Multidonor FMT capsules improve symptoms and decrease fecal calprotectin in ulcerative colitis patients while treated - an open-label pilot study.

Background: Growing evidence indicates that gut dysbiosis is a factor in the pathogenesis of ulcerative colitis (UC). Fecal microbiota transplantation (FMT) appears to be promising in inducing UC remission, but there are no reports regarding administration using capsules. Methods: Seven patients with active UC, aged 27-50 years, were treated with 25 multidonor FMT capsules daily for 50 days as a supplement to their standard treatment in an open-label pilot study. The primary objective was to follow symptoms through the Simple Clinical Colitis Activity Index (SCCAI). Secondary objectives were to follow changes in fecal calprotectin and microbial diversity through fecal samples and quality of life through the Inflammatory Bowel Disease Questionnaire (IBDQ). Participants were followed through regular visits for six months. Results: From a median of 6 at baseline, the SCCAI of all participants decreased, with median decreases of 5 (p = .001) and 6 (p = .001) after 4 and 8 weeks, respectively. Three of the seven patients had flare-up/relapse of symptoms after the active treatment period. The median F-calprotectin of ≥1800 mg/kg at baseline decreased significantly during the treatment period, but increased again in the follow-up period. The median IBDQ improved at all visits compared to baseline. The fecal microbiota α-diversity did not increase in the study period compared to baseline. All participants completed the treatment and no serious adverse events were reported. Conclusion: Fifty days of daily multidonor FMT capsules temporarily improved symptoms and health-related life quality and decreased F-calprotectin in patients with active UC.

SIRT1 is increased in affected brain regions and hypothalamic metabolic pathways are altered in Huntington disease.

AIMS: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. METHODS: Quantitative real-time polymerase chain reactions were used to assess levels of SIRT1-3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno-associated viral vectors in mice. RESULTS: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin-concentrating hormone (MCH) in the LHA and of brain-derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. CONCLUSIONS: We show that SIRT1 expression is increased in HD-affected brain regions and that metabolic pathways are altered in the HD hypothalamus.

Detailed assessment of hypothalamic damage in craniopharyngioma patients with obesity.

BACKGROUND/OBJECTIVES: Hypothalamic obesity (HO) occurs in 50% of patients with the pituitary tumor craniopharyngioma (CP). Attempts have been made to predict the risk of HO based on hypothalamic (HT) damage on magnetic resonance imaging (MRI), but none have included volumetry. We performed qualitative and quantitative volumetric analyses of HT damage. The results were explored in relation to feeding related peptides and body fat. SUBJECTS/METHODS: A cross-sectional study of childhood onset CPs involving 3 Tesla MRI, was performed at median 22 years after first operation; 41 CPs, median age 35 (range: 17-56), of whom 23 had HT damage, were compared to 32 controls. After exclusions, 35 patients and 31 controls remained in the MRI study. Main outcome measures were the relation of metabolic parameters to HT volume and qualitative analyses of HT damage. RESULTS: Metabolic parameters scored persistently very high in vascular risk particularly among HT damaged patients. Patients had smaller HT volumes compared to controls 769 (35-1168) mm3 vs. 879 (775-1086) mm3; P < 0.001. HT volume correlated negatively with fat mass and leptin among CP patients (rs = -0.67; P < .001; rs = -0.53; P = 0.001), and explained 39% of the variation in fat mass. For every 100 mm3 increase in HT volume fat mass decreased by 2.7 kg (95% CI: 1.5-3.9; P < 0.001). Qualitative assessments revealed HT damage in three out of six patients with normal volumetry, but HT damage according to operation records. CONCLUSIONS: A decrease in HT volume was associated with an increase in fat mass and leptin. We present a method with a high inter-rater reliability (0.94) that can be applied by nonradiologists for the assessment of HT damage. The method may be valuable in the risk assessment of diseases involving the HT.

Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma.

BACKGROUND: Both subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. OBJECTIVE: We aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. METHODS: BALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). RESULTS: We find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models.

Pollen derived low molecular compounds enhance the human allergen specific immune response in vivo.

