Characterization of Ras k 1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species.

BACKGROUND: Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. OBJECTIVE: The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). METHODS: Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. RESULTS: Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. CONCLUSIONS AND CLINICAL RELEVANCE: Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV.

Pru p 3, a marker allergen for lipid transfer protein sensitization also in Central Europe.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.

Factors affecting the relationship between quantitative polymerase chain reaction (qPCR) and culture-based enumeration of Enterococcus in environmental waters.

AIMS: To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class (qPCR or culture) and (iii) between-class difference. METHODS AND RESULTS: We analysed 306 samples using two culture-based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both qPCR methods indicated beach posting while both culture methods did not. CONCLUSION: Measurement target difference (DNA vs growth) accounted for the majority of the qPCR-vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to quantify the frequency with which culture-vs-qPCR discrepancies can be attributed to target difference - vs - method variability.

Oxidative stress markers and micronutrients in maternal and cord blood in relation to neonatal outcome.

BACKGROUND/OBJECTIVES: Oxidative stress and micronutrient deficiencies have been related to lower birth weight (BW), small for gestational age (SGA) offspring and preterm delivery. SUBJECTS/METHODS: The relation between neonatal outcome (BW, head circumference, SGA, preterm delivery) with markers of oxidative stress and micronutrients in maternal and cord blood was to be examined. Oxidative stress markers (protein carbonyls (PrCarb), 3-nitrotyrosine (3NT), malondialdehyde (MDA)), total protein concentration and lipid-soluble micronutrients (carotenoids, retinol, tocopherols) were measured in 200 newborns (11% preterms, 13% SGA) and 151 mothers. Associations between target parameters in cord plasma and maternal serum with BW, head circumference and risk of being SGA or preterm were explored. RESULTS: Maternal protein concentration, PrCarb, MDA and all lipid-soluble micronutrients were significantly higher compared with newborns, except for 3NT, which was significantly elevated in newborns. Newborn parameters correlated positively with those of mothers. Preterms had lower proteins and retinol but higher PrCarb than terms. Maternal PrCarb and retinol were inversely associated with BW and head circumference. Mothers with PrCarb, MDA and retinol in the highest quintile had a 3.3-fold (0.9; 12.1), 2.1-fold (0.7; 6.4) and 3.3-fold (1.2; 9.4) risk, respectively, for delivering an SGA newborn, whereas the lowest quintile of retinol in cord blood was associated with an increased risk for preterm delivery. CONCLUSIONS: Oxidative stress (elevated PrCarb) was associated with lower BW/head circumference and SGA. Inadequate hemodilution may explain the inverse relation of maternal retinol with BW and head circumference, and the association between highest maternal retinol and risk for SGA.

5-Methyltetrahydrofolate and thiamine diphosphate in cord-blood erythrocytes of preterm versus term newborns.

BACKGROUND/OBJECTIVES: A low folate or low thiamine status may be associated with the risk of preterm delivery, small for gestational age (SGA) offspring and adverse pregnancy outcomes. SUBJECTS/METHODS: 5-Methyltetrahydrofolate (5MTHF) and thiamine diphosphate (TDP) were measured directly in cord-blood erythrocytes (CBEs) of early preterm (n=26; <32 weeks gestational age; including 50% multiple births), late preterm (n=38; 32 to <37 weeks; including 24% multiple births) and term newborns (n=60, 37-42 weeks) via high-performance liquid chromatography and fluorescence detection. Associations between 5MTHF and TDP with gestational age, newborn anthropometrics (birth weight, newborn's length and head circumference) and risk of being SGA were explored. RESULTS: Group comparison as well as multivariate linear regression analysis of cord-blood vitamins revealed that 5MTHF was significantly lower in late preterms compared with terms but did not differ between singletons and multiples. TDP tended to be higher in preterms than in terms and lower in multiples than in singletons in both early and late preterms. Multivariate analysis on birth outcomes showed that 5MTHF was significantly positively associated with gestational age, birth weight and newborn's length. 5MTHF, increasing gestational age and parity were associated with a significantly reduced risk for being SGA, while TDP, multiple births and gender were not associated with the risk for being SGA. CONCLUSIONS: Higher CBE concentrations of 5MTHF were associated with improved birth outcomes. Lower TDP concentrations were observed in multiple births. Future studies evaluating cord-blood vitamin concentrations and their associations with birth outcomes should additionally include dietary intakes and maternal blood concentrations at different stages of pregnancy.

[Telemedicine in acute stroke care--a health economics view]. / Telemedizin in der Schlaganfall-Akutversorgung--eine gesundheitsökonomische Bestandsaufnahme.

