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Background ESR1 mutations have been identified as mechanism for endocrine resistance and are also associated with a decreased overall survival. We assessed ESR1 mutations in circulating tumor DNA (ctDNA) for impact on outcome to taxane-based chemotherapy in advanced breast cancer patients. Methods ESR1 mutations were determined in archived plasma samples from patients treated with paclitaxel and bevacizumab (AT arm, N = 91) in the randomized phase II ATX study. Samples collected at baseline (n = 51) and at cycle 2 (n = 13, C2) were analyzed using a breast cancer next-generation sequencing panel. This study was powered to detect a benefit in progression-free survival (PFS) at six months for patients treated with paclitaxel/bevacizumab compared to historical trials with fulvestrant. PFS, overall survival (OS), and ctDNA dynamics were exploratory analyses. Results PFS at six months was 86% (18/21) in patients with an ESR1 mutation detected and 85% (23/27) in wildtype ESR1 patients. In our exploratory analysis, median progression-free survival (PFS) was 8.2 months [95% CI, 7.6–8.8] for ESR1 mutant patients versus 8.7 months [95% confidence interval (CI), 8.3–9.2] for ESR1 wildtype patients [p = 0.47]. The median overall survival (OS) was 20.7 months [95% CI, 6.6–33.7] for ESR1 mutant patients versus 28.1 months [95% confidence interval (CI), 19.3–36.9] for ESR1 wildtype patients [p = 0.27]. Patients with ≥ two ESR1 mutations had a significantly worse OS, but not PFS, compared to those who did not [p = 0.003]. Change in ctDNA level at C2 was not different between ESR1 and other mutations. Conclusions Presence of ESR1 mutations in baseline ctDNA might not be associated with inferior PFS and OS in advanced breast cancer patients treated with paclitaxel/bevacizumab.

Background Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient–derived plasma samples. Methods Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. Results Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane–based methods, samples extracted with magnetic beads–based kits revealed an overall lower total ccfDNA yield (−29%; P Conclusions In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane–based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

Our aim is to find sufficient conditions for weak convergence of stochastic integrals with respect to the state occupation measure of a Markov chain. First, we study properties of the state indicator function and the state occupation measure of a Markov chain. In particular, we establish weak convergence of the state occupation measure under a scaling of the generator matrix. Then, relying on the connection between the state occupation measure and the Dynkin martingale, we provide sufficient conditions for weak convergence of stochastic integrals with respect to the state occupation measure. We apply our results to derive diffusion limits for the Markov-modulated Erlang loss model and the regime-switching Cox–Ingersoll–Ross process.

IntroductionThe locoregional failure (LRF) rate in human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) remains disappointingly high and toxicity is substantial. Response prediction prior to or early during treatment would provide opportunities for personalised treatment. Currently, there are no accurate predictive models available for correct OPSCC patient selection. Apparently, the pivotal driving forces that determine how a OPSCC responds to treatment, have yet to be elucidated. Therefore, the holistiC early respOnse assessMent for oroPharyngeaL cancer paTiEnts study focuses on a holistic approach to gain insight in novel potential prognostic biomarkers, acquired before and early during treatment, to predict response to treatment in HPV-negative patients with OPSCC.Methods and analysisThis single-centre prospective observational study investigates 60 HPV-negative patients with OPSCC scheduled for primary radiotherapy (RT) with cisplatin or cetuximab, according to current clinical practice. A holistic approach will be used that aims to map the macroscopic (with Intra Voxel Incoherent Motion Diffusion Kurtosis Imaging (IVIM-DKI); before, during, and 3 months after RT), microscopic (with biopsies of the primary tumour acquired before treatment and irradiated ex vivo to assess radiosensitivity), and molecular landscape (with circulating tumour DNA (ctDNA) analysed before, during and 3 months after treatment). The main end point is locoregional control (LRC) 2 years after treatment. The primary objective is to determine whether a relative change in the mean of the diffusion coefficient D (an IVIM-DKI parameter) in the primary tumour early during treatment, improves the performance of a predictive model consisting of tumour volume only, for 2 years LRC after treatment. The secondary objectives investigate the potential of other IVIM-DKI parameters, ex vivo sensitivity characteristics, ctDNA, and combinations thereof as potential novel prognostic markers.Ethics and disseminationThe study was approved by the Medical Ethical Committee of Erasmus Medical Center. The main results of the trial will be presented in international meetings and medical journals.Trial registration numberNL8458.

