Dendritic cell phenotype in severe asthma reflects clinical responsiveness to glucocorticoids.

BACKGROUND: Subsets of patients with severe asthma remain symptomatic despite prolonged, high-dose glucocorticoid therapy. We hypothesized that the clinical glucocorticoid sensitivity of these asthmatics is reflected in differences in peripheral blood dendritic cell subsets. OBJECTIVE: To compare peripheral blood leucocyte populations using flow cytometry at baseline and after 2 weeks of systemic glucocorticoid (steroid) treatment to identify immunological differences between steroid-sensitive (SS) and steroid-resistant (SR) asthmatics. METHODS: Adult severe asthmatics (SS n = 12; SR n = 23) were assessed for their response to 2 weeks of therapy with oral prednisolone. Peripheral blood was obtained before and after therapy and stained for lymphocyte (CD3, CD19, CD4, CD8 and Foxp3) and dendritic cell markers (Lineage negative [CD3, CD14, CD16, CD19, CD20, CD56], HLA-DR+, CD304, CD11c, ILT3 and CD86). RESULTS: A higher median frequency of myeloid DCs (mDCs) but not plasmacytoid DCs (pDCs) was observed in the blood of SR as compared to SS asthmatics (P = .03). Glucocorticoid therapy significantly increased median B cell, but not T cell numbers in both cohorts, with a trend for increased numbers of Foxp3+ Tregs in SS (P = .07), but not SR subjects. Oral prednisolone therapy significantly reduced the median numbers and frequencies of total DCs and pDCs in both SS and SR asthmatics. Interestingly, the expression of HLA-DR and ILT3 was also reduced on pDCs in all patients. In contrast, therapy increased the median frequency of mDCs in SS, but reduced it in SR asthmatics. CONCLUSIONS: Myeloid DC frequency is elevated in SR compared with SS asthmatics, and mDC shows a differential response to oral prednisolone therapy.

Influence of a single oral dose of vitamin D(2) on serum 25-hydroxyvitamin D concentrations in tuberculosis patients.

SETTING: Newham Chest Clinic, London, UK. OBJECTIVE: To determine the safety and efficacy of the administration of bolus-dose vitamin D(2) in elevating serum 25-hydroxyvitamin D (25[OH]D) concentrations in tuberculosis (TB) patients. DESIGN: A multi-ethnic cohort of TB patients was randomised to receive a single oral dose of 2.5 mg vitamin D(2) (n = 11) or placebo (n = 14). Serum 25(OH)D and corrected calcium concentrations were determined at baseline and 1 week and 8 weeks post-dose, and compared to those of a multi-ethnic cohort of 56 healthy adults receiving an identical dose of vitamin D(2). RESULTS: Hypovitaminosis D (serum 25[OH]D < 75 nmol/l) was present in all patients at baseline. A single oral dose of 2.5 mg vitamin D2 corrected hypovitaminosis D in all patients in the intervention arm of the study at 1 week post-dose, and induced a 109.5 nmol/l mean increase in their serum 25(OH)D concentration. Hypovitaminosis D recurred in 10/11 patients at 8 weeks post-dose. