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The effects of disrupting the muscarinic or nicotinic systems on short-term spatial memory were investigated using a delayed matching to position (DMTP) procedure. Rats were trained on the DMTP until stability and then divided into two groups: one group was implanted with an indwelling cannula aimed at the lateral ventricle. The cannulated group received injections of selective muscarinic antagonists (pirenzepine, M1; AFDX 116, M2; UH-AH 37, M1/M3) or hemicholinium-3 (a choline uptake inhibitor). The remaining animals were treated with conventional muscarinic antagonists (scopolamine, methyl scopolamine) or nicotinic channel blockers (mecamylamine, hexamethonium). Scopolamine, methyl scopolamine and UH-AH 37 disrupted all performance parameters in a non-specific but dose related manner. Pirenzepine disrupted accuracy in a delay, but not dose dependent manner, and exerted no other negative effects on performance. Hemicholinium-3-induced performance deficits showed some elements of effects seen following pirenzepine and scopolamine (delay dependent effects on accuracy, some negative effects on other motoric aspects of performance). AFDX 116 and hexamethonium had no significant effects on performance with respect to control. Mecamylamine reduced accuracy and increased response latencies at the highest dose tested. These data indicate that muscarinic antagonists are more effective at disrupting mnemonic performance than nicotinic blockers, and moreover, that distinct muscarinic receptors may have differential effects on cognitive performance.

The effects of the tricyclic antidepressant imipramine and the atypical antidepressant mirtazapine were compared on the performance of rats in three operant procedures: a differential reinforcement of low rates schedule (DRL), a delayed matching to position (DMTP), and simultaneous visual discrimination tasks. Both compounds improved performance in the DRL task in a similar dose-related manner. Imipramine, but not mirtazapine, disrupted performance in the visual discrimination task. Imipramine reduced accuracy and increased response latencies and missed trials. Neither compound effected accuracy in the DMTP; both compounds caused some slowness of responding and imipramine caused several animals to fail to respond in a manner similar to that observed in the visual discrimination task. These data suggest that although imipramine and mirtazapine are similarly effective in putative tests of antidepressant activity, imipramine has a greater tendency to disrupt other aspects of cognitive performance, as well as exert generally depressive effects on operant responding.

Histamine-releasing factors (HRF) that release mediators from human basophils by interacting with IgE have been identified from different cell sources, including lymphocytes, monocytes, thrombocytes and endothelial cells. These factors are studied in view of their potential importance as a stimulus in chronic inflammation. In this report we investigated the qualitative variability of the histamine-releasing activity in the supernatants of activated mononuclear cells. Purified human mononuclear cells of 8 donors were activated with streptokinase/streptodornase (SK/SD) and the supernatants (HRF-MN) were tested for histamine-releasing activity (HRA) in both allergic (RAST positive for inhalant allergens) and nonallergic individuals. Four of the eight HRF-MN supernatants were discriminating, i.e. showing no histamine-release response with nonallergic individuals, whereas four supernatants were not. Two of the HRF-MN supernatants that exhibited discriminating properties were studied in more detail. The response to HRF-MN was tested (1) in a direct bioassay on basophils of allergic (RAST positive for inhalant allergens) and nonallergic individuals and (2) in an indirect bioassay with 70% pure basophils of RAST-negative donors after passive sensitization with sera of allergic donors. An association was found between the response to HRF-MN and the RAST for inhalant allergens: none (0/12) of the RAST-negative but 15/22 of the RAST-positive individuals were HRF-MN responders. The IgE dependency of HRF-MN was shown e.g. by inhibition of passive sensitization by preincubating a responder serum with monoclonal antibody (moAb) anti-IgE MH25-1. Our results are in contrast with findings of other investigators who use pooled supernatants and demonstrated HRF-MN responsiveness with both allergic and nonallergic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

A three-part strategy has been developed to study molecular interactions in biological systems. Isotopically labelled carotenoids or other substances are prepared by organic total synthesis in specific isotopically labelled form. These small molecules are used to assemble the active biological system, which is then studied by physical techniques. A general synthetic scheme for the synthesis of carotenoids, both symmetrical and asymmetrical, selectively labelled with l3C at predetermined positions in the C10-central part, is presented. The synthetic scheme is based on the C15 + Clo + C15 strategy. For symmetrical carotenoids the (210-dialdehyde, 2,7- dimethylocta-2,4,6-tiene- 1 ,%dial 1, is used. For asymmetrical carotenoids two novel synthons are introduced: 2,7-dimethylocta-2,4,6-triene-8-alnitrile 2 and N-methoxy- N,2,7-trimethylocta-2,4,6-triene-8-alamide 3. The use of these synthons is illustrated by the synthesis of labelled p-carotene 4, astaxanthin 5 and spheroidene 6. Some of the structural and functional results that have been obtained by this strategy in the fields of photosynthesis and vision, are given.

To determine seasonal variability in mineralization dynamics of mussel biodeposits, we applied a multiple-element approach measuring mineralization rates of carbon (C), nitrogen (N), phosphorus (P) and silicate (Si) during three periods (March, August and November). The results of this study showed that mineralization rates vary between seasons and between elements and that mineralization dynamics were influenced by both temperature and biodeposit nutrient composition. Mineralization rates were 3.2 ± 0.4 mmol C, 0.17 ± 0.04 mmol N, 0.06 ± 0.02 mmol P and 3.91 ± 3.75 mmol Si per gram biodeposit (DW) per day, which represented 24 % of the particulate organic C and 17 % of the particulate organic N in mussel biodeposits. Seasonal variability was largest for Si mineralization with 60–80-fold higher rates measured in March compared to August and November. This difference is most likely related to the difference in biodeposit nutrient composition. It was furthermore shown that the labile fraction of biodeposits became mineralized after, respectively, 18, 9 and 13 days during the experimental periods in March, August and November. This indicates that temperature enhances biodeposit decomposition with approximately 2–3 times faster turnover at a 10 °C temperature interval (Q10).

Recent work has indicated the significance of IL-4- and IL-5-secreting allergen-specific human Th2 lymphocytes in the control of immune responses to allergens in atopic individuals. The precise allergenic epitopes that activate these allergen-specific Th2 cells are, however, hardly known. We analyzed the epitope-specificity of T lymphocytes specific for Der p II, one of the major allergens of house dust mite Dermatophagoides pteronyssinus. Using a panel of overlapping synthetic peptides that span the entire Der p II molecule, we could demonstrate that polyclonal Der p II-specific T cell lines prepared from the peripheral blood of five atopic patients can react with at least 10 different epitopes of the molecule. Each donor showed a different pattern of reactivity with the synthetic peptides, suggesting that Der p II contains multiple T cell epitopes that may differ from individual to individual. We studied the specificity of the T cell response to Der p II in more detail in one atopic patient using a short term polyclonal T cell line that strongly reacted to one single peptide (116-129) of the allergen. From this patient we established a panel of 11 Der p II-specific TLC. Ten TLC were of the CD3+ CD4+ phenotype and showed a high IL-4/IFN-gamma production ratio, whereas another TLC expressed CD3 and CD8 and failed to secrete substantial IL-4 and IFN-gamma. The use of at least four different TCR V beta gene segments was shown within this panel TLC. All TLC tested recognized the allergen in an HLA-DR1-restricted manner. Although this patient reacted to only one peptide on the polyclonal level, two T cell epitopes were identified on the clonal level by using synthetic peptides and autologous APC to stimulate the TLC. Combining data of CD4/CD8 expression, TCR V beta usage, and epitope specificity, at least six different types of Der p II-specific TLC could be identified within this patient. Binding of IgE to all synthetic peptides of Der p II is low and of low affinity, which may be of particular importance with respect to possible desensitization protocols using such peptides.

The all-E isomers of (12,12′-13C2)-, (13,13′-13C2)-, (14,14′-13C2)-, (15,15′-13C2)- and (20,20′-13C2)astaxanthin were prepared by total synthesis starting from commercially available 99% 13C-enriched acetonitrile, acetic acid and methyl iodide. The astaxanthins were obtained in high purity and with high isotope incorporation (> 99% for every position). For this synthesis, the C15 + C10 + C15 strategy was used. The central C10-synthon, 2,7-dimethylocta-2,4,6-triene-1,8-dial, was symmetrically dilabelled at any position using two new synthetic schemes. The 13C2-enriched C10-dialdehydes were then converted in one step to the 13C2-enriched astaxanthins.

Raising safety levees and reinforcing dykes is not a sufficient and sustainable solution to the intense winter and summer floods occurring with increasing frequency in Eastern Europe. An alternative, creating permanently flooded floodplain wetlands, requires improved understanding of ecological consequences. A 9 month mesocosm study (starting in January), under natural light and temperature conditions, was initiated to understand the role of previous land use (fertility intensity) and flooding water quality on soil biogeochemistry and vegetation development. Flooding resulted in severe eutrophication of both sediment pore water and surface water, particularly for more fertilized soil and sulphate pollution. Vegetation development was mainly determined by soil quality, resulting in a strong decline of most species from the highly fertilized location, especially in combination with higher nitrate and sulphate concentrations. Soils from the less fertilized location showed, in contrast, luxurious growth of target Carex species regardless water quality. The observed interacting effects of water quality and agricultural use are important in assessing the consequences of planned measures for ecosystem functioning and biodiversity in river floodplains.

Echocardiography shows that multiphasic septal movement and a septal to lateral apical systolic left ventricular (LV) motion have a high predictive value for dyssynchrony and the response to cardiac resynchronisation therapy (CRT). Presence of dyssynchrony is also the major marker for CRT response in the presence of scar tissue, provided the interventricular (V-V) pacing interval is optimalised. For atrioventricular (AV) interval optimisation, the velocity-time integral of the transmitral flow has an excellent correlation with invasive LVdP/dt(max). In acute haemodynamic measurements, LVdP/dt(max) shows strongly the effect of AV and V-V optimisation. It also illustrates that the haemodynamic effect of LV pacing when associated with intrinsic conduction over the right bundle is equal to or better than biventricular pacing. We found that once AV and V-V interval were optimised, QRS morphology could be used as a template for optimal therapy. Automated continuous optimisation of the pacing intervals will be the big challenge for the future. (Neth Heart J 2008;16(Suppl1):S32-S35.).

One of the focal points of our group is to reveal the macromolecular organisation of cells by means of cryo-electron tomography. This is the only method that can be used to obtain molecular resolution information of intact cells constituencies in a near-native situation. The tomogram contains a 3D map of the cellular proteome and we are just beginning to explore its potential after we worked for a few years on some technical hurdles of specimen preparation. The other central point of our group is to localize gene product in cellular structures by EM at the highest resolution with gold probes on cryosections. Our main focus is on understanding the molecular machinery and organization within the endomembrane system. We concentrate our work on the transport mechanism within the endocytic pathway and the link to pathogenesis. Cryo-electron tomography of unfixed frozen hydrated cryo-sections and cryo immunogold-EM of aldehyde fixed cells are our main techniques, which allow the subcellular detection and visualization of molecular machines at the highest resolution.

We describe three patients with progressive fibrosing interstitial lung disease (ILD) as the first and only manifestation of systemic sclerosis. In one patient the presence of anti-Scl-70 autoantibodies suggested systemic sclerosis to be the underlying cause of the disease. In the two other subjects, however, anti-Scl-70 antibodies were negative. In these patients the lung disease preceded other manifestations of systemic sclerosis by several years. Diagnosis, prognosis and treatment of systemic sclerosisassociated ILD is discussed.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies.

