Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity.

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross-reactivity.

BACKGROUND: Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity. METHODS: A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide-specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross-reactivity between the allergens was investigated by IgE inhibition studies. RESULTS: Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N- and C-terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross-reactivity could be detected. CONCLUSION: Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope-containing area located on similar portions of their structure, but they lack relevant IgE cross-reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen-specific immunotherapy of HDM allergy.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.

[Importance of helicopter rescue]. / Stellenwert der Hubschrauberrettung.

Helicopter emergency medical service (HEMS) have become a main part of prehospital emergency medical services over the last 40 years. Recently, an ongoing discussion about financial shortage and personal shortcomings question the role of cost-intensive air rescue. Thus, the value of HEMS must be examined and discussed appropriately. Since the number of physician-staffed ground ambulances may decrease due to the limited availability of qualified physicians, HEMS may fill the gap. In addition patient transfer to specialized hospitals will require an increasing number of air transports in order to minimize prehospital time. The higher risk ratio for HEMS missions when compared with ground rescue requires a rigorous quality management system. When it comes to missions in remote and exposed areas, the scope of medical treatment must be adjusted to the individual situation. Medical competence is key in order to balance guideline compliant or maximal care versus optimal care characterized as a mission-specific, individualized emergency care concept. Although, medical decision making and treatment is typically based on the best scientific evidence, personal skills, competence, and the mission scenario will determine the scope of interventions suitable to improve outcome. Thus, the profile of requirements for the HEMS medical crew is high.

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors.

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.

Nitric oxide inhibits growth of glomerular mesangial cells: role of the transcription factor EGR-1.

UNLABELLED: Nitric oxide inhibits growth of glomerular mesangial cells: Role of the transcription factor Egr-1. BACKGROUND: In previous studies, we found a close link of early growth response gene-1 (Egr-1) expression to mesangial cell (MC) proliferation. Antiproliferative agents inhibited mitogen-induced Egr-1 expression. Here we investigated the effect of S-nitrosoglutathione (GSNO) on the proliferation of MCs, specifically asking how GSNO regulates the transcription factor Egr-1, which we have previously shown to be critical for the induction of MC mitogenesis. METHODS: The proliferation of MCs was measured by thymidine incorporation and cell counting. Egr-1 mRNA and protein levels were detected by Northern and Western blots. Electrophoretic mobility shift assays (EMSAs) and chloramphenicol acetyltransferase (CAT) assays were performed to test whether GSNO modulates DNA binding and transcriptional activation of Egr-1. RESULTS: GSNO strongly inhibited serum-induced MC proliferation (-84% at 1 mmol/L). A mild inhibition of serum-induced Egr-1 mRNA was observed at GSNO concentrations from 50 to 200 micromol/L, whereas mRNA levels increased again at concentrations above 500 micromol/L. This increased mRNA expression, however, was not translated into Egr-1 protein. Instead, Egr-1 protein induction was inhibited (-40%). EMSAs indicated that GSNO inhibited specific binding of Egr-1 to its DNA consensus sequence. Moreover, transcriptional activation by Egr-1 in CAT assays using a reporter plasmid bearing three Egr-1 binding sites was strongly suppressed by GSNO. CONCLUSIONS: Our data identify GSNO as a potent inhibitor of MC growth with potential beneficial effects in proliferative glomerular diseases. This antimitogenic property is mediated at least in part by inhibitory effects of GSNO on Egr-1 protein levels and by reducing the ability of Egr-1 to activate transcription by impairing its DNA binding activity.

Whole-body PET imaging with [18F]fluorodeoxyglucose in management of recurrent colorectal cancer.

