although other authors already reported tolerance to baked CM proteins in CM allergic children [7] , [19] , [20] , we herein prove for the first time how CM allergic children can tolerate a dairy product as PR cheese, lacking CM high temperature processing.

Although all CM allergenic proteins extracted from PR in its various maturation stages largely retained their ability to bind IgE, the clinical findings prompted us to investigate the clinical tolerance mechanism of this non-baked product. Among the CM proteins, CAS seems to behave differently from the others, as the proteolytic enzymes activity during the maturation process reduces, at least partially, their IgE binding capability.

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The presence of CAS derived peptides indicate that an intense proteolytic process on the CAS fraction is continuously acting, degrading the full proteins and the peptides derived from them, making aged PR very poor in CAS in their native structure. The absence of CAS in the extracts might thus be due in part to the poor CAS solubility in water, but mostly to their proteolytic degradation which takes place during PR manufacturing and maturation; actually, older samples were accordingly poorer in CAS than younger ones.

Conversely, no peptides derived from whey proteins were observed, indicating that the proteolysis does not affect these proteins, and actually whey protein content seems not to be affected by the maturation time. Whey proteins were unexpectedly present in all WSE, albeit the fact that during cheese making only CAS precipitate in the curd and become part of the cheese, whereas most of the whey proteins should remain in solution and used for other diary production purposes.

Anyway, since some whey is always included in the curd, it is quite obvious to presume that part of the whey proteins become trapped in the PR. Overall, PR maturation combined with gastro-duodenal digestion brought to a marked decrease of the IgE binding inhibition capability of WSE. Reduction in the antigenicity of CM proteins can be also achieved using fermentation with lactic acid bacteria [25] , the same microorganisms added to produce PR, though in experimental models BLG seems to be fully degraded [26] , [27].

The clinical outcome of our study clearly shows the beneficial effect of the PR production procedures, though the clinical tolerance seems to be achieved combining the partial proteolysis degrading some CM proteins in highly aged PR, the gut digestion and a specific IgE reactivity to allergenic molecule defined by diagnostic profiling with the ISAC microarray.

BLG IgE reactivity seems to play a critical role at this regard as shown by their presence in sera of PR-reactive patients. These findings are in line with those previously reported of preserved IgE binding by BLG peptides [28]. It provides a preliminary estimate of allergen presence in the source, though the result largely depends on concentration, affinity, and avidity of the allergenic molecules under study.

These IgE binding structures might not trigger mast cell activation. Nevertheless, the usefulness of a multiplexed IgE inhibition retains its validity as it gives the full profile of proteins present or not in a preparation. Notably, in our experiments the availability of fractionated CAS on ISAC 89 lead to differentiate the sensitivity to digestion of each of them. As reported by Bernard et al.

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The biochemical and immunochemical experiments run in the present study show an unexpected presence of seemingly intact whey proteins in matured PR. Recognition of still present whey proteins could thus explain PR allergy. Even in those favourable cases, where a large rate of success is expected when giving patients with fully matured PR, oral food challenge in a supervised setting to assess PR tolerability in CM allergic children is strongly suggested. PR allergic reaction, though rarer, could be very severe, as shown in the present study, being PR a CM protein concentrates.

Conversely, specific markers of PR clinical reactivity are still to be identified. The tested PR tolerant children are going ahead consuming PR without any problem. Further studies are required to verify if regular PR ingestion might anticipate CM tolerance as reported in studies using baked CM products [7]. Published studies on children outgrowing CM allergy have shown levels of CM specific IgE antibodies lower than those of persistent allergic patients [9] , [33] , therefore a low IgE response to all CM proteins on microarray testing, as reported in the present study, could be a good marker of PR tolerability toward the following CM reintroduction.

Beside the problematic CM allergy and not being tolerant to PR ingestion, group C had also a higher number of additional food sensitization, which, with a timely diagnosis, could prevent major allergic reactions to food sources other than CM. A possible explanation is the CAS partial hydrolysis induced by the 36 months PR maturation process, additionally digested in the gastro-intestinal tract.

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Our combined biochemical, immunological, and diagnostic methods should be encouraged in study protocols exploring CM protein clinical tolerance to allow comparability among different approaches. Reintroduction of a tasty food as PR was highly appreciated by tolerant children and their relatives leading to an improvement of the overall family quality of life. Hence, PR could be added to baked products for CM allergic children management. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

Objective and Methods To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Download: PPT. Table 1. Table 2. Table 3. Ninety two PR water soluble extracts from 46 samples as provided by six producers at different maturation ages. Notably, cross-reactions with other members of the antigenic complex Semliki Forest, including Mayaro virus Hassing et al.

Therefore, in regions where these viruses circulate, additional tests may be required to confirm the infection. Pharmacological treatment of pain during the acute phase — The following recommendations were based on a protocol developed by a multiprofessional group for the treatment of pain in chikungunya Brito et al.

The reference protocol is based on the visual analogue scale VAS , in which pain intensity varies from 0—10, with 0 signifying the absence of pain and 10 indicating its maximum expression. Occasionally, the stress caused by the disease tends to lead the patients to overstate their pain intensity. For this reason, we recommend complementing VAS with a clinical examination by a physician.


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  • Technical guidelines are additionally provided in a document developed by a structured work group at the request of the French Ministry of Health Simon et al. Before beginning treatment during this phase, the physician should heed certain precautions to prevent undesirable reactions:.

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    Ask the patient about any history of allergies or adverse reactions to the medication that will be used e. Investigate the existence of any comorbidity that may cause adverse reactions to the medication used during this treatment stage, such as diabetes, arterial hypertension, glaucoma, renal insufficiency, and cardiomyopathies. Avoid corticosteroids during this phase, except for specific manifestations, such as neuritis or encephalopathy. Recall that hydration and absolute rest are crucial components of the integrative approach to the patient.

    Address special attention to monitoring the risk of toxicity due to medication, whether due to overdosage, prolonged use, or self-medication. Mild-intensity pain VAS from 1 to 3 — The two most commonly used analgesics are dipyrone and paracetamol, which offer quite satisfactory results when they are correctly prescribed. For an adult individual weighing more than 60 kg, dipyrone is recommended at a dosage of 1. Paracetamol may be prescribed at dosages of — mg every 4—6 h, not exceeding the maximum daily dosage of 4. Moderate-intensity pain VAS from 4 to 6 — For pain defined as moderate-intensity, i.

    In cases of allergy to dipyrone, tramadol hydrochloride should be used. However, the use of these two drugs must be considered carefully in pregnant or breastfeeding patients. When moderate pain does not subside with the use of dipyrone with paracetamol, the 4—Question Neuropathic Pain Diagnostic Questionnaire DN4 should be applied Bouhassira et al.

    For patients with severe neuropathic pain, 25 or 50 mg of amitriptyline hydrochloride may be combined with the analgesic being used dipyrone or paracetamol. Strict precautions must be followed regarding the use of antidepressants and anticonvulsants. For example, the use of amitriptyline should be avoided in older adults, for whom gabapentin is recommended and must be prescribed in progressive doses.

    Additionally, the use of amitriptyline is not recommended for patients with either a past or existent history of cardiac arrhythmia.