It is presently unknown which factors determine the occurrence and persistence of asthma in house dust mite-allergic individuals. The level of allergen-specific IgE antibodies does not seem to be decisive for asthmatic symptoms. Moreover, levels of exposure to mite allergens do not seem to differ significantly between asthmatic and non-asthmatics individuals.It was hypothesized that the presence or absence of asthmatic symptoms in house dust mite-allergic patients is associated with quantitative or qualitative differences in the cellular bronchial inflammatory response during the late phase of the allergic reaction. This hypothesis was tested in the bronchial allergen challenge model.Whole lung challenges with house dust mite extract were performed in 52 house dust mite-allergic subjects, of whom 26 had asthma and 26 had perennial rhinitis without asthmatic symptoms. Primary outcomes were parameters for bronchial inflammation in serial samples of induced sputum (cell differentials, eosinophil cationic protein (ECP), interleukin-8 (IL-8), myeloperoxydase (MPO)). In addition, lung function, non-specific bronchial hyper-responsiveness and serial blood samples (eosinophils and IL-5) were analysed.At baseline sputum eosinophils and ECP were similar in both groups but neutrophils and IL-8 were higher in asthmatics. The early bronchoconstriction after allergen challenge was similar in asthma and non-asthmatic rhinitis (median decrease in FEV1: asthma -31.7% vs. non-asthmatics -29.1%, P0.1). The late phase bronchoconstriction was significantly greater in asthma (median decrease in FEV1: asthma -27.6% vs. non-asthmatics -18.9%, P = 0.02). Induction of bronchial hyper-responsiveness was similar in both groups. Bronchial allergen challenge elicited significant increases in sputum eosinophils and ECP, which were indistinguishable for both groups (P0.1 and P = 0.07, respectively). In contrast, higher numbers of neutrophils persisted in asthma 24h after challenge and were accompanied by significant increases in IL-8 and MPO, which were absent in non-asthmatics (difference between groups P = 0.007 and P = 0.05, respectively).Allergen challenge inducedvery similar increases in eosinophils and ECP in induced sputum in allergic asthmatics and in allergic non-asthmatic patients. The difference in bronchial inflammation between asthma and non-asthmatic rhinitis appeared to be more closely related to indices for neutrophilic inflammation.

Analysis of sputum as a specimen containing inflammatory indices has gained considerable interest during the last decade with focus on chronic bronchitis (CB) with or without airway obstruction, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. The nature of the specimens requires expert skill for handling them and for performing analyses. The analysis of spontaneously produced sputum has been performed less frequently than that of induced sputum. This is surprising as several studies have shown that the collection procedures and the assay methods are highly reproducible. There are several valid parameters of sputum analysis that can be applied in research on airway inflammation and in monitoring therapy of patients. Spontaneously produced sputum can be obtained in a simple and non-invasive way, which warrants further efforts to expand the range of applications of its analysis. Here, we will critically review the procedures for collecting spontaneously produced sputum, for handling the samples, and the requirements for assay of sputum components. This will imply the reproducibility of the analysis, the recovery of solutes, the validity of assays in terms of reproducibility and of linearity of the response, the validity of the assays with respect to association with other inflammatory parameters and with clinical parameters, and the usefulness of assays with respect to their response upon treatment of patients.

The purpose of this review is to describe the present state of knowledge regarding host susceptibility factors that may determine the occurrence, development and severity of interstitial lung disease (ILD) caused by exogenous agents. First, host susceptibility may pertain to differences in the delivery and/or persistence of the noxious agent in the lung. The deposition and clearance of inhaled particles or fibres may vary depending on innate anatomical or physiological characteristics, and on acquired changes, such as nasal disease or smoking-induced alterations. Genetically- or environmentally-induced interindividual differences in the expression of pulmonary biotransformation enzymes may form the basis for, or contribute to the risk of, drug-induced interstitial lung disease. Secondly, there are genetic and acquired variations in various enzymatic and nonenzymatic defence systems that protect cells and tissues against oxidative stress, which is often involved in the pathogenesis of interstitial lung disease caused by particles, fibres, metals, organic agents and drugs. Thirdly, the occurrence of immunological sensitization is dependent on both genetic and environmental factors. This has been demonstrated in chronic beryllium lung disease and in hypersensitivity pneumonitis. Fourthly, the propensity of individuals to develop particular types of inflammation, such as granulomas, is probably under genetic control. The regulation and resolution of inflammation and fibrogenesis caused by dust particles are also partly determined by genetic factors, involving cytokine networks and growth factors. In conclusion, although the issue of genetics pervades the entire discussion of host susceptibility, genes are not the only determinants of health and disease. Environmental factors may be equally important in shaping host susceptibility. Therefore, research must be focused on both the genetic bases and the environmental determinants of interstitial lung disease, in order to provide mechanism-based prevention strategies, early detection of, and improved therapy for these conditions.

Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.

A 33-year-old woman presented with a non-specific inflammation of the respiratory passages, which occurred two months after partial colectomy and sigmoidectomy for local stenosis caused by an unclassified inflammatory bowel disease. After other causes of the respiratory symptoms had been ruled out, it was concluded that these were a complication of the bowel disease. Due to the osteoporosis, the patient was given a prolonged treatment with high doses of inhaled corticosteroids instead of systemic corticosteroids. She was treated successfully.

Immature mast cells migrate into tissues where they differentiate into mature mast cells under the influence of local factors. In the airways of asthmatics increased numbers of chronically activated mast cells are located nearby the airway epithelium.The aim of this study was to evaluate whether and, if so, which products released by epithelial cells may affect mast cell proliferation and differentiation.We performed in vitro studies using the human lung mucoepidermoid carcinoma-derived H292 cell line and the immature human mast cell line, HMC-1. Proliferation was assessed by 3H-thymidine incorporation. Differentiation of HMC-1 cells was inferred from tryptase production.Exposure of HMC-1 cells to medium conditioned for 48 h by H292 cells resulted in a reduction of proliferation with 65 +/- 4.9% (mean +/- SEM, n = 9) at day 5. Culturing HMC-1 cells for 8 days in the presence of H292-conditioned medium resulted in morphological changes indicative of differentiation, and in a 3.0 +/- 0.4-fold increase of tryptase production (P = 0.0039, n = 9). Conditioned medium from H292 cells that were stimulated by LPS also inhibited HMC-1 proliferation. Inhibitory antibodies against two mediators from H292 cells, interleukin-6 (IL-6) and stem cell factor (SCF), abolished the increase in HMC-1 tryptase production induced by H292-conditioned medium. Recombinant human (rh) IL-6, but not rhSCF, reduced HMC-1 proliferation with 44% and 13% at day 3 and 5, respectively. Surprisingly, rhIL-6 did not increase HMC-1 tryptase production significantly whereas incubation with rhSCF did (1.5 +/- 0.1-fold, P = 0.002, n = 10) although the increase was less than observed for conditioned medium.Epithelial-derived IL-6 and SCF are implicated in differentiation of HMC-1 cells but additional factors are not excluded. As activated primary bronchial epithelial cells also express IL-6 and SCF, it should be considered that these cells are involved in mast cell differentiation within the airways, particularly in diseases where epithelial cells are activated, such as asthma.

Recently acquired insights into the complex functions of the immune system in the airways and lungs shed new light on the importance of local immunologic defence mechanisms in the maintenance of a normal lung function. Prolonged exposure to inhaled allergens or to aspecific injurious substances in the environment, in particular, may give rise to the onset of immunopathology that may manifest itself only years later. Clinically, the most common of such diseases is allergic asthma. This results from the interaction of an unknown number of genes with specific and aspecific environmental factors. In chronic obstructive pulmonary disease, exposure to such aspecific factors (cigarette smoking) is the most important aetiological factor. Immune-mediated diseases may also develop in the presence of congenital or acquired immunodeficiencies and in idiopathic diffuse pulmonary disorders. In addition, pulmonary disease is often a component of collagen diseases, occupational diseases and idiopathic pulmonary fibrosis. Recently developed investigational techniques such as bronchoalveolar lavage (BAL), cloning experiments on cells obtained by BAL, and ex vivo studies on cytokine production patterns are making an important contribution to the acquired understanding of the immunopathogenesis of pulmonary diseases.

Changes in airway inflammation can be studied with bronchoalveolar lavage, but the widespread use of this procedure is limited by its invasiveness. The aim of this study was to evaluate the usefulness of induced sputum as a non-invasive alternative to bronchoalveolar lavage for studying changes in airway inflammation in patients with asthma. Thirty patients were treated for 12 weeks with an inhaled corticosteroid (fluticasone propionate (FP), 250 microg twice daily) or a short-acting beta-agonist (salbutamol (Sb), 400 microg twice daily) in a double-blind, double-dummy, randomized parallel group study. Sputum induction with hypertonic saline solution was performed twice before treatment and after 4, 8, 10, and 11 weeks of treatment. Bronchoalveolar lavage fluid divided into two pools (first 60 mL portion as bronchoalveolar lavage/bronchial wash (BAL/BW) and subsequent 80 mL as bronchoalveoalar lavage (BAL)) was obtained before and after 12 weeks of treatment. Changes in cell differentials and plasma-protein leakage (alpha2-macroglobulin, albumin, and their ratio (relative coefficient of excretion, RCE)) were analyzed in induced sputum and were compared with changes in BAL/BW and BAL. During treatment with FP, the PC20histamine (interpolated concentration of histamine that caused a fall in FEV1 of 20% of the baseline value) increased (P < .0001), and the percentage of eosinophils (P = .004), levels of (alpha2-macroglobulin (P = .09) and RCE (P = .007) decreased in sputum. These changes were different from those in the Sb group (PC20histamine P

Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma.To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge.Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated.We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge.The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-gamma, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4 h with 1 ng/ml PMA and 1 microg/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN-gamma positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4+ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-gamma+ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-gamma+. We conclude that this method allows detection of intracellular cytokine proteins in both CD4+ and CD8+ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.

Increasing insights into the mechanism underlying the allergen-induced late asthmatic response (LAR) have been gained with implication of activated eosinophils and CD4+ T lymphocytes. However, the patient characteristics that indicate the individual capacity to develop a LAR are not well-defined.In 22 subjects with mild to moderate house dust mite-allergic asthma, we investigated the relationship between the LAR and two other models of late-phase allergic inflammation, i.e. the allergen-specific proliferative response of peripheral blood T lymphocytes in vitro and the late cutaneous response. Non-specific bronchial responsiveness (PC20histamine), lung function (FEV1), peripheral blood eosinophil count, early phase allergic skin sensitivity, and levels of total and specific immunoglobulin E (IgE) were determined prior to bronchial allergen challenge. Serum levels of interleukin-5 (IL-5) were measured before and at several time points after allergen inhalation.A significant correlation was found between the magnitude of the LAR and the allergen-specific proliferative response of peripheral T lymphocytes (r = 0.44, P = 0.04) but not the late cutaneous response. Stepwise-multiple linear regression of the magnitude of the LAR on the parameters analysed at baseline, resulted in a model combining PC20 histamine, early phase allergic skin sensitivity, and the allergen-specific proliferative response of peripheral T lymphocytes (R2 = 0.84, P0.001). No contribution of the late cutaneous response to the prediction of the LAR was found. Serum levels of IL-5 increased significantly at 6 h (P = 0.01) and 24 h (P = 0.003) after bronchial allergen challenge and correlated with the allergen-specific proliferative response of peripheral T lymphocytes in vitro (rho = 0.48, P = 0.02).The findings in this study point to a role of TH2-lymphocyte responses in the development of the allergen-induced LAR. In allergic asthmatic patients, allergen-specific responsiveness of peripheral T-lymphocytes in vitro may serve as a model to determine the individual capacity to develop a LAR after allergen inhalation.

Carotenoproteins, commonly found in marine invertebrate animals, are responsible for green, purple or blue colours. In contrast, free carotenoid exhibit yellow, orange or red colours. An example is provided by the lobster Homarus gammarus. Astaxanthin, which is responsible for the colour, is deep-blue when the lobster is alive and becomes bright orange-red after it is being cooked. The nature of the interactions by which the protein causes the large bathochromic shift of ca 150 nm in the absorption spectrum of astaxanthin is still a matter of debate. This energy shift is comparable to the opsin shift in the visual retinal pigments and bacteriorhodopsin. In these complexes retinal is covalently bound to the protein as a protonated Schiff base whereas in carotenoproteins, the interactions are non-covalent. Previous studies indicated that the β-ring keto groups are essential for the formation of complexes, and favoured a polarisation mechanism [1].

The formation of conjugated diene hydroperoxides and hexanal was compared to the development of aroma profiles during initial lipid oxidation of a vegetable oil and its 40% oil-in-water emulsion at 60°C. The aroma profiles of the oil and the emulsion with and without addition of ascorbic acid or ascorbyl palmitate were compared. The aroma compounds were isolated under a model mouth system and analyzed by gas chromatography/sniffing port analysis. Detectable odors were found and corresponded to 11 and 14 volatile compounds in the oil and the emulsion, respectively. The emulsion had higher lipid oxidation rates than the oil. Addition of ascorbic acid and ascorbyl palmitate had little influence on the aroma composition of the oil. In the emulsion, addition of these compounds resulted in diminished generation of odor active compounds. Results of measurements of conjugated diene hydroperoxides and headspace hexanal corresponded to that of the lipid oxidation rate in general, but predicted insufficiently the alterations in the aroma compositions by antioxidants.