HYPOTHESIS: Metabolic imaging by positron emission tomography (PET) using [18F]fluorodeoxyglucose will be more accurate than anatomic imaging by computed tomography (CT) for detection of recurrent colorectal cancer. More accurate staging of recurrent tumor by PET will lead to more appropriate management decisions. DESIGN: Prospective blinded study comparing PET with CT, using histologic diagnosis, serial CT imaging, and clinical follow-up as criterion standards, with a fully blinded, retrospective reinterpretation of PET studies. Changes in diagnosis resulting from PET findings were correlated with subsequent treatment and surgical findings. Potential cost savings resulting from use of PET for preoperative staging were calculated. SETTING: Private practice in an outpatient tertiary referral center. PATIENTS: A group of 155 consecutive patients with imaging for diagnosis or staging of recurrent colorectal cancer. Twenty-one patient (14%) were excluded due to lack of a criterion standard. Computed tomographic scans were available for comparison for 115 patients. RESULTS: Positron emission tomographic scan sensitivity and specificity were 93% and 98%, respectively, compared with 69% and 96% for CT. Ninety-five percent confidence intervals for the differences between the modalities were 16% to 32% for sensitivity and 1% to 5% for specificity. The sensitivity of both modalities varied with anatomic site of recurrence. Positron emission tomographic scans were true positive in 12 (67%) of 18 patients with elevated serum carcinoembryonic antigen levels and negative CT findings. In 23 (29%) of 78 preoperative studies in which CT showed a single site of recurrence, PET showed tumor at additional sites. At surgery, nonresectable, PET-negative tumor was found in 7 (17%) of 42 patients who had PET evidence of localized recurrence only. Potential savings resulting from demonstration of nonresectable tumor by PET were calculated at $3003 per preoperative study. CONCLUSIONS: Positron emission tomography was more sensitive and specific than CT for detection of recurrent colorectal cancer. Preoperative detection of nonresectable tumor by PET may avoid unnecessary surgery, and thereby reduce the cost of patient treatment.

Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins.

During inflammatory processes of the kidney, lesions of the glomerulus lead to aggregation of thrombocytes and infiltration of macrophages, which can release bioactive mediators. One of these important signalling molecules is lysophosphatidic acid (LPA). Incubation of rat mesangial cells with LPA induced mRNA and protein expression of the early-response genes pghs-2 (for prostaglandin G/H synthase-2/cyclo-oxygenase-2) and egr-1. As shown by antisense experiments, induction of egr-1 was related to the strong mitogenic effect of LPA. LPA-mediated gene expression was inhibited by pertussis toxin, indicating coupling to G-proteins of the Gi family. Specific inhibition of proteins of the small G-protein subfamily Rho with toxin B from Clostridium difficile led to changes in mesangial cell morphology without induction of apoptosis. LPA-mediated expression of pghs-2 and egr-1 was reduced to base-line levels by toxin B, indicating a role for Rho proteins in LPA-mediated gene induction. Of the two mitogen-activated protein kinase (MAPK) pathways investigated, the MAPK kinase-extracellular signal-regulated kinase pathway was involved in the induction of both pghs-2 and egr-1 mRNA expression, as shown by the inhibitory effect of PD98059. Activation of the MAPK p38, however, was only related to pghs-2 expression, whereas egr-1 expression was not affected by treatment of mesangial cells with the specific inhibitor SB203580. Taken together our data provide evidence that LPA-mediated activation of MAPK kinase and Rho proteins leads to the induction of the functionally distinct early-response genes pghs-2 and egr-1, whereas activation of MAPK p38 revealed considerable differences between the regulation of these two genes.

Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.

The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.

Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH.

Elevation of intracellular pH (pHi) enhances the activity of native L-type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L-type Ca2+ channels comprised of either the alpha1c subunits alone or of alpha1c plus beta2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of alpah1c and beta2a subunits. This alkalosis-induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of alpha1c alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L-type Ca2+ channels is conferred by the beta subunit of the channel complex.

Estimating the number of channels in patch-clamp recordings: application to kinetic analysis of multichannel data from voltage-operated channels.

Important kinetic information of voltage-operated ion channels can be obtained by estimating the open probability, the availability, and the first latency, and by applying run analysis. In the case of multichannel patches, estimation of the number of available channels is a prerequisite for the above analysis. Here we describe a method for calculation of the a posteriori probability of the number of available channels in each sweep by using the Bayes formula. This probability serves as a measure for the number of channels and allows for first latency determination and run analysis. The methods described were applied to simulated and experimental data obtained from L-type Ca2+ channel recordings.

