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.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

BACKGROUND: Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. METHODS: Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. RESULTS: Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. CONCLUSION: Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.

Clinical characteristics and risk profile of patients with elevated baseline serum tryptase.

BACKGROUND: The clinical relevance of elevated basal serum tryptase (eBST ≥ 11.4 ng/ml) often remains unclear. METHODS: BST was assessed in 15,298 patients attending our outpatient clinic. Frequency and severity of anaphylaxis was compared in 900 patients with eBST and 900 patients with normal BST. The prevalence of eBST was evaluated in patients with drug reactions, urticaria, gastrointestinal symptoms or venom allergy. Mast cell-associated symptoms were recorded prospectively in 100 patients with eBST and 100 controls using a standardised questionnaire. RESULTS: 5.9% (n=900) of 15,298 patients had eBST ≥11.4 ng/ml (mean 20 ± 21 ng/ml, 11.4-390 ng/ml). In 47% of them BST was <15.0 and in 78% <20.0 ng/ml. In patients with normal BST (4.5 ± 2.1 ng/ml), mean levels increased continuously with age (0.28 ng/ml per decade; p<0.001). Fatigue, meteorism, muscle/bone ache, vertigo, tachycardia, flush, palpitations, diarrhoea and oedema were associated with eBST (p<0.05 to <0.0001) without significant differences between slightly (11.4-20 ng/ml) or strongly (>20 ng/ml) eBST. eBST was significantly associated with adverse reactions to drugs (34%), radio contrast media (15%) and insect stings (24%) (p<0.05). Anaphylaxis was more common in patients with eBST (21% vs. 14%, p<0.001). The relative role of insect stings, drugs and food as the most important triggers was similar in patients with elevated and normal BST. Severe reactions (grade 3/4) occurred most often in subjects with BST >20 ng/ml (BST <11.4 mg/ml: 2.8%; 11.5-20 ng/ml: 5.9%; >20 ng/ml: 12.4%). CONCLUSIONS: In clinical practice it appears reasonable to assess BST, besides after anaphylactic reactions also in patients suffering repeatedly from vertigo, flush, tachycardia, palpitations, oedema and nausea. Even patients with slightly eBST have a higher risk of anaphylaxis and experience more severe reactions.

[Cross reactions between Hymenoptera venoms from different families, genera and species]. / Kreuzreaktionen zwischen den Giften von Hymenopteren unterschiedlicher Familien, Gattungen und Arten.

Simultaneous reactivity with the venoms of different Hymenoptera is commonly seen in patients allergic to insect venoms. Strong, though individually variable, cross-reactivity occurs between the venoms of different Vespinae species (Vespula, Dolichovespula, Vespa). In Middle Europe, anaphylaxis after European hornet stings is nearly always due to cross-reactivity with Vespula venom. The identification of the primary venom in patients testing positive for Vespula and Polistes (paper wasps) is particularly important in Mediterranean areas. Component-resolved diagnosis with the marker allergens Ves v 5 and Pol d 5 may directly identify the causative venom in the majority of patients. There is substantial cross-reactivity between honeybee and bumblebee venom, sometimes causing allergic symptoms in patients allergic to honeybee venom after accidental bumblebee stings. However, subjects strongly exposed to bumblebees may show bumblebee-specific sensitization and require immunotherapy with bumblebee venom. More than half of all venom-allergic patients show double-positive test results to honeybee and vespid venoms. This may be due to true double sensitization or due to cross-reactivity between homologous allergens present in both venoms and sharing around 50 % sequence identity, i.e. hyaluronidases (Api m 2/Ves v 2), dipeptidyl peptidases (Api m 5/Ves v 3), and vitellogenins (Api m 12/Ves v 6). The clinical relevance of these cross-reactions is unknown. In up to 50 % the double-positivity is caused by clinically irrelevant IgE antibodies against CCDs. Many (though not all) patients with true double sensitization may be identified by means of the species-specific marker allergens Api m 1 and Ves v 1/5. Some Vespula venom-allergic patients may clinically cross-react to fire ant stings (Solenopsis), but otherwise allergen relationships with other ant species are not well studied.

Determination of sIgE to rPhl p 1 is sufficient to diagnose grass pollen allergy.