The Netherlands, May 1994 to May 1996.1) To estimate to what extent drug-resistant tuberculosis was acquired or recently transmitted in The Netherlands, 2) to assess the relevance of drug resistance data as routinely collected, and 3) to describe case management.Prospective descriptive study. Patients diagnosed with drug-resistant tuberculosis were interviewed. Information on patient management and contact tracing was collected. IS6110 restriction fragment length polymorphism (RFLP) patterns of all strains were compared with those of the National RFLP library and clusters were analyzed.In total 193 cases were included in the study. Acquired drug resistance (ADR) was rare. Dutch ADR patients reported receiving treatment a long time previously (mean age 58, mean treatment interval 23 years). Most foreign ADR patients had been treated recently in their country of origin. Of 151 primary drug-resistant (PDR) cases, 129 (85%) were foreign-born, of whom few (8%-19%) had been infected in The Netherlands. Few Dutch PDR cases had been infected recently (mean age 49 years). Rifampicin resistance was more frequently observed in foreign ADR cases than in foreign PDR cases (28% vs 5%; P0.001). One third of cases had not been treated according to treatment guidelines.Only a small proportion of drug-resistant cases resulted from recent infection or treatment in The Netherlands. General rates of ADR and PDR do not reflect current Dutch programme performance. For programme monitoring, ADR/PDR rates and their trends must be reported and evaluated in Dutch and foreign patients separately.

Previous studies have shown the usefulness of divalent cobalt isotopes to visualize cerebral damage after stroke. The site of accumulation of cobalt ion is unknown but may be explained by neuronal influx, analogous to that of calcium ion. Additionally, uptake may be due to infiltrating leukocytes or protein-bound cobalt. The aims of this study were to compare 57Co-SPECT with leukocyte SPECT and to compare the SPECT findings with clinical outcome as scored by the Orgogozo scale.Ten patients with a CT scan positive for middle cerebral artery infarcts were included in the study (7 men, 3 women; mean age 70 yr). Technetium-99m leukocyte and cobalt-SPECT (interval 2-4 days) were made with a double-headed gamma camera, after the injection of 10-15 mCi 99mTc-HMPAO-labeled leukocytes and 0.4 mCi 57Co, respectively. Scans were performed within 5-30 days after onset of the first symptoms. Regions of interest (ROI) containing the area of infarction in the slices displaying enhanced radioactivity or the middle cerebral artery (MCA) region in four successive slices were defined for calculating enhancement ratios. The 99mTc leukocyte enhancement ratio (LER) and cobalt enhancement ratio (CER) were defined as the quotient of radioactivity in the ROI and an identical contralateral ROI. The MCA stroke-scale according to Orgogozo was used to assess neurological deficits at the time of scanning and discharge.Cobalt-57 and 99mTc-HMPAO showed uptake in the infarcted brain area in five patients; the quantitative uptake in the infarcted brain area of the two tracers correlated significantly (p0.05). Both the LER and the CER correlated significantly (p0.05) with the Orgogozo score at the time of scanning. Only the LER correlated significantly (p0.05) with the Orgogozo score at discharge.Uptake of cobalt and leukocytes in the peri-infarct tissue suggests that 57Co may visualize a component of the inflammatory response. Divalent 57Co may be convenient to predict clinical prognosis after stroke.

Multiple Sclerosis (MS) is an auto-immune central nervous system (CNS) inflammation. At this moment, MRI is the most accurate paraclinical test in MS to monitor disease activity, although poorly correlated with clinical impairment. PET using Co-55 as a Ca-tracer may visualize Co-transport across the neuronal membrane, Ca-mediated inflammatory processes and passive leakage through a breach in the blood-brain barrier. Co-registration of MRI and Co-PET may actually allow identification of clinically active lesions. MRI and Co-PET were performed as described elsewhere. Based on a statistic parametric mapping (SPM-96)-software package, MRI and Co-PET were superimposed. A semi-automated technique was used to count the MS-lesions. We included four groups of eight MS-patients with relapsing-remitting (RR), primary progressive (PP), progressive relapsing (PR) and secondary progressive (SP) courses and eight healthy volunteers. MS was assessed in terms of impairment using Kurtzke's Expanded Disability Status Scale (EDSS) and Scripps Neurological Rating Scale (NRS). Co-PET displayed focal uptake throughout the MS brain, both in the grey and white matter. All four patients groups (as compared to controls) demonstrated a more inhomogeneous distribution of Co-spots with a tendency to show clustering, most evident in RR-MS. SPM-analysis revealed an essentially different distribution pattern of MS spots on MRI and Co-PET. (Merging of) Co-PET and MRI may eventually form complementary tools for identifying clinically relevant lesions, thus providing a more reliable secondary outcome measure in MS.

Several studies have shown the feasibility of Co-isotopes (55Co and 57Co) in imaging of neuronal damage in stroke, multiple sclerosis, cerebral tumors and trauma. These studies indicate that Co-isotopes allow visualization of brain pathology related to inflammatory processes, reactive gliosis and cell death. Until now, it is not clear if 55Co accumulation occurs in the core of infarction or in the penumbra. Therefore, in the present study, we compared 55Co-PET with functional parameters such as cerebral bloodflow (rCBF) using C15O2, oxygen metabolism (rCMRO2) using 15O2 and cerebral bloodvolume (CBV) using C15O in PET and with the anatomical parameter Gd-MRI.Seventeen patients (11 male; 6 female) age 43 to 84 (mean 69) with middle cerebral artery (mca) stroke, as proven by CT or MRI, were examined with 55Co-PET (0.5-1.0 mCi 55CoCl2), C15O2-, 15O2- and C15O-PET in one session 0-30 days after stroke-onset. Regions of infarction were defined by rCMRO2 being smaller than 65% or rCBF below 45% of the contralateral value and were subsequently superimposed on the cobalt scan. To compare the Cobalt uptake with the Gd-MRI, a realignment program was used that matches the MRI with the bloodflow images. Clinical status was established using the Orgogozo stroke scale at admission and at discharge (at least 6 weeks after admission) and the Barthel index.Eight patients showed a positive Co-PET scan and were used for further analysis. It appeared that Co accumulates in areas with a diminished oxygen metabolism and with a preserved bloodflow. We found Co-uptake in only a part of the Gd enhanced brain tissue with a tendency to be located peripherally or outside the Gd demarcated brain tissue.The results of the present study suggest that Co accumulates into infarcted brain tissue with a rather preserved flow independently of blood-brain barrier breakdown.

The usefulness of prostate specific antigen (PSA) in screening for prostatic carcinoma was studied in 262 inpatients of the department of internal medicine. All patients underwent a rectal digital examination and determination of PSA by the Tandem-E method (Hybritech). The plan was to perform biopsies if there were suspicious findings on the rectal examination or if the PSA value was more than 10 ng/ml. The PSA values wereor = 4 ng/ml in 219 patients (83.6%),4 to 10 ng/ml in 27 patients (10.3%) and10 ng/ml in 16 men (6.1%). In consideration of the severity of disease which limited life expectancy we did not perform a biopsy on 37.5% of the patients with PSA10 ng/ml. 7 patients with prostatic carcinoma were found. Their PSA values varied between 11.2 and 875 ng/ml. The cancer detection rate was highest for the combination of a suspicious rectal examination and a PSA value10 ng/ml (70%).

This study examined the clinical relevance of the determination of free PSA (f-PSA) in addition to total PSA (t-PSA).Both total PSA- and free PSA-values of frozen sera obtained pretherapeutically from 80 patients with carcinoma (PC) and 171 patients with benign hyperplasia of the prostate (BPH) were analysed by means of PSA IRMA and FREE PSA IRMA (IMMUNOCORP/IBL).At 95% specificity (true negative test results), a cut-off value of 16.8 [micrograms/L] was obtained for total PSA (9 patients with BPH [5%] were above this value). For this cut-off value we calculated a sensitivity (true positive test results) of 41%. Using the same criteria for the ratio Q = f-PSA:t-PSA a cut-off of 0.083 was found again at a specificity of 95%. In a second step only patients with total PSA values below the cut-off level of 16.8 [micrograms/L]) were considered. Of these patients 11 of 160 with BPH (missing values = 1) and 13 of 33 with PC (missing values = 2) were below the above mentioned ratio (Q = 0.083). Considering both steps (total PSA and Q) 46 patients with PC were detected correctly and 20 patients with BPH would have been biopsied unnecessarily (positive biopsy rate: 70%).High total PSA levels are a very good indicator for the presence of prostate cancer. There is still concern to improve the differentiation between the diagnosis between BPH and PC, when an intermediate or low value (or = 95% specificity) is observed. The determination of Q is only useful in this range and might be helpful for the clinician's decision to apply or avoid biopsy.

We have developed an in vitro airway epithelial cell model for dimeric immunoglobulin (Ig) A (dIgA) transcytosis that allows the assessment of polymeric Ig receptor (pIgR) gene expression and actual dIgA transport. Tight monolayers of human lung-derived Calu-3 adenocarcinoma cells grown on permeable membranes expressed pIgR mRNA and released more secretory component (SC; P < 0.01) and secretory IgA (sIgA; P < 0.02) into the apical medium than into the basolateral medium. Transcytosis of dIgA was not due to paracellular leakage and was inhibited to approximately 20 and 30% of control values by anti-pIgR antibodies and the competitive ligand pentameric IgM, respectively. Interferon-gamma (IFN-gamma; 200 U/ml) induced pIgR mRNA expression and increased apical release of free SC and sIgA in a dose-dependent fashion (P < 0.0001). Basolateral addition of increasing amounts of dIgA dose dependently increased apical sIgA release (P < 0.0001). These data indicate that Calu-3 monolayers are capable of translocating dIgA through the pIgR. In addition, we show the integrated stimulatory effect of IFN-gamma on pIgR mRNA and protein expression and dIgA transcytosis.

b Abstract: a-Crustacyanin, the 320 kDa astaxanthin-protein from the carapace of the lobster, Homarus gammarus, is the best known of the blue-purple carotenoproteins found in marine invertebrate animals. Reconstituted a-crustacyanin complexes have been prepared from a range of natural and synthetic carotenoids. Only normal C~O carotenoids in the all-E configuration fit into the binding site, though some variation in the ring size, shape and methylation pattern is tolerated. The C(20) and C(20') methyl groups must be present; presumably these are involved in essential steric interactions. The main structural requirement is the presence of keto groups at C(4) and C(4'); these must be conjugated with the main polyene chain. Circular dichroism shows that the carotenoid chromophore experiences a chiral twist, but this is not a major factor in the spectral shift, and that the two astaxanthin molecules in the P-crustacyanin dimer are close together and show some interaction in the excited state. Resonance Raman and NMR spectroscopy of complexes containing I3C-labelled astaxanthins shows that the blue colour can be attributed to perturbation of the ground-state electronic structure of the carotenoid, caused by polarization of the chromophore. The results are consistent with protonation of the C(4) and C(4') keto groups, but the magnitude of the polarization effect is not the same in the two halves of the molecule.

The isotopes 55Co and 57Co have been evaluated for PET and SPECT imaging in several clinical brain studies. For clinical application of cobalt, it is important to know the delivered radiation dose. The biodistribution of 55Co in both rat and humans after intravenous (bolus)-administration was studied. Based on pharmacokinetic data, radiation dose calculations according to the MIRD system are presented. By combining present measurements with literature data on 60CoCl2, the radiation dose delivered by 56CoCl2 (T1/2 78.8 days) and 57CoCl2 (T1/2 = 270 days) could be assessed.Whole-body Co-PET was performed in two healthy volunteers and one rat after intravenous injection of 37 and 3.7 MBq (1 resp. 0.1 mCi) 55Co, respectively. Blood samples were withdrawn during 300 min in humans. In seven rats the 55Co-biodistribution was determined by postmortem analysis. The residence time of the liver (critical organ) was determined in rats and humans. Blood partition-data of 55Co were assessed resulting in basic pharmacokinetic data in humans. Based on these kinetic data, radiation dose was calculated using the MIRD protocol.In both the humans and the rat, the liver and bladder retained the highest fractions of 55Co (about 50% resp. 40% of the administered dose). The liver residence time in humans was 8.6 hr. The free fraction 55Co in the human plasma was at maximum 12%. The total-body mean transit time was 152 min. The volume of the central compartment = 2.8 liter and the steady-state distribution volume = 48 liter.From these results, according to the WHO recommendations for class II studies, 22.2 MBq (0.6 mCi) 55Co and 14.8 MBq (0.4 mCi) 57Co (excluding any radionuclide contamination) can be used.