[Egr-1 transcription factor regulates the growth of glomerular mesangium cells]. / Der Transkriptionsfaktor Egr-1 reguliert das Wachstum glomerulärer Mesangiumzellen.

BACKGROUND: The transcriptional regulator Early growth response gene-1 (Egr-1) is rapidly and transiently induced by various mitogens in cultured rat mesangial cells (MCs). METHOD AND RESULTS: Here we show Egr-1 induction in an in vivo model of mesangioproliferative glomerulonephritis (GN). A 14.9-fold increase in Egr-1 mRNA was observed 6 days after disease induction. A concomitant increase in Egr-1 protein was demonstrated by immunocytochemistry. Egr-1 was mainly localized to the nuclei of cells in mesangial localization. To test whether Egr-1 directly regulated MC proliferation, we preincubated cultured MCs with antisense oligonucleotides directed against Egr-1. The platelet-derived growth factor (PDGF)-induced increase in Egr-1 mRNA and protein levels was inhibited by 75% and 74%, respectively. At the same time Egr-1 antisense oligonucleotides dose-dependently inhibited MC-proliferation as determined by thymidine-uptake by up to 75%. Control oligonucleotides were without effects on Egr-1 mRNA, protein or MC growth. CONCLUSION: We conclude that Egr-1 induction is a necessary step in the mitogenic signaling cascade in glomerular MCs.

LDL-mediated interaction of Lp[a] with HepG2 cells: a novel fluorescence microscopy approach.

We studied the topography of Lp[a]-LDL-cell interactions by means of fluorescence microscopy, using fluorescence-labeled lipoproteins. In contrast to known methods which are based on noncovalent labeling of lipoproteins by positively charged amphiphiles, the protein moiety of LDL and Lp[a] was covalently labeled with either BODIP-succinimide-ester (green) or rhodamine X iodoacetamide (red). The interaction of the fluorescent lipoproteins with cultured HepG2 cells was studied using a confocal laser scanning fluorescence microscope. LDL and Lp[a], each labeled with a different dye, could be examined separately within a mixture of both lipoproteins during their interaction with HepG2 cells. At 4 degrees C, the majority of both fluorescent particles co-localized and only a few separate LDL- or Lp[a]-binding domains could be observed. Quantification of the amount of fluorescent lipoprotein associated with the cell surface at 4 degrees C showed that binding of Lp[a] was increased in the presence of LDL under these conditions, probably via formation of an Lp[a]-LDL complex. At 37 degrees C, LDL and Lp[a] were taken up by the cells within 10 min. Again the majority of LDL and Lp[a] particles co-localized intracellularly. Only minor amounts of LDL and Lp[a] could be observed separately. As the entire fluorescence of labeled Lp[a] co-localized with excess of LDL in cells, and taking into account the high tendency of LDL-Lp[a] association in solution and on cell surfaces, it is concluded that a significant portion of the internalized Lp[a] is taken up into the cells by the LDL receptor via LDL by a hitchhiking-like process.

Transcription factor Egr-1 regulates glomerular mesangial cell proliferation.

Increase of glomerular mesangial cells (MCs) is a prominent histopathological finding in many types of glomerulonephritis. We have shown previously that expression of the zinc-finger transcription factor, early growth response gene-1 (egr-1), is closely correlated with the proliferation of cultured MCs. To elucidate whether Egr-1 is required for MC proliferation, we inhibited serum-induced Egr-1 expression by phosphothioate-modified antisense oligonucleotides (ODNs). Uptake of antisense ODNs into MCs was demonstrated, and five different egr-1 antisense ODNs were tested for their impact on serum-induced egr-1 mRNA and protein levels and on MC growth. The most potent egr-1 antisense ODN inhibited serum-induced egr-1 mRNA by 68%, protein induction by 58%, and MC replication as measured by [3H]thymidine uptake and cell counts by 78 and 46%, respectively. The effects of antisense ODNs on MC growth correlated closely with their ability to inhibit Egr-1 protein. ODNs acted in a dose-dependent manner, the minimal effective concentration being 1 microM. Control ODNs had no significant effects. In addition, antisense ODNs against egr-1 potently inhibited endothelin-1-induced Egr-1 expression and MC growth. Heparin, a known inhibitor of MC growth, suppressed serum-induced [3H]thymidine uptake by 39% and egr-1 mRNA expression by 44%. We conclude that Egr-1 is an essential part of the mitogenic signal transduction cascade in cultured MCs.