Quantification of tumor-specific variants (TSVs) in cell-free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next-generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell-free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell-free DNA to its full potential.

International audience; This paper studies Ornstein-Uhlenbeck (OU) processes in a random environment. The OU model has found widespread use in networking, as a Gaussian approximation of the user-level dynamics that allows explicit analysis; adding modulation to it allows incorporating phenomena in which the users' activity level is affected by exogenous factors. The focus lies on rare-event analysis: under a specific scaling of the parameters involved , we establish the large deviations asymptotics of the probability that the process reaches an extreme value. The decay rate of this probability is generally only implicitly available (as the solution to a variational problem), but specializing to the case of Markov modulation we succeed in devising efficient numerical procedures.

A vulnerable elderly man with prednisone-induced delirium Background Delirium always has an underlying cause, for example, medication. This article describes the case of a man with severe delirium caused by prednisone. Case description A 91-year-old vulnerable man developed severe delirium with agitation, verbal and physical aggression and visual hallucinations 3 days after starting treatment with low-dose prednisone. The delirium disappeared completely after prednisone was stopped. The delirium led to a long hospital stay of 36 days, weight loss of 10 kg due to undernourishment and extension of home care on discharge. Conclusion This case illustrates that delirium is always lurking around the corner in patients with multiple vulnerabilities. The provoking factor can be slight, for example 15 or 5 mg of prednisone. So be careful when starting prednisone in vulnerable elderly patients.

Many bivalve species occur in aggregations, and locally cover large partsof the seafloor. Above a certain density they provide a distinct, three dimensional structure and the aggregations are called bivalve beds or reefs. These persistent aggregations form a biogenic habitat for many other species. Bivalve beds, therefore, often have, in comparison with the surrounding areas, a high biodiversity value and can be seen as hotspots for biodiversity. Bivalve have a wide global distribution, on rocky and sedimentary coasts. Different processes and mechanisms influence the presence of associated benthic fauna. This paper reviewed the main drivers that influence the biodiversity, such as the bivalve species involved, the density, the size and the age of the bed, the depth or height in the tidal zone and the substratum type. Bivalve beds not only occur naturally in many subtidal and intertidal areas around the world, but mussels and oysters are also extensively cultured. Addition of physical cultivation structures in the water column or on the bottom allows for development of substantial and diverse communities that have a structure similar to that of natural beds. Dynamics of culture populations may however differ from naturalbivalve reefs as a result of culture site and/or maintenance and operation likeharvesting of the bivalve cultures. We used the outcome of the review on the drivers for wild assemblages to evaluate trade-offs between bivalve aquaculture and biodiversity conservation. Studies comparing natural and cultured assemblages proved to allow for a better understanding of the effect of the culture strategies and, consequently, to forward sustainable bivalve cultures. This is illustrated by a case study in the Dutch Wadden Sea.

This paper studies the diffusion limit for a network of infinite-server queues operating under Markov modulation (meaning that the system's parameters depend on an autonomously evolving background process). In previous papers on (primarily single-node) queues with Markov modulation, two variants were distinguished: one in which the server speed is modulated, and one in which the service requirement is modulated (i.e., depends on the state of the background process upon arrival). The setup of the present paper, however, is more general, as we allow both the server speed and the service requirement to depend on the background process. For this model we derive a Functional Central Limit Theorem: we show that, after accelerating the arrival processes and the background process, a centered and normalized version of the network population vector converges to a multivariate Ornstein-Uhlenbeck process. The proof of this result relies on expressing the queueing process in terms of Poisson processes with a random time change, an application of the Martingale Central Limit Theorem, and continuous-mapping arguments., Comment: 20 pages

In this paper we consider an Ornstein–Uhlenbeck (OU) process (M(t))t≥0 whose parameters are determined by an external Markov process (X(t))t≥0 on a finite state space {1, . . ., d}; this process is usually referred to as Markov-modulated Ornstein–Uhlenbeck. We use stochastic integration theory to determine explicit expressions for the mean and variance of M(t). Then we establish a system of partial differential equations (PDEs) for the Laplace transform of M(t) and the state X(t) of the background process, jointly for time epochs t = t1, . . ., tK. Then we use this PDE to set up a recursion that yields all moments of M(t) and its stationary counterpart; we also find an expression for the covariance between M(t) and M(t + u). We then establish a functional central limit theorem for M(t) for the situation that certain parameters of the underlying OU processes are scaled, in combination with the modulating Markov process being accelerated; interestingly, specific scalings lead to drastically different limiting processes. We conclude the paper by considering the situation of a single Markov process modulating multiple OU processes.