Cytokines produced by T-lymphocytes play an important regulatory role in inflammation in the airways of asthmatic patients. Our aim was to analyse the cytokine production by T-cell clones from bronchoalveolar lavage fluid (BAL) of patients with allergic asthma and the cytokine production of clones from the patients' peripheral blood (PB), as well as from BAL and blood from healthy controls. In 75 randomly selected CD4+ T-cell clones, we assessed the production of interleukin (IL)-2, IL-4 and interferon-gamma (IFN-gamma). After stimulation with anti-CD3, the clones from the asthmatic patients' BAL (A-BAL) produced significantly more IL-4 and IFN-gamma (median 0.32 and 4.17 ng.mL-1, respectively) than clones from A-PB (0.11 and 1.12 ng.mL-1, respectively). No evidence was found for a dominance of a type 1 or type 2 T-helper cell (Th1- or Th2)-cytokine profile in any of the groups. In three out of nine clones tested, the stimulation with anti-CD2/CD28/phorbol myristate acetate (PMA) induced a shift of the IFN-gamma/IL-4 ratio towards a Th2-type cytokine profile. Our results suggest that the clones from the asthmatic patients' bronchoalveolar lavage were derived from a more differentiated T-cell population. In several clones, the cytokine profile was still modulated by the stimulus applied. Similarly, local conditions in the airways may be involved in directing the cytokine production of T-cells.

BACKGROUND: In an outpatient population, a high frequency of positive skin prick test responses to dog dander was found in the absence of detectable IgE to dog dander in the RAST. The majority of these patients were sensitized to house dust mites (Dermatophagoides pteronyssinus) and had no obvious dog-related allergic symptoms. These findings prompted us to investigate whether dog dander skin test preparations are contaminated with house dust mite allergens in amounts sufficient to cause false-positive skin prick test responses in patients sensitized to house dust mites. METHODS: Antigen detection assays with monoclonal and polyclonal antibodies were used to determine concentrations of the major allergen Can f 1 from dog dander and the major allergens Der p 1 and Der p 2 from house dust mites in five commercially available dog dander skin prick test preparations (A to E). RESULTS: Can f 1 concentrations varied for the different extracts (A: 170 μg/ml, B: 11.1 μg/ml, C: 13.3 μg/ml, D: 3.8 μg/ml, and E: 59.4 μg/ml). Der p 1 was detectable in all extracts (A: 33.4 ng/ml, B: 5.1 ng/ml, C: 29.6 ng/ml, D: 0.4 ng/ml, and E: 1.9 ng/ml), and Der p 2 was detectable in some of the commercially available dog dander skin prick test preparations tested (A: 31.3 ng/ml, B: 3.0 ng/ml, and C: 7.5 ng/ml). The median house dust mite threshold in the skin prick test was found to be 5.8 ng/ml of Der p 1 (range, 3.5 to 20.8 ng/ml) in nine patients tested. CONCLUSION: Contamination of commercially available dog dander skin prick test preparations with the major allergens (Der p 1 and Der p 2) of the house dust mite (D. pteronyssinus) was demonstrated. These contaminations cause false-positive responses to skin prick tests with dog dander in patients sensitized to house dust mite. (J Allergy Clin Immunol 1996;98:1028-34.)

We investigated the relation between IgE reactive with histamine-releasing factor (HRF) and clinical status in patients with asthma or rhinitis. Sera were used to passively sensitize purified, lactic-acid treated basophils. IgE-independent HRF due to chemokines was removed from mononuclear cell supernatants with heparin-Sepharose. IgE-dependent HRF was determined by measuring the increase in histamine release between 1 min and 60 min, which was designated delta HRF. HRF-reactive IgE was demonstrable in nine of 18 patients with allergic asthma, three of 19 patients with nonallergic asthma, five of 17 patients with allergic rhinitis, and none of 19 control patients. The presence of HRF-reactive IgE was associated with: (1) IgE to inhalant allergens; 40% of radioallergosorbent test (RAST)-positive individuals versus 8% of RAST-negative individuals were positive (OR = 7.8, p < 0.005); (2) bronchial sensitivity to histamine in all asthmatic patients (geometric mean PC20: 1.50 versus 0.51 mg/ml; p < 0.005); and (3) bronchial sensitivity to histamine in allergic asthmatic patients (geometric mean PC20: 1.27 versus 0.37 mg/ml, p < 0.02). These findings support the hypothesis that IgE-dependent HRF might contribute to the chronic allergic reaction

Objectives The new ACS prostate-specific antigen (PSA) assay was methodologically and clinically compared with the established Tandem-E PSA assay. We intended to find a possible advantage in primary diagnosis and monitoring the recurrence of prostate cancer due to the additional better recognition of the free PSA form by the ACS PSA assay. Methods Sera of 51 healthy men, 127 patients with hyperplasia, and 82 untreated patients with prostate cancer were analyzed by means of the Tandem-E PSA assay (Hybritech) and the ACS PSA assay (Ciba Corning). Follow-up was done on 12 cancer patients with recurrences. Results Both assays correlated very well (r = .98 for all studied men or hyperplasia patients or cancer patients). However, both assays did not yield comparable values: The ACS assay was characterized by nearly doubled values compared with the Tandem-E assay. At 95% specificity versus patients with benign hyperplasia, cutoff values were obtained as follows: 28.8 ng/mL for the ACS PSA assay and 15.2 ng/mL for the Tandem-E PSA assay. At 95% specificity versus hyperplasia patients, we calculated sensitivities of 60% (ACS PSA) and 63% (Tandem-E PSA). Our longitudinal study revealed more prominent slopes for the ACS assay in patients with recurrent cancer disease. However, using either the ACS assay or the Tandem-E assay, the PSA increase started at the same time. In 1 patient, the increasing ACS PSA accelerated 3 months earlier than the increasing Tandem-E PSA did. Conclusions The ACS assay has obviously higher PSA levels. The clinician is not familiar with such high PSA levels. The specificity-sensitivity profile nonetheless remains unchanged. If the PSA concentration is measured by the ACS assay, patients who relapse will reveal a more rapid PSA increase. Then, recurrent cancer disease may be detected earlier in some cases.