Oxidation of fluorescent glycero- and sphingophospholipids in human plasma lipoproteins: alkenylacyl subclasses are preferred targets.

OBJECTIVES: Oxidation of polyunsaturated fatty acids in lipoproteins is supposed to play a crucial role at the early stages of atherogenesis. The polyunsaturated lipids (PUFAs) become oxidized and, thus, the degree and rate of lipid oxidation depend on their concentration and, probably, on the lipid moiety to which they are attached. DESIGN AND METHODS: To determine the relative oxidation susceptibilities of sphingo- and glycerolipid-bound fatty acyl chains, we used fluorescent analogs of the respective compounds, in which one natural fatty acyl chain was replaced by fluorescent diphenylhexatriene propionic acid. RESULTS: Oxidation susceptibilities of the fluorescent acyl chains in the presence of Cu2+ or AAPH depended, in general, on the phospholipid to which they were bound and the lipoprotein. Phospholipids were oxidized faster in HDL than in LDL or Lp(a). Plasmalogens were more susceptible to oxidation than phosphatidylcholine and sphingomyelin. CONCLUSION: Thus, HDL and plasmalogens may be considered as preferred targets of lipid oxidation before the bulk of polyunsaturated phospholipids (mainly phosphatidylcholine) in LDL is subject to free radical attack.

Concentrations of various arachidonic acid metabolites in menstrual fluid are associated with menstrual pain and are influenced by hormonal contraceptives.

In a pilot study we investigated the association between concentrations of various eicosanoids in menstrual blood with pain and oral contraceptive use. Menstrual fluid was collected on tampons by 12 women who did not use an oral contraceptive but suffered from slight primary dysmenorrhea and by three pain-free women who used an oral contraceptive. Eicosanoids (cyclooxygenase products: 6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin E2, prostaglandin F2 alpha, 13,14-dihydro-15-ketoprostaglandin F2 alpha, 12-hydroxy-heptadecatrienoic acid; lipoxygenase products: 5-, 12-, 15-hydroxy-eicosatetraenoic acid (HETE), leukotriene B4, leukotriene C4, leukotriene D4, leukotriene E4) and female sex steroids (17 beta-estradiol and progesterone) were analyzed by the combined use of high-performance liquid chromatography and radioimmunoassay. 12-HETE was the main arachidonic acid metabolite. An increased metabolism of arachidonic acid was associated with pain, especially when synthesis of 12-HETE was elevated. Oral contraceptive use decreased the synthesis of prostaglandins as well as leukotrienes. The concordant changes of cyclooxygenase and lipoxygenase products in dysmenorrhea or in oral contraceptive use may be explained by an increased or decreased phospholipid metabolism, respectively.

Staging non-small cell lung cancer by whole-body positron emission tomographic imaging.

BACKGROUND: A need exists for an accurate, noninvasive means of staging non-small cell lung cancer. METHODS: A prospective evaluation of regional and whole-body positron emission tomography (PET) imaging for staging lung cancer was carried out in 99 patients. Mediastinal PET and computed tomography findings were compared with results of surgical staging in 76 patients. Those PET and computed tomography findings that indicated possible distant metastasis were compared with biopsy results and the results of clinical and imaging follow-up. RESULTS: Sensitivity and specificity for the diagnosis of N2 disease were 83% and 94% for PET and 63% and 73% for computed tomography, respectively. Positron emission tomography showed previously unsuspected distant metastasis in 11 patients (11%), with no demonstrated false-positive results. Normal PET findings were obtained at distant sites of computed tomography abnormality in 19 patients (19%). Clinical and imaging follow-up in 14 of these patients showed no evidence of metastasis. In 1 case, the PET result proved to be falsely negative. CONCLUSIONS: Imaging with PET was more accurate than computed tomography for diagnosis of mediastinal and distant metastasis. Detection of unsuspected metastatic disease by PET may permit reduction in the number of thoracotomies performed for nonresectable disease.