BACKGROUND: New diagnostic tools such as the basophil activation test (BAT) and component-resolved diagnosis (CRD) are promising for Hymenoptera venom or food allergy. A clear benefit for inhalant allergens has not yet been shown. Our aim was to compare new and established tests for grass pollen allergy. METHODS: Forty-nine patients with grass pollen allergy and 47 controls were prospectively enrolled in the study. A symptom score was calculated for each patient. Conjunctival provocation tests (CPT), skin prick tests (SPT), BAT, and sIgE determination including CRD were performed. Sensitivity and specificity were compared and results were correlated with the symptom score. RESULTS: Single determination of sIgE to rPhl p 1 showed the best balance between sensitivity (98%) and specificity (92%). Use of additional components, such as rPhl p 2 and 5, did not increase sensitivity. Generally, sensitivity of tests was high: SPT 100%, ISAC-112 100%, sIgE to timothy grass 98%, BAT 98%, ISAC-103 84%, and CPT 83%. Specificity ranged from 79% (SPT) to 96% (CPT). All test results and calculated values (e.g. ratio sIgE/tIgE) did not correlate with symptom severity. Asymptomatic sensitization to timothy grass in controls was rare in the CAP (11%) and predominantly due to Phl p 1 sensitization. CONCLUSION: rPhl p 1 was sufficient to diagnose grass pollen allergy, and sIgE patterns were the same in symptomatically and asymptomatically sensitized subjects. The testing of multiple components was of minor importance, and no test correlated with symptom severity.

Inhibition of IgE binding to cross-reactive carbohydrate determinants enhances diagnostic selectivity.

BACKGROUND: Allergy diagnosis by determination of allergen-specific IgE is complicated by clinically irrelevant IgE, of which the most prominent example is IgE against cross-reactive carbohydrate determinants (CCDs) that occur on allergens from plants and insects. Therefore, CCDs cause numerous false-positive results. Inhibition of CCDs has been proposed as a remedy, but has not yet found its way into the routine diagnostic laboratory. We sought to provide a simple and affordable procedure to overcome the CCD problem. METHODS: Serum samples from allergic patients were analysed for allergen-specific IgEs by different commercial tests (from Mediwiss, Phadia and Siemens) with and without a semisynthetic CCD blocker with minimized potential for nonspecific interactions that was prepared from purified bromelain glycopeptides and human serum albumin. RESULTS: Twenty two per cent of about 6000 serum samples reacted with CCD reporter proteins. The incidence of anti-CCD IgE reached 35% in the teenage group. In patients with anti-CCD IgE, application of the CCD blocker led to a clear reduction in read-out values, often below the threshold level. A much better correlation between laboratory results and anamnesis and skin tests was achieved in many cases. The CCD blocker did not affect test results where CCDs were not involved. CONCLUSION: Eliminating the effect of IgEs directed against CCDs by inhibition leads to a significant reduction in false-positive in vitro test results without lowering sensitivity towards relevant sensitizations. Application of the CCD blocker may be worthwhile wherever natural allergen extracts or components are used.

Identification of Bet v 1-related allergens in fig and other Moraceae fruits.

BACKGROUND: Allergy to fig fruit (Ficus carica) has been described in patients allergic to Ficus benjamina or rubber latex but may occur also in pollen-allergic patients. OBJECTIVE: To study the potential cross-reactivity between fig and taxonomically related fruits with the major birch pollen allergen Bet v 1. METHODS: One hundred and eighty-eight patients with or without birch pollen allergy were prick-to-prick tested with fig (F. carica), mulberry (Morus alba), jackfruit (Artocarpus heterophyllus; all family Moraceae) and other pollen-associated foods. Moraceae fruit extracts were separated by SDS-PAGE and tested with patient sera and polyclonal antisera against Mal d 1. Western blot inhibition was performed with Moraceae fruit extracts, birch pollen and recombinant Bet v 1. Putative Bet v 1 homologs in Moraceae fruits were analysed by liquid chromatography-ion trap mass spectrometry. RESULTS: Among 85 patients with isolated birch pollen allergy, 78% had a positive skin test to fresh fig, 10% to dried fig, 91% to mulberry, 91% to jackfruit, 77% to Rosaceae fruits and 83% to hazelnut. Sixty-six per cent of birch pollen-allergic patients positive for fig, reported symptoms after consumption of fresh figs, whereas dried figs were mostly well tolerated. In 60 patients with isolated Ficus benjamina sensitization, the reactivity rates to the same foods were 83-40-0-0-0-0%. None of 32 mugwort pollen-allergic patients reacted to Moraceae fruits. Rabbit anti-Mal d 1 and patient sera reacted to a 17 kDa band in all Moraceae extracts. IgE binding to these proteins was completely inhibited by birch pollen and rBet v 1. Mass spectrometry identified several peptides from the 17 kDa fig, mulberry and jackfruit allergen with respectively 60%, 56% and 76% homology to Bet v 1. CONCLUSION: Fig and other Moraceae fruits contain allergens homologous to Bet v 1 and represent clinically relevant birch pollen-associated foods.