Specialized stroke units offer optimal treatment of patients with an acute stroke. Unfortunately, their installation is limited by an acute lack of experienced neurologists and the small number of stroke patients in sparsely populated rural areas. This problem is increasingly being solved by the use of telemedicine, so that neurological expertise is made available to basic and regular care. It has been demonstrated by national and international pilot studies that solidly based and rapid decisions can be made by telemedicine regrading the use of thrombolysis, as the most important acute treatment, but also of other interventions. So far studies have only evaluated improvement in the quality of care achieved by networking, but not of any lasting effect on any economic benefit. Complementary to a medical evaluation, the qualitative economic assessment presented here of German and American concepts of telemetric care indicate no difference in efficacy between various ways of networking. Most noteworthy, when comparing two large American and German studies, is the difference in their priorities. While the American networks achieved targeted improvements in efficacy of care that go beyond the immediate wishes of the doctors involved, this was of only secondary importance in the German studies. Also, in contrast to several American networks, the German telemetry networks have not tended to be organized for future growth. In terms of economic benefits, decentralized organized networks offer a greater potential of efficacy than purely local ones. Furthermore, the integration of inducements into the design of business models is a fundamental factor for achieving successful and lasting existence, especially within a highly competitive market.

The source matters: no impact of the CCL2/MCP-1-1-2518G polymorphism of the donor on renal allograft outcome during the first year after transplantation.

Chemokines are involved in the recruitment of inflammatory cells to vascularized allografts. The chemokine CCL2/MCP-1 is expressed during allograft dysfunction, which is associated with the recruitment of inflammatory cells. Both intrinsic renal cells (donor origin) as well as infiltrating inflammatory cells (recipient origin) can be a source of CCL2/MCP-1. We previously demonstrated that the recipient MCP-1-2518G polymorphism is associated with increased CCL2/MCP-1 production by inflammatory cells and decreased renal allograft survival. We evaluated the impact of the MCP-1-2518G polymorphism in donor cells on renal allograft outcomes. We enrolled 252 recipients of kidney allografts in this retrospective study who had received grafts from 152 cadaveric donors. The CCL2/MCP-1 genotype was assessed using genomic DNA isolated from cryopreserved donor splenocytes. Outcome parameters studied were acute biopsy proven rejection (Banff criteria), serum creatinine, and glomerular filtration rate (GFR) at 1 year after transplantation, allograft loss, and death. MCP-1-2518 genotypes were in HW equilibrium. A/A was present in 125 (49.6%), A/G in 107 (42.5%), and G/G in 20 (7.9%) donor kidneys. There were no significant differences in the number of rejection episodes, the number of allograft losses, serum creatinine, GFR, or overall survival 1 year after transplantation. In contrast with the detrimental effect of the CCL2/MCP-1 polymorphism of the recipient, the CCL2/MCP-1 polymorphism of the donor has no impact on the allograft outcome during the first year after transplantation. The impact on the long-term outcomes needs further evaluation.

Association of low-grade inflammation with nephropathy in type 2 diabetic patients: role of elevated CRP-levels and 2 different gene-polymorphisms of proinflammatory cytokines.

BACKGROUND: Chronic inflammatory processes are thought to play a key role in the development of micro- and macrovascular complications in type 2 diabetes mellitus. An association between low -grade inflammation and type 2 diabetes has been described in some studies. We assayed the association of two frequent polymorphisms in proinflammatory cytokines: the interleukin 6 G(-174)C promoter polymorphism [IL-6G(-174)C], the exon 2 interleukin receptor antagonist insertion deletion polymorphism [IL1RA]) and serum CRP levels with the prevalence of diabetic nephropathy in patients suffering from type 2 diabetes mellitus. SUBJECTS AND METHODS: A total of 141 patients with type 2 diabetes mellitus, with and without diabetic nephropathy was genotyped for the above mentioned polymorphisms: 66 with normoalbuminuria, 31 with microalbuminuria and 44 with macroalbuminuria. CRP levels were analysed by a high sensitivity - immunnephelometric assay. RESULTS: While a significant association be-tween macroalbuminuria and CRP could be observed (p<0,015), no associations were found between IL-6G(-174)C or IL1RA genotype and any stage of nephropathy. CRP-levels were similar in the 3 different IL-6G(-174)C genotypes as well as in the 2 IL1RA genotypes. CONCLUSIONS: In type 2 diabetic subjects elevated CRP levels are associated with an increased prevalence of albuminuria. The two investigated proinflammatory polymorphisms do not seem to contribute to initiation of nephropathy in type 2 diabetic patients but we cannot exclude effects of these polymorphisms on course of nephropathy.

