Silencing the major apple allergen Mal d 1 by using the RNA interference approach

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.     

Effect of reduced exposure on natural rubber latex sensitization in health care workers.

BACKGROUND: Knowledge about the long-term course of allergy or sensitization to natural rubber latex (NRL) is insufficient. OBJECTIVE: To investigate the long-term effect of preventive measures on sensitization variables in health care workers who had been diagnosed as having NRL allergy (NRLA) or NRL sensitization (NRLS) without clinical symptoms. METHODS: Repeated follow-up investigations, skin prick tests, and NRL specific IgE serum antibodies were performed in 88 health care workers-33 with NRLA and 55 with NRLS. All workers had been instructed to avoid NRL exposure. At the workplace, powder-free NRL gloves for all other employees were gradually introduced. Re-evaluations were done at 14 +/- 3.7 (N = 86) and 38 +/- 4.0 (N = 78) months after the first examination. RESULTS: At the last follow-up, a loss of skin prick test reactivity to NRL was observed in 1 of 29 subjects with NRLA (3.4%) and 16 of 35 with NRLS (45.7%) with previous skin test reactions (P < .001). Among those subjects who demonstrated a kU/L level (CAP class) equal to or greater than class I to NRL at the initial examination, NRL-specific IgE was absent at the last follow-up in 8 (32.0%) of 25 subjects with NRLA and 14 (38.9%) of 36 with NRLS. At the final examination, we could no longer demonstrate sensitization to NRL by any method in 24 (27.3%) of 88 health care workers. Complete loss of NRL sensitization was less frequent in subjects with NRLA than in those with NRLS (1 of 33 or 3.0% vs 23 of 55 or 41.8%; P < .001). CONCLUSIONS: Implementation of simple preventive measures lowers markers of sensitization to NRL quickly in many health care workers with NRLA or NRLS.     

Identification and characterization of linear B-cell epitopes of beta-globulin, a major allergen of sesame seeds

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.     

Requirement for tumor necrosis factor receptor 2 expression on vascular cells to induce experimental cerebral malaria

Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.     

cDNA arrays: gene expression profiles of Hodgkin's disease and anaplastic large cell lymphoma cell lines

cDNA arrays are a powerful tool for the identification of differentially expressed genes in malignant tumors. We used this technique to study the gene expression profiles of anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD). Gene expression of 11 lymphoma cell lines was analyzed covering 1176 cDNA sequences. Comparing these data to the expression profiles of B- and T-lymphocytes, we identified 27 genes that were deregulated in all cell lines or in a particular entity. For the establishment of gene expression profiles the 27 genes were assigned to four groups composed of genes deregulated in (i) all lymphoma cell lines, (ii) ALCL and HD, (iii) only HD, and (iv) ALCL exclusively. Our results indicate that ALCL and HD share the differential expression of at least five genes. In addition, both entities are characterized by the differentially deregulated expression of four genes in HD and seven genes in ALCL. Because the expression profiling was performed on cell lines, further studies are needed to clarify the biological significance of the differentially expressed genes.     

Randomized Controlled Clinical Trial of Blood Glucose Awareness Training (BGAT III) in Switzerland and Germany

Although both diabetes and the efficacy of medical management are international issues, psycho-educational interventions might be culturally bound. Blood Glucose Awareness Training (BGAT) is a psycho-educational program for patients with type 1 diabetes mellitus. It is focused on improving recognition and management of extreme blood glucose levels, and is the best documented American psycho-educational program for this purpose. A randomized controlled clinical trial of BGAT's long-term benefits in a non-American setting has been lacking. One hundred and eleven adults with type 1 diabetes mellitus from Switzerland and Germany participated. After a 6 months baseline assessment, subjects were randomly assigned to receive either 2 months of BGAT (n = 56) or a physician-guided self-help control intervention (n = 55). BGAT improved recognition of low (p = 0.008), high (p = .03), and overall blood glucose (p = 0.001), and reduced frequency of severe hypoglycemia (p = 0.04), without compromising metabolic control. BGAT reduced both the external locus of control (p < 0.02) and fear of hypoglycemia (p < 0.02). BGAT was efficacious in reducing adverse clinical events and achieving clinically desirable goals in a European, as well as American setting.     

Recruitment of fibre types and quadriceps muscle portions during repeated, intense knee-extensor exercise in humans

To investigate recruitment of slow-twitch (ST) and fast-twitch (FT) muscle fibres, as well as the involvement of the various quadriceps femoris muscle portions during repeated, intense, one-legged knee-extensor exercise, 12 healthy male subjects performed two 3-min exercise bouts at ~110% maximum thigh O₂ consumption (EX1 and EX2) separated by 6 min rest. Single-fibre metabolites were determined in successive muscle biopsies obtained from the vastus lateralis muscle (n=6) and intra-muscular temperatures were continuously measured at six quadriceps muscle sites (n=6). Creatine phosphate (CP) had decreased (P<0.05) by 27, 73 and 88% in ST fibres and 25, 71 and 89% in FT fibres after 15 and 180 s of EX1 and after 180 s of EX2, respectively. CP was below resting mean–1 SD in 15, 46, 84 and 100% of the ST fibres and 9, 48, 85 and 100% of the FT fibres at rest, after 15 and 180 s of EX1 and after 180 s of EX2, respectively. A significant muscle temperature increase (T_m) occurred within 2–4 s at all quadriceps muscle sites. T_m varied less than 10% between sites during EX1, but was 23% higher (P<0.05) in the vastus lateralis than in the rectus femoris muscle during EX2. T_m in the vastus lateralis was 101 and 109% of the mean quadriceps value during EX1 and EX2, respectively. We conclude that both fibre types and all quadriceps muscle portions are recruited at the onset of intense knee-extensor exercise, that essentially all quadriceps muscle fibres are activated during repeated intense exercise and that metabolic measurements in the vastus lateralis muscle provide a good indication of the whole-quadriceps muscle metabolism during repeated, intense, one-legged knee-extensor exercise.     

Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba,Brazil

Carbapenem-resistant Acinetobacter baumannii isolates were obtained from eight patients in two hospitals in Curitiba, Brazil. The isolates were multiresistant, belonged to a single strain, and produced the OXA-23 carbapenemase. Treatment options were limited, although the isolates were susceptible to polymyxin B in vitro. The strain contributed to the deaths of five patients.