Intranasal mupirocin prophylaxis in elective surgery. A review of published studies.

BACKGROUND: Mupirocin is a natural antibiotic from Pseudomonas fluorescens which is available as a 2% ointment. The drug has been used mainly for topical treatment of the nasal vestibulum in patients carrying methicillin-resistant Staphylococcus aureus (MRSA). However, mupirocin is also active against methicillin-sensitive S. aureus. Nasal colonization with S. aureus has been identified as a significant risk factor for surgical site infection (SSI). METHODS: Randomized trials and sequential cohort studies investigating mupirocin nasal treatment for prophylaxis of SSI in elective surgery in comparison with placebo or no treatment were found by Medline review and additional manual search. Evaluable studies were analyzed regarding the influence of mupirocin on the rate of all SSIs and, specifically, of SSIs due to S. aureus. The effect in cardiosurgical patients was analyzed in detail. RESULTS: Four randomized and seven sequential open cohort studies were analyzed. Study design and mupirocin application schemes varied considerably. Three out of 5 studies carried out in cardiac surgery patients showed a significant reduction in sternotomy site infections. However, all three studies were open sequential cohort studies. By contrast, the only prospective, randomized, double-blind study in cardiosurgical patients showed no benefit of mupirocin. In other surgical disciplines, results were inconclusive or negative. Two studies specifically addressing the prevention of SSIs due to MRSA showed a significant effect of mupirocin on postsurgical infections due to this organism. CONCLUSIONS: Because of the heterogeneity of the studies and the variability of results, no recommendation can be given for the general use of mupirocin in elective surgical patients. Specifically, because of the negative result of a recently published high-quality study, no recommendation can be made for the use of mupirocin in cardiosurgical patients. By contrast, eradication of MRSA before surgery appears to lower SSI rates due to MRSA and is therefore recommended.

[Cross-reactivity to honeybee and wasp venom]. / Kreuzreaktivität auf Bienen- und Wespengift.

About 30-40% of patients with insect venom allergy have IgE antibodies reacting with both honeybee and Vespula venom. Apart from true double sensitization, IgE against cross-reactive carbohydrate determinants (CCDs, alpha1,3-fucosylated N-glycans) with low clinical relevance is the most frequent and often only cause for the multiple reactivity. Venom hyaluronidases have been identified as the most important allergens displaying CCDs, whereas cross-reactions through the hyaluronidases' peptide backbones are less common. If IgE binding to CCDs is disregarded, Vespula venom hyaluronidase is only a minor allergen. In-vitro tests using fucosylated plant glycoproteins (e.g. assessment of specific IgE antibodies by CAP-FEiA to bromelain) are helpful in identifying sera containing CCD-specific IgE, although a positive result (occurring in 70-80% of all double-positive sera) does not reliably exclude true double-sensitization. Reciprocal in-vitro inhibition including non-venom inhibitor proteins rich in CCDs is the method of choice to discriminate between double-sensitization and cross-reactivity. Future in-vitro diagnosis will be markedly improved when recombinant allergens lacking CCDs become commercially available.

[New developments for surgical ablation of atrial fibrillation]. / Zukunftsvisionen und neue Technologien in der chirurgischen Behandlung des Vorhofflimmerns.

The Cox-Maze procedure and less complex modifications have gained widespread use as a treatment modality for patients with concomitant atrial fibrillation. Hypothermic or hyperthermic energy sources play a significant role in rapidly creating linear lesions. Endocardial ablation is easy to perform and effective with different energy sources. Epicardial techniques may simplify the procedure by allowing surgery on a beating heart. But epicardial fat and the heat sink effect of the flowing endocardial blood are obstacles to effective ablation. New devices using bipolar (irrigated) radiofrequency, microwave or laser energy, cryoablation or focused ultrasound are in clinical or pre-clinical stages and permit lesions to be created on a beating heart without cardiopulmonary bypass. Minimally invasive or video-assisted surgical techniques and effective devices for epicardial ablation will enable the treatment of patients suffering from lone atrial fibrillation. To facilitate the comparison between different techniques and devices, guidelines for reporting clinical results are necessary.

Inhibition of cross-reactive carbohydrate determinants (CCDs) enhances the accuracy of in vitro allergy diagnosis.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) as they occur on natural allergens from plants and insects influence the measurement of antigen-specific IgE-antibodies in the context of in vitro allergy diagnosis. When positive results are based solely on the reaction of CCDs with anti-CCD IgE, results must be rated as false-positive. A generally applicable solution to this problem has not yet been presented. METHODS/PATIENTS: Sera of patients for whom an assumed allergy should be verified or ruled out were tested with three methods for specific IgE determination (a multiallergen teststrip format, a single allergen test and an allergen-component array) in the absence and presence of a novel, semi-synthetic CCD-blocker. The study was not prospective and for many patients unequivocal clinical data were missing; the data section thus focusses on few, well-defined patient sera. RESULTS: More than 20% of all patients were tested positive for IgE-anti-CCD antibodies and hence against a multitude of similarly glycosylated allergen extracts in a strip-based multiallergen test. Incubation of these positive sera with the CCD-blocker led to significant reductions of read-out values and in many cases to negative test results. The inhibitory efficiency was highest for the allergen strip test and for the component array. Results remained positive for relevant allergens for which a true sensitization had been indicated by skin tests or other means. The CCD-blocker did not alter the read-outs for unglycosylated allergens or - with CCD-negative sera - for all allergens. CONCLUSION: Elimination of CCD-specific IgE antibodies by means of a synthetic CCD-blocker drastically reduced the number of false-positive in vitro test results without compromising the sensitivity for relevant IgE interactions. Thus, the herein described CCD-blocker constitutes a valuable tool for increasing the test specificity of routine in vitro allergy diagnosis.

Autophosphorylation of creatine kinase: characterization and identification of a specifically phosphorylated peptide.

We report that several different chicken and rabbit creatine kinase (CK)1 isoenzymes showed an incorporation of 32P when incubated with [gamma-32P]ATP in an autophosphorylation assay. This modification was was shown to be of covalent nature and resulted from an intramolecular phosphorylation reaction that was not dependent on the CK enzymatic activity. By limited proteolysis and sequence analysis of the resulting peptides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.5 kDa peptide, spanning a region that is very likely an essential part of the active site of creatine kinase. Homologous peptides were found to be autophosphorylated in chicken muscle-type CK and a mitochondrial CK isoform. Phosphopeptide as well as mutant enzyme analysis provided evidence that threonine-282(2), threonine-289 and serine-285 are involved in the autophosphorylation of CK. Thr-282 and Ser-285 are located close to the reactive cysteine-283. Thr-289 is located within a conserved glycine-rich region highly homologous to the glycine-rich loop of protein kinases, which is known to be important for ATP binding. Thus, it seems likely that the described region constitutes an essential part of the active site of CK.

Identification of creatine kinase isoenzymes in the guinea-pig. Presence of mitochondrial creatine kinase in smooth muscle.