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.

L-(3a-13C)- And L-(6-13C)tryptophan have been synthesized from simple labelled compounds via a single reaction scheme based on the conversion of 1,3-cyclohexanedione into indole. The labelled indoles have been converted in one step into the corresponding L-tryptophans using transformed Escherichia coli cells with large amounts of the enzyme, tryptophan synthetase. The same reaction scheme has been used for the synthesis of 4- and 7-indolol. These hydroxyindoles together with 5-indolol have been converted into 4-, 7- and 5-hydroxy-L-tryptophan, respectively, using the Escherichia coli cells. The latter compound is the immediate precursor of the neurotransmitter, serotonin. It appears that 7-indolol is the only indole derivative which is converted faster than unsubstituted indole by the enzyme, tryptophan synthetase. With the preparation of L-(3a-13C)- and L-(6-13C)tryptophan, we have completed the series of indoles and L-tryptophans with a stable isotope (13C, 15N or 2H) in the aromatic ring. In this paper, we also discuss the NMR parameters of these mono-isotopically labelled systems.

International audience; In this paper, we generalize the upper bound in Varadhan's Lemma. The standard formulation of Varadhan's Lemma contains two important elements, namely an upper semicontinuous integrand and a rate function with compact sublevel sets. However, motivated by results from queueing theory, in this paper we do not assume that rate functions have compact sublevel sets. Moreover, we drop the assumption that the integrand is upper semicontinuous and replace it by a weaker condition. We prove that the upper bound in Varadhan's Lemma still holds under these weaker conditions. Additionally, we show that only measurability of the integrand is required when the rate function is continuous. Keywords. Varadhan's Lemma exponential integrals large deviations principle upper bound

Summary Background 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. Aim 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. Material and methods 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. Results 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%, P > 0.1). 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 (P > 0.1 and P = 0.07, respectively). In contrast, higher numbers of neutrophils persisted in asthma 24 h 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). Conclusion Allergen challenge induced very 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.

The spectroscopic properties of open-chain, all-trans-C{sub 30} carotenoids having seven, eight and nine {pi}-electron conjugated carbon-carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation and time-resolved absorption spectroscopy. These diapocarotenes were purified by high performance liquid chromatography (HPLC) prior to the spectroscopic experiments. The fluorescence data show a systematic crossover from dominant S{sub 1} {yields} S{sub 0} (2{sup 1}Ag{yields} 1{sup 1}Ag) emission to dominant S{sub 2} {yields} S{sub 0} (1{sup 1}Bu {yields} 1{sup 1}Ag) with increasing extent of conjugation. The low temperatures facilitated the determination of the spectral origins of the S{sub 1} {yields} S{sub 0} (2{sup 1}Ag {yields} 1{sup 1}Ag) emissions, which were assigned by Gaussian deconvolution of the experimental line shapes. The lifetimes of the S{sub 1} states of the molecules were measured by transient absorption spectroscopy and were found to decrease as the conjugated chain length increases. The energy gap law for radiationless transitions is used to correlate the S{sub 1} energies with the dynamics. These molecules provide a systematic series for understanding the structural features that control the photochemical properties of open-chain, diapocarotenoids. The implications of these results on the roles of carotenoids in photosynthetic organisms are discussed.