BACKGROUND: Besides allergens, pollen release bioactive, low molecular weight compounds that modulate and stimulate allergic reactions. Clinical relevance of these substances has not been investigated to date. OBJECTIVE: To elucidate the effect of a non-allergenic, low molecular weight factors from aqueous birch pollen extracts (Bet-APE < 3 kDa) on the human allergic immune response in vivo. METHODS: Birch and grass pollen allergic individuals underwent skin prick testing with allergen alone, allergen plus Bet-APE < 3 kDa, or allergen plus pre-identified candidate substances from low molecular pollen fraction. Nasal allergen challenges were performed in non-atopic and pollen allergic individuals using a 3 day repeated threshold challenge battery. Subjects were either exposed to allergen alone or to allergen plus Bet-APE< 3 kDa. Local cytokine levels, nasal secretion weights, nasal congestion and symptom scores were determined. RESULTS: Skin prick test reactions to pollen elicited larger weals when allergens were tested together with the low molecular weight compounds from pollen. Similar results were obtained with candidate pollen-associated lipid mediators. In nasal lining fluids of allergic patients challenged with allergen plus Bet-APE < 3 kDa, IL-8 and IgE was significantly increased as compared to allergen-only challenged patients. These patients also produced increased amounts of total nasal secretion and reported more severe rhinorrhea than the allergen-only challenged group. CONCLUSIONS: Low molecular compounds from pollen enhance the allergen specific immune response in the skin and nose. They are therefore of potential clinical relevance in allergic patients.

Lixisenatide for the treatment of type 2 diabetes.

Lixisenatide (trade name Lyxumia®), a short-acting glucagon-like peptide 1 receptor (GLP-1R) agonist, was approved for the treatment of type 2 diabetes by the European Medicines Agency in early 2013. In preclinical investigations, acceptable toxicity and carcinogenicity profiles were demonstrated, as well as pancreatic beta cell-preserving actions and favorable effects on glycemic control. Following subcutaneous administration in humans, lixisenatide displays linear pharmacokinetics and an absorption-dependent elimination half-life of 2-3 hours. In clinical trials of up to 1 year duration in patients with type 2 diabetes, treatment with lixisenatide alone and in combination with insulin and various oral antidiabetics conferred significant reductions in HbA1c, fasting and postprandial plasma glucose. In direct comparison with the other GLP-1R agonists on the market (exenatide and liraglutide), lixisenatide appears to be less efficient, or at best non-inferior in terms of reducing HbA1c, fasting plasma glucose and body weight. Nevertheless, lixisenatide confers fewer adverse events than the other currently marketed GLP-1R agonists, while exhibiting a clinically valuable effect on postprandial hyperglycemia.

Comparison of Timothy grass pollen extract- and single major allergen-induced gene expression and mediator release in airway epithelial cells: a meta-analysis.

BACKGROUND: Seasonal allergic rhinitis (AR) is a global health problem and its prevalence has increased considerably in the last decades. As the allergic response with its clinical manifestations is triggered by only a few proteins within natural extracts, there is an increasing tendency for single-component-resolved diagnosis and immunotherapy. OBJECTIVE: As natural exposure is not to single proteins, but to complex mixtures of molecules, we were interested in comparing the activation of respiratory epithelial cells induced by the purified major allergen Phl p 1 with the induction caused by a complete extract of Timothy grass pollen (GPE). METHODS: NCI-H292 cells were exposed to GPE or Ph1 p 1 for 24 h, isolated RNA and cell culture supernatants were used for microarray analysis, multiplex enzyme-linked immunosorbant assay (ELISA) and subsequent analysis. RESULTS: We found 262 genes that showed a GPE-induced change of at least 3-fold, whereas Ph1 p 1-stimulation resulted in 71 genes with a fold induction of more than 3-fold. Besides genes that were regulated by both stimuli, we also detected genes displaying an opposite response after stimulation, suggesting that GPE might be more than purified major allergens with regard to induced immune responses. CONCLUSIONS AND CLINICAL RELEVANCE: Additional components within GPE and the resulting modulation of general processes affecting gene transcription and signalling pathways might be crucial to maintain/overcome the diseased phenotype and to induce the influx of cells contributing to late-phase allergic responses. When the initial process of sensitization is the matter of interest or late-phase allergic responses, one might miss important immune modulatory molecules and their interaction with allergens by applying single components only.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells.

BACKGROUND: Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. OBJECTIVE: To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. METHODS: NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. RESULTS: Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. CONCLUSION AND CLINICAL RELEVANCE: Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment.

Hypothalamic and neuroendocrine changes in Huntington's disease.

Huntington's disease (HD) is neither a fatal hereditary neurodegenerative disorder without satisfactory treatments nor a cure. It is caused by a CAG repeat expansion in the huntingtin gene. The clinical symptoms involve motor-, cognitive- and psychiatric disturbances. Recent studies have shown that non-motor symptoms and signs, such as mood changes, sleep disturbances and metabolic alterations often occur before the onset of overt motor impairments. The hypothalamus is one of the main regulators of emotion, sleep and metabolism, and it is therefore possible that dysfunction of the hypothalamus and neuroendocrine circuits may, at least partly, be responsible for these non-motor symptoms in HD. Several hypothalamic and neuroendocrine changes have now been identified in clinical HD as well as in rodent models of the disease. These changes could be important both in the pathogenesis of HD, constitute biomarkers to track disease progression as well as to provide novel therapeutic targets for this devastating disease. The current state of knowledge in the area of hypothalamic and neuroendocrine changes in both patients and rodent models of HD is summarized in this review, and their potential as targets for novel treatment paradigms are discussed.

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy.

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase.

BACKGROUND: Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens. METHODS: Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients' sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients. RESULTS: Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients' sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients' sera. CONCLUSIONS: Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust.

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen.

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.

Monitoring the content and intake of trace elements from food in Denmark.

The content of cadmium, lead, nickel, mercury and selenium in 83 foods was monitored from 1993 to 1997. In comparison with similar results from 1988 to 1992, a general decrease in lead levels had occurred, whereas the contents of cadmium, nickel, mercury and selenium were stable or declined only slightly. The distribution in dietary intake of the five trace elements was estimated by combining the mean trace element concentrations with food consumption data from 1837 Danes aged 15-80 years. The lead intake for 1993-97 showed a decrease in comparison with similar estimates from the previous monitoring cycles: 1983-87 and 1988-92. The intake of cadmium and mercury decreased to a lesser extent, whereas the intake of selenium and nickel remained unchanged in the same period. The dietary intake of trace elements was compared with the provisional tolerable weekly intake (PTWI). The 95th percentile of the distribution in cadmium intake amounts to 34% of PTWI, which is relatively high, and therefore calls for a more detailed future risk assessment. The intakes of lead and mercury were 11% of PTWI and, like the intake of nickel, did not cause any health concern in the adult population. The Danes ingest close to 100% of the Nordic Nutrition Recommendation for selenium at 50 microg day(-1), and no individuals had an intake less than the lower limit of 20 microg day(-1).

Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen.

The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation. The fragments were differentially recognized by individual patients' sera. By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment. It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule. Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5. rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Effects of beta-hydroxy-beta-methylbutyrate on muscle damage after a prolonged run.

This study examined the effects of supplemental beta-hydroxy-beta-methylbutyrate (HMB) on muscle damage as a result of intense endurance exercise. Subjects (n = 13) were paired according to their 2-mile run times and past running experience. Each pair was randomly assigned a treatment of either HMB (3 g/day) or a placebo. After 6 wk of daily training and supplementation, all subjects participated in a prolonged run (20-km course). Creatine phosphokinase and lactate dehydrogenase (LDH) activities were measured before and after a prolonged run to assess muscle damage. The placebo-supplemented group exhibited a significantly greater (treatment main effect, P = 0.05) increase in creatine phosphokinase activity after a prolonged run than did the HMB-supplemented group. In addition, LDH activity was significantly lower (treatment main effect, P = 0.003) with HMB supplementation compared with the placebo-supplemented group. In conclusion, supplementation with 3.0 g of HMB results in a decreased creatine phosphokinase and LDH response after a prolonged run. These findings support the hypothesis that HMB supplementation helps prevent exercise-induced muscle damage.

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.