Isoenzymes of creatine kinase (CK, EC 2.7.3.2) in guinea-pig smooth, cardiac and skeletal muscles as well as in brain were analyzed by cellulose acetate electrophoresis and FPLC gel permeation chromatography. In crude tissue extracts of smooth muscles brain type BB-CK and the hybrid form MB-CK were detected, but in enriched mitochondrial fractions from different guinea-pig smooth muscles, mitochondrial type Mi-CK was unambiguously identified. Smooth muscle Mi-CK displayed the same electrophoretic mobility as Mi-CK from brain, which migrates slower than cardiac Mi-CK. Identical to parallel experiments with Mi-CK from cardiac muscle and brain, smooth muscle Mi-CK could be resolved into dimeric and octameric species, the latter being remarkably stable. In contrast to guinea-pig smooth muscles, Mi-CK was not detected in chicken gizzard tissue extracts nor in enriched mitochondrial fractions thereof. The presence of Mi-CK, predominantly in octameric form, in guinea-pig smooth muscles, but not in chicken gizzard, may represent a clue for the different physiological properties of these muscles and may provide the molecular basis for the dependence of the PCr production on oxidative metabolism observed in the guinea-pig taenia caeci.

Effect of okadaic acid on protein phosphorylation patterns of chicken myogenic cells with special reference to creatine kinase.

Okadaic acid and other agents affecting cellular phosphorylation and dephosphorylation processes profoundly changed the phosphoprotein pattern of 32Pi-labelled chicken embryonic skeletal muscle cells. The phosphorylation states of proteins in the lower molecular weight range were especially increased. Immunoprecipitation of cellular extracts with anti-creatine kinase antibodies enabled us to identify creatine kinase (CK) phosphoproteins. B-CK was phosphorylated after treating the cultures with 1-oleoyl-2-acetyl-sn-glycerol, dibutyryl-cAMP, okadiac acid and combinations thereof, but not with 1,2-dioleoyl-sn-glycerol. M-CK was also shown to be phosphorylated. The results indicated that in vivo, CK isoforms in muscle are subjected to control mediated by phosphorylation and dephosphorylation processes.

Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo.

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Median fibrillation frequency in cardiac surgery: influence of temperature and guide to countershock therapy.

OBJECTIVE: This study was designed (1) to investigate the effects of normothermic and hypothermic perfusion on the median frequency of the fibrillating myocardium, and (2) to elucidate whether frequency-guided countershock therapy improves countershock success during the reperfusion phase of cardiac surgery. DESIGN: Prospective, randomized study. SETTING: University hospital cardiac surgery room. PATIENTS: Thirty patients (first part of the study) and 38 patients (second part of the study) scheduled for elective coronary artery bypass surgery. METHODS AND RESULTS: During cardiopulmonary bypass, ventricular fibrillation (VF) was induced at a core body temperature of 34.1+/-0.2 degrees C (normothermia) (n=15) or at a core body temperature of 29.8+/-0.2 degrees C (hypothermia) (n=15). Using fast Fourier transformation of the ECG signal, median fibrillation frequency was recorded continuously for a period of 120 s. At the end of surgery, countershock was performed as soon as VF was recognized on the ECG monitor (X Hz group; n=19) or countershock was not performed until median fibrillation frequency had increased to the threshold of at least 5 Hz (5 Hz group; n=19). Median fibrillation frequency in the normothermic fibrillation group was statistically higher than in the hypothermic group. In the X Hz and 5 Hz countershock group, median fibrillation frequency before the first countershock attempt was 3.6+/-0.2 Hz and 5.4+/-0.1 Hz (p<0.0001), respectively. In the X Hz group, six countershocks resulted in supraventricular rhythm, 10 in VF, two in electromechanical dissociation, and one in asystole. In the 5 Hz group, 16 countershocks resulted in supraventricular rhythm, two in VF, and one in asystole (p=0.008). CONCLUSIONS: During normothermia, median fibrillation frequency is significantly higher than during hypothermic perfusion conditions. During the reperfusion phase of cardiac surgery, countershock success rate is significantly higher when a threshold of at least 5 Hz had been reached before the first countershock attempt.

Leaflet escape in a TEKNA and an original duromedics bileaflet valve.

We report a case of leaflet escape in an Edwards-TEKNA bileaflet valve, in the mitral position. The examination findings of the explanted valve are compared with a similar case of leaflet escape in an original Edward-Duromedics prosthesis. Based on our findings alone, it is not certain whether the TEKNA valve continues to have a higher risk for fracture.

High-dose diltiazem prevents migration and proliferation of vascular smooth muscle cells in various in-vitro models of human coronary restenosis.