Profilin is a protein found in all eukaryotic cells. Profilin in pollen from grasses, trees, and weeds was found to be largely responsible for crossallergenicity between pollen and vegetable foods. 1, 2 Valenta et al. 3 described cross-reactivity for human IgE antibodies between profilin from birch pollen and profilin from human platelets, using immunoblots as an assay system. We identified 11 sera with IgE to a human profilin-containing fraction in a preliminary investigation. 4 In this study we re-investigated the occurrence of IgE against human profilin in view of the possibility that human profilin, acting as an autoallergen, might be implicated in IgE-dependent histamine-releasing activity. 5 Human profilin was isolated from human platelets by its affinity to poly1-proline. 3 IgE to profilin, as described by others a, 3 and by us, 1 reacts with profilin from pollen and vegetable foods. It was therefore unexpected to find IgE binding to the fraction containing human profilin in sera that were negative for IgE to pollen allergens. 4 Because all of these sera were positive in the RAST forAspergillusfumigatus, other molds, and yeast, we assumed that this might indicate cross-reactivity between profilin from molds and human profilin; alternatively, we considered the possibility that tissue damage by the mold was

The present results support a role for epithelial cells in the activation of T cells in an apparent antigen-independent manner. The transient expression of CD25 indicates a short acting T cells activation. Possibly, this event primes T cells to respond swiftly upon antigen-specific stimulation or to synthesize mediators that affect the local milieu. The molecular mechanism of interaction, although not well defined possibly involves LFA3-CD2 interactions. In T cell activation, via LFA3-CD2 interaction, the density of presented LFA3 molecules is critical. With the increase in the level of expression of LFA3 by epithelial cells this critical density may have been reached. However, based on what is known about T cell activation and CD25 expression in particular it is likely that additional signals such as soluble mediators are required for T cell activation by epithelial cells. Whether this mode of activation occurs in vivo remains to be established by studying ex vivo and in situ material. Not much is known about the expression of LFA3 by epithelial cells in vivo, nor about the stimuli that induce the upregulation of LFA3. In preliminary experiments with fluorescence microscopy we found that neither TNF-alpha nor IL-1 beta induce LFA3 in the same fashion as IFN-gamma. In conclusion, T cell activation by epithelial cells could be an important feature in inflammatory and immunological processes in mucosal systems such as the bronchi and deserves further research.

α-Crustacyanin is the 320 kDa carotenoprotein complex isolated from the carapace of the lobster, Homarus gammarus. It is a water-soluble complex consisting of the carotenoid astaxanthin stoichiometrically bound to the protein. Upon binding to the protein, the astaxanthin, which is orange in solution, (λmax = 488 nm), undergoes a very large bathochromic shift in the visible spectrum resulting in the blue colour of the α-crustacyanin complex (λmax = 632 nm). A detailed resonance Raman (RR) spectroscopic study of unbound astaxanthins and of α-crustacyanin complexes containing astaxanthins labelled with 13C in selected positions has been carried out.

Fel d I, the major cat dander allergen, is recognized by serum IgE of more than 80% of all cat-allergic patients. Because IgE synthesis by B lymphocytes is under the control of T lymphocytes, we studied the specificity and lymphokine production profiles of cat dander-specific T lymphocytes. Polyclonal cat dander-specific T cell lines were found to react with purified Fel d I, but not with cat albumin, the only other characterized cat allergen. Similarly, within a panel of CD4+ T lymphocyte clones (TLC) that was generated from these cat dander-specific T cell lines, 5 of 16 TLC were found to react with Fel d I, and 0 of 16 with cat albumin. The remaining 11 TLC were shown to recognize at least two different proteins. In general, the TLC had a high IL-4/IFN-gamma production ratio, and could recognize the cat dander extract in an HLA-DR, HLA-DQ, or HLA-DP restricted manner. In addition, five distinct T cell epitopes of Fel d I were identified by using a panel of overlapping synthetic peptides of both chains of Fel d I. The data presented here indicate that, even though multiple proteins in cat dander extract are recognized by T lymphocytes of allergic patients, Fel d I, the major IgE binding allergen, is also important in T cell activation. The fact that the cat-specific TLC are Th2-like indicates that these cells may play an important role in the pathophysiology of allergic responses to cat allergens. However, the diversity of HLA-class II restriction of cat dander- and Fel d I-specific TLC and the presence of multiple T cell epitopes in the allergen may complicate future immunotherapies.

In animal experiments, the radionuclide 55Co2+ has been shown to accumulate in degenerating cerebral tissue similar to Ca2+.The potential role of 55Co2+ for in vivo brain PET imaging was investigated in four patients after ischemic stroke.PET showed uptake of 55Co2+ in damaged brain tissue irrespective of blood-brain barrier integrity, as affirmed by CT and MRI.Our preliminary results indicate that 55CoCl2 may prove to be a useful and relatively inexpensive PET radiopharmaceutical for visualization of degenerative processes in brain tissue.

67Ga lung uptake, obtained by a standardized computer-assisted quantitative method of 67Ga scintigraphy, was compared to carbon monoxide diffusion capacity (DLCO) in 45 patients with biopsy proven pulmonary sarcoidosis. Increased 67Ga lung uptake was found in 24 (53%) patients and DLCO was decreased in only 16 (36%) patients. An inverse relationship (r = -0.53; p0.001) was demonstrated between 67Ga lung uptake and DLCO. Eleven patients had an increased 67Ga lung uptake whereas the DLCO values were normal. There was no correlation between 67Ga lung uptake or DLCO and either chest radiographic stage or mode of clinical presentation. On the basis of the normal limits for 67Ga lung uptake and DLCO, 4 subgroups of patients could be identified. The use of the combined investigations may open an opportunity for an early identification of those patients who require therapy. An increased 67Ga accumulation within the lung seems to be considered as a factor indicating risk for pulmonary disability, which is supported by the follow-up of the 4 subgroups of patients.