Haem oxygenase-1 genotype and cardiovascular adverse events in patients with peripheral artery disease.

BACKGROUND: A functional GT dinucleotide length polymorphism in the haem oxygenase-1 (HO-1) gene promoter is thought to be involved in the pathogenesis of cardiovascular disease. Short (< 25) (GT)n repeats are suggested to facilitate enhanced HO-1 up-regulation in response to injury and confer potent anti-inflammatory and antioxidative effects. MATERIALS AND METHODS: We investigated the association between the HO-1 GT-polymorphism and cardiovascular outcome in 472 patients with advanced peripheral artery disease. Cardiovascular risk profile and DNA samples for determination of the HO-1 genotype (carrier vs. noncarrier of a short (GT)n repeat allele) were obtained at baseline, and patients were followed for median 21 months for the occurrence of coronary events (myocardial infarction, percutaneous coronary interventions and coronary artery bypass graft), cerebrovascular events (stroke or carotid revascularization) and all-cause mortality. RESULTS: Coronary events occurred in 48 patients (9%), cerebrovascular events in 40 patients (9%) and 59 patients (13%) died. In total, 173 major adverse cardiovascular events (MACE) occurred in 133 patients (28%). Carriers of the short (GT)n repeat allele had a 0.46-fold reduced adjusted hazard ratio for coronary events (P = 0.016) as compared to noncarriers. No significant difference was found for cerebrovascular events, mortality and overall MACE. CONCLUSION: Apparently, the HO-1 genotype exerts potentially protective effects against coronary adverse events in patients with peripheral artery disease. Homozygous and heterozygous carriers of < 25 (GT)n repeats had lower rates of myocardial infarction, percutaneous coronary interventions and coronary bypass operations compared to patients with longer (GT)n repeats.

[The concept of myocardial infarct rehabilitation in phase III]. / Konzept der Herzinfarktrehabilitation in der Phase III.

The benefit of outpatient rehabilitation in coronary artery disease is well documented in literature. Despite this, there is evident lack of rehabilitation facilities during phase III WHO in our country. Departments of Physical Medicine and Rehabilitation seem to be well suited to run ambulant rehabilitation programmes. Therefore we developed a comprehensive rehabilitation programme including physical training, dietary counselling and teaching lessons for patients. Physical training is strictly aerobic according to the guidelines of medical training therapy. Training time will rise systematically. To keep risks low, precise in- and exclusion criteria are defined. Regular training for the staff is mandatory. Uniformity of therapeutic interventions and documentation provide quick evaluation of therapeutic success.

Sister chromatid exchange (SCE) induced by laser-UV-microirradiation: correlation between the distribution of photolesions and the distribution of SCEs.

Small, medium, and large nuclear areas comprising approximately 5, 30, or 80% of the total area of the interphase nuclei of Chinese hamster cells (M3-1) cultivated in vitro were irradiated with a laser-UV-microbeam of wavelength 257 nm. The DNA of the cells was substituted with 5-bromodeoxyuridine (BrdUrd) for 1 cell cycle in one set of experiments. After microirradiation the cells were grown for a second cycle in medium without BrdUrd (protocol A). In a second set, cells with nonsubstituted DNA were microirradiated and grown for 2 additional cycles, the first in the presence, the second in the absence of BrdUrd (protocol B). In situ chromosome preparation and differential chromatid staining was subsequently performed. The induction of sister chromatid exchanges (SCEs) was found to be dependent on both the ultraviolet (UV) dose and the spatial distribution of the UV energy within the cell nucleus. Following both protocols the average number of chromosomes with SCEs was significantly higher after microirradiation of a large nuclear area as compared to microirradiation of a small nuclear area. In the latter case, multiple SCEs were noted on individual chromosome arms at the first postirradiation mitosis (protocol A). In other cells, especially at higher doses, protocol A resulted in shattering of a few closely neighbored chromosomes which were surrounded by intact ones with normal SCE levels. Microirradiation of medium-sized nuclear areas produced high levels of SCEs over a number of chromosomes which still appeared spatially related in a part of the metaphase spread. Finally, high SCE levels could be observed over most or all chromosomes when a large nuclear area (up to 100%) was exposed to the microbeam. Following protocol B the increase of SCEs was much less pronounced. Microirradiation of a small part of the cytoplasm in addition to the nuclei did not induce SCEs. Our results support the concept (i) that interphase chromosomes occupy distinct nuclear domains and indicate (ii) that the induction of SCEs by UV light is restricted to microirradiated chromatin.