Nonencapsulated Haemophilus influenzae often causes chronic infections of the lower respiratory tract in both nonobstructive and obstructive chronic bronchitis. We assessed airway inflammation in clinically stable, chronically H. influenzae-infected patients with nonobstructive (CB-HI, n = 10) and in patients with obstructive chronic bronchitis (COPD-HI, n = 10) by analyses of the sol phase of spontaneously expectorated sputum (SSP). As compared with the CB-HI group, the COPD-HI group had significantly higher (p < 0.05) levels of myeloperoxidase (MPO) and tumor necrosis factor (TNF)-alpha in their SSP, whereas the degree of plasma protein leakage (SSP-to-serum ratio of plasma proteins) and the levels of interleukin (IL)-8, secretory IgA, and lactoferrin were similar in the two groups. These findings point to differences in pathophysiology in CB-HI and COPD-HI. The high level of TNF-alpha in the SSP of COPD-HI patients is in accord with the proposed role of TNF-alpha in the development of airway obstruction in COPD patients. In apparent contradiction, low levels of TNF-alpha were found in the SSP of noninfected but otherwise similar COPD patients (n = 9). This finding, however, does not exclude an exaggerated TNF-alpha response to infection or another stimulus in the airways of COPD patients. The SSP levels of MPO and IL-8, and the degree of plasma protein leakage in the COPD-HI group, were retrospectively compared with and found significantly higher than those of noninfected COPD patients, suggesting a more marked inflammatory response in COPD-HI. Whether this reflects a direct cause-and-effect relationship should be addressed in a future long-term prospective study involving repeated measurements in the same patients

Background 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. Objective To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. Methods 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. Results 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. Conclusion The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.

The all-E isomer of (4,4′-13C2)astaxanthin (1a) has been prepared by total synthesis starting from commercially available 99% 13C enriched acetonitrile. The labelled astaxanthin was obtained in high purity and with high isotope incorporation. For this synthesis, the C15 + C10 + C15 strategy was used. The central C10-synthon, 2,7-dimethylocta-2,4,6-triene-1,8-dial (3), was coupled with 13C-enriched C15-phosphonium salt 2a. The new synthetic scheme for the preparation of the C15-phosphonium salt is discussed in this paper; the same scheme can be used to label all positions and combinations of positions of the C15-phosphonium salt.

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.

A recent study observed that numerical chromosome abnormalities in Hodgkin's disease (HD) are detected not only in morphologically abnormal Hodgkin/Reed-Sternberg cells, but also in a fraction of morphologically normal cells. However, the phenotypic constitution of these genetically abnormal, morphologically normal cells and their relationship to the malignant Hodgkin/Reed-Sternberg cells could not be established in the earlier cases studied, because of the low frequency of these cells. The present study investigated two cases of classical Hodgkin's disease containing a relatively large population of such apparently normal cells with aberrant chromosome copy numbers. The phenotype and their position within the developmental route of the malignant compartment were examined by a combined in situ hybridization and immunocytochemistry approach. Numerical abnormalities for chromosome 1 in one case and for chromosomes X, Y, and 1 in the other case were observed not only in CD30-positive Hodgkin/Reed-Sternberg cells, but also in CD30-negative, morphologically normal cells. It was shown that these genetically aberrant cells expressed the B-cell antigen CD19, thus confirming their B-cell nature. These studies indicate a relationship between the genome aberrations in these genetically abnormal, morphologically normal B-cells and the Hodgkin/Reed-Sternberg cells, suggesting that they are progenitor cells of the malignant cell fraction.

A series of apocarotenes with 5 to 11 conjugated double bonds were synthesized and all-trans isomers were isolated using HPLC techniques. Absorption, fluorescence, and fluorescence excitation spectra were obtained in 77 K glasses. As previously noted for other polyenes and carotenoids, fluorescence spectra of the apocarotenes exhibit a systematic crossover from S1(21Ag) → S0(11Ag) to S2(21Ag) → S0(11Ag) emissions and a sharp decrease in fluorescence yields with increasing conjugation. The apocarotene spectra have sufficient resolution to accurately locate the dominant vibronic bands of the S0(11Ag) → S2(11Bu) and S1(21Ag) → S0(11Ag) transitions, thus leading to an accurate catalog of S1 and S2 electronic energies as a function of conjugation length. We also have obtained the low-temperature absorption and fluorescence spectra of several model polyenes and diapocarotenes. Comparisons between these series allow a systematic exploration of the influence of terminal cyclohexenyl rings on the energies of carot...