BACKGROUND: Restenosis after coronary angioplasty is considered to be caused mainly by increased migration and proliferation of smooth muscle cells (SMC). The concept of local, site-specific delivery of pharmacologic therapies has opened the door for new, high-dose drug regimes. METHODS AND RESULTS: SMC were isolated by enzymatic disaggregation with collagenase/elastase from human coronary plaque tissue of 29 patients (pSMC) and post mortem from the coronary media of 33 corpses (mSMC). Endothelial cells were isolated from human umbilical veins by enzymatic disaggregation with collagenase/dispase. By positive reaction with antibodies against smooth muscle alpha-actin and von Willebrand factor cells were identified as SMC or endothelial cells. In proliferation studies 5-150 micrograms/ml diltiazem was added to the culture media of pSMC, mSMC and endothelial cells. After 5 days there was a significant dose-dependent inhibition of cell proliferation (for pSMC with > 50 micrograms/ml, for mSMC with > 25 micrograms/ml, and for endothelial cells with > 5 micrograms/ml). In migration studies the effect of 5-150 micrograms/ml diltiazem on the velocity of migration of pSMC was investigated over a period of 48 h. Administration of diltiazem at concentrations of 100 and 150 micrograms/ml caused a significant inhibition of the migration of pSMC. The cytoskeletal components smooth muscle alpha-actin, vimentin, and alpha-tubulin of pSMC and the expression of von Willebrand factor of endothelial cells were investigated after an incubation period of 5 days with 50 and 150 micrograms/ml diltiazem. In the transfilter coculture model the effect of 50 micrograms/ml diltiazem on mSMC was investigated after mechanical injury of cocultured endothelial cells. Administration of diltiazem at a concentration of 50 micrograms/ml inhibited the development of a neointimal proliferate in the transfilter coculture model significantly (P < 0.001). CONCLUSIONS: A high dose of diltiazem inhibited the migratory and proliferative activities of coronary SMC significantly. In further experimental studies the effect of locally applied high doses of diltiazem on postangioplasty restenosis should be elucidated.

Expression of intercellular adhesion molecule-1 in human coronary endothelial and smooth muscle cells after stimulation with tumor necrosis factor-alpha.

BACKGROUND: The intercellular adhesion molecule-1 (ICAM-1) is one of several human cell adhesion molecules that play a critical role in the early stages of postangioplasty restenosis. In this study, the in-vitro expression of ICAM-1 in human coronary endothelial cells and human coronary smooth muscle cells (SMC) after stimulation with tumor necrosis factor-alpha (TNF-alpha) was investigated. METHODS AND RESULTS: SMC were isolated from the media of normal human coronary arteries (n = 26) up to 10 h post mortem (HCMSMC) and from human atherosclerotic coronary arteries (HCPSMC) that were extracted by thrombendarterectomy (n = 25). Endothelial cells of human coronary arteries (HCAEC) were purchased from Clonetics (Cell System, Remagen, Germany), and endothelial cells from human umbilical cord veins (HUVEC) were isolated after vaginal delivery. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the proliferative activity of HUVEC, HCAEC, HCPSMC, and HCMSMC, serum-free media was used. After 24 h cell number and cell size distribution were measured in a cell analyzer system. The proliferation of HCPSMC and HCMSMC was increased by TNF-alpha; however, significant differences compared with controls were not reached. The proliferation of HUVEC and HCAEC was significantly reduced by TNF-alpha. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the surface expression of ICAM-1, monoclonal anti-ICAM-1 antibodies (84H10) were used. The expression of ICAM-1 was analyzed using an immunofluorescence microscope. For flow cytometry analysis, 5 x 10(3) cells (100% gated) were analyzed using a fluorescence-activated cell sorter. In control cultures with no stimulation, the expression of ICAM-1 was positive in HCAEC, HCPSMC, HCMSMC, and HUVEC. TNF-alpha stimulated the expression of ICAM-1 in a time- and dose-dependent manner. After maximal stimulation with TNF-alpha (20 ng/ml for 24 h), the expression of ICAM-1 was stronger in HCMSMC than in HCPSMC. CONCLUSIONS: These results suggest that the cytokine TNF-alpha regulates the expression of ICAM-1 in both human coronary endothelial cells and SMC, and could therefore play an important role in the pathophysiology of inflammatory and immune processes in restenosis after angioplasty.