Background 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. Methods 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 (PC20 histamine), 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. Results 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, P

A three-part strategy has been developed to study molecular interactions in biological systems at the atomic level. First, isotopically labeled carotenoids and retinoids are prepared by organic total synthetic schemes with labels at predetermined atomic positions and combinations of positions. Subsequently, the labeled compounds are incorporated in the biological system. Finally, the system is studied by isotope sensitive spectroscopic techniques. In this paper, the synthesis of 10-fold 13 C-labeled retinal palmitate and b-carotene a- crustacyanin carotene for nutritional studies is discussed. Also, the scheme to label the end positions of astaxanthin and canthaxanthin with 13 C for spectroscopic investigations of a- crustacyanin with isotope labels in the chromophore is given. The synthesis of 10-methyl retinal is discussed, starting from isotopically labeled synthons obtained via schemes to 13 C- labeled natural retinal. Finally, the possibility for spectroscopic studies of caroteno and retino proteins via an expression of apoproteins by way of genetic techniques in the post-genomic era is discussed.

Semiempirical AM1 calculations have been carried out for β-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of β-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12′; 13,13′; 14,14′; 15,15′; 15, and 20,20′ were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310–321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115–178; Spectrochim Acta 1996, 53, 381–392; Biochim Biophys Acta 1994, 1185, 188–196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 19–33, 1999

Background: Segmental allergen challenge is a powerful tool to study inflammatory reactions in asthmatic airways. There is little information on the early events at 5 min and 4 h after allergen challenge with respect to the cell influx and the chemokine interleukin–8 (IL–8). Methods: Seven mild to moderate allergic asthmatics (AA group), 5 allergic nonasthmatics (ANA group) and 5 nonallergic controls underwent segmental allergen challenge, with allergen doses based upon skin reactivity. Bronchoalveolar lavage (BAL) samples were obtained before, 5 min and 4 h postchallenge, and were analyzed for cell numbers and differential counts, eosinophil and neutrophil chemotactic activity, and levels of IL–8. Results: At 5 min postchallenge, no changes were observed compared to baseline. At 4 h postchallenge, an increase was found in the number of neutrophils and the levels of IL–8, which was dependent on the dose of allergen in the AA and ANA group. At the same allergen dose, the increases in neutrophils and levels of IL–8 were calculated to be 91 and 67 times higher, respectively, in AA than in ANA. Levels of IL–8 correlated with the number of neutrophils and with the in vitro neutrophil chemotactic activities in BAL fluid. Conclusions: Neutrophil chemotactic activity is increased in BAL fluid at 4 h after segmental allergen challenge. We suggest that apart from IgE–mediated mast cell degranulation, additional local factors in the airways determine the degree of IL–8 increase and neutrophil influx.

Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of alpha-crustacyanin reconstituted with specifically 13C-labeled astaxanthins at the positions 15, 15,15', 14,14', 13,13', 12,12', or 20,20' were recorded. This approach enabled us to obtain information about the effect of the ligand-protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the C==C stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250-1400 cm(-1) spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in alpha-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the pi-antibonding C==O orbitals and the HOMO-LUMO gap.

Spheroidene and a series of spheroidene analogues with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh, R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57...

Hodgkin and Reed-Sternberg cells are considered to represent the malignant fraction in Hodgkin's disease. Several studies have shown that the Hodgkin and Reed-Sternberg cells are chromosomally abnormal, but genetic data about the morphologically normal cell population in Hodgkin's disease are very limited. This latter cell population has therefore been examined for chromosomal aberrations, using the in situ hybridization (ISH) procedure, making use of DNA probes for chromosomes 1, 7, 8, 9, 11, 12, 15, 17, and 18. Nuclei were isolated from freshly frozen (10 cases) and paraffin-embedded (16 cases) biopsy samples and 1000 nuclei per case were evaluated. The cases of Hodgkin's disease were compared with reactive lymph nodes, which show aberrant chromosome copy numbers in less than 1 per cent of the cells. Using strict scoring criteria, nuclei in the tumour were found to show an abnormal genotype, in the range of 1-12 per cent, with trisomies occurring most frequently. No characteristic numerical chromosome abnormality was observed. ISH on 4 microns thick paraffin sections of six cases of Hodgkin's disease revealed numerical aberrations for chromosome 1 in cells which appeared to be morphologically normal. The genomically abnormal nuclei did not differ in morphology or size from the nuclei of morphologically normal cells, but differed considerably in size when compared with the nuclei of Hodgkin/Reed-Sternberg cells after the ISH procedure. Three of these six cases revealed a population of apparently normal cells with an aberrant copy number which differed notably from the fraction observed in reactive lymph nodes. It is concluded, therefore, that a subset of morphologically normal cells, next to the Hodgkin/Reed-Sternberg cells, are chromosomally aberrant and may participate in the malignant cell fraction of Hodgkin's disease.

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15′-cis-spheroidene, were studied by absorption, fluorescence, circular dichroism, fast transient absorption and electron spin resonance spectroscopies in solution and after incorporation into the reaction center of Rhodobacter (Rb.) sphaeroides R-26.1. HPLC purification of the synthetic molecule reveals the presence of several di-cis geometric isomers in addition to the mono-cis isomer of locked-15,15′-cis-spheroidene. In solution, the absorption spectrum of the purified mono-cis sample was red-shifted and showed a large cis-peak at 351 nm compared to unlocked all-trans spheroidene. Molecular modeling and semi-empirical calculations reveal how geometric isomerization and structural factors affect the room temperature spectra. The spectroscopic studies of the purified locked-15,15′-mono-cis molecule in solution reveal a more stable manifold of excited states compared to the unlocked spheroidene. Reaction centers of Rb. sphaeroides R-26.1 in which the locked-15,15′-cis-spheroidene was incorporated show no difference in either the spectroscopic properties or photochemistry compared to reaction centers in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 reaction centers which naturally contain spheroidene. The data suggest that the natural selection of a cis-isomer of spheroidene for incorporation into native reaction centers of Rb. sphaeroides wild type strain 2.4.1 is more determined by the structure or assembly of the reaction center protein than by any special quality of the cis-isomer of the carotenoid that would affect its ability to participate in triplet energy transfer or carry out photoprotection.

Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation.

By selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in a-crustacyanin, the major blue astaxanthin binding carotenoprotein complex from the carapace of the lobster Homarus gammarus, have been analysed. 13C Magic Angle Spinning (MAS) NMR spectra were obtained after reconstitution with astaxanthins labelled in the centre of the molecule or at the two keto groups. The MAS data reveal electrostatic polarizations of the conjugated chain. In addition, solid state NMR results for pure unlabelled astaxanthin can be compared with natural abundance I3C MAS data for canthaxanthin and p-carotene, to address the effect of the ring functionalities on the electronic properties of the polyene chain. Quantum chemical calculations were performed to reconcile the MAS data with one of several simple and straightforward mechanisms for the colour shift. The results point towards a colour shift mechanism in which the astaxanthin may be doubly charged, possibly by a double protonation of the two ring keto groups.

In IgE allergic diseases both mast cells and T lymphocytes play an important role. Whereas mast cels have been implicated in immediate allergic responses, T lymphocytes mediate subsequent late phase responses and chronic inflammation. Here we review possible links between the early mast cell activation and the later T lymphocyte stimulation. Products from mast cells were found to exert effects on T lymphocytes. Human Mast Cell line-1 (HMC-1) mast cells modulated proliferation and cytokine production of a human CD8+ T-cell clone in vitro. Activated mast cells seemed to drive this CD8+ T-cell clone towards a more pronounced T (helper) 1 type of response, simultaneously decreasing T-cell numbers. It is hypothesized that this might be a negative feed back mechanism operating in allergic subjects, by which the Th2-driven IgE production and eosinophilia are counteracted.

Objective To compare a one-step (rapid) application of negative pressure (vacuum) with conventional stepwise application for ventouse extraction following a prolonged second stage of labour. Design Randomised controlled study. Setting Teaching Hospital. Participants Forty-seven women were randomised to the rapid vacuum group and 47 to the stepwise group. Main outcome measures Duration of ventouse procedure, effectiveness of methods of application, morbidity of mother and infant. Results There was no significant difference in frequency of detachment of the cup after rapid or stepwise application of vacuum. A reduction in mean duration of the ventouse procedure of 6 min was realised without significant difference in maternal or neonatal morbidity. Conclusion Rapid application of vacuum significantly reduces the duration of a ventouse extraction procedure without compromise to efficiency and safety.

Patients with allergic asthma have higher levels of nonspecific bronchial responsiveness than patients with allergic rhinitis. The aim of the study was to investigate whether this is caused by differences in the degree of allergy to inhalant allergens between asthmatics and rhinitics. Therefore, bronchial responsiveness to histamine was measured in 25 allergic patients with isolated upper airways symptoms. Nonspecific bronchial responsiveness in this group was compared with nonspecific bronchial responsiveness in a group of 136 patients with allergic asthma, with allergy and % predicted FEV1 as confounding variables. In addition, a matched pair analysis was performed. Twenty-five patients with nonallergic rhinitis served as controls to evaluate the influence of an IgE-independent inflammatory reaction in the upper respiratory tract on the level of bronchial responsiveness. Furthermore, we investigated the level of nonspecific responsiveness in 18 healthy controls. In the patients with allergic asthma, a correlation was found between nonspecific bronchial responsiveness and IgE against indoor allergens (n = 136, r = 0.34, p < 0.001) and % predicted FEV1 (n = 136, r = 0.37), p < 0.001). Patients with allergic asthma and patients with allergic rhinitis differed with respect to the level of bronchial responsiveness (p < 0.001), and the amount of specific IgE antibodies against indoor allergens (p = 0.01). The difference in level of bronchial responsiveness remained (p < 0.001) after correction with % predicted FEV1 and specific IgE against indoor allergens as confounding variables. Similarly, after matching of patients with allergic rhinitis (n = 25) with patients with allergic asthma (n = 25) regarding the specific IgE, total IgE and age of the patients, the difference in level of bronchial responsiveness remained (p < 0.001). Patients with nonallergic rhinitis had higher levels of nonspecific bronchial responsiveness than healthy controls and did not differ from patients with allergic rhinitis. In conclusion, the results confirm that IgE against common indoor allergens plays an important role in the mechanism underlying nonspecific bronchial hyperresponsiveness. However, differences in bronchial responsiveness between patients with asthma and patients with rhinitis are not merely explained by differences in the IgE antibody concentrations

BACKGROUND: Study of the relationship between skin test results and IgE antibody levels is seriously hampered by the use of conventional allergen extracts because the precise amount of relevant allergen for each patient is unknown. OBJECTIVE: This study was designed to investigate skin reactivity with purified major allergens and to assess the relation with serum levels of IgE antibodies and to determine which additional factors contribute to the skin test result. METHODS: We used five purified major allergens Der p 1 , Der p 2 , Fel d 1 , Lol p 1 , and Lol p 5 ) in skin tests, RASTs, and histamine release tests in 43 multisensitized patients with asthma or rhinitis. RESULTS: The differences in biologic activity of the five major allergens at a given level of specific IgE are within one order of magnitude. A significant residual variation remains in the correlation between skin test results and levels of IgE antibodies, which cannot be explained by imprecision of both tests (Pearson log skin test vs log specific IgE: r=-0.46-0.92). With similar levels of specific IgE, the amount of allergen that is required for a positive skin test result may differ by as much as a factor of 100 between patients. The amount of total IgE in serum contributes significantly to the skin test result. High values of total IgE are accompanied by a lower skin reactivity for allergen. Within individuals, allergens that cause skin test results that deviate from the prediction based on IgE antibody level often show a similar deviation in the histamine release test. This indicates that the type of IgE response (i.e., affinity or epitope recognition pattern) contributes significantly to the skin test result. Skin reactivity for histamine does not significantly influence the skin reactions expressed as allergen threshold. However, increased skin reactions with higher allergen dosages depend on histamine reactivity. CONCLUSION: The major allergens tested show similar biologic activities. In addition to IgE antibody level, total serum IgE and type of IgE antibody response contribute significantly to the skin test threshold for allergens. Even in a system with purified allergens, IgE antibody levels and skin test results are not interchangeable as an indicator of the degree of allergic sensitization.(J ALLERGY CLIN IMMUNOL 1996;97:16-25.)

MAS (magic angle spinning) 13C NMR has been used to study protein-chromophore interactions in alpha-crustacyanin, the blue astaxanthin-binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14' or 15,15' positions. Two signals are seen for alpha-crustacyanin containing [14,14'-13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With alpha-crustacyanin containing [15,15'-13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in alpha-crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176-191]. The splitting of the 14 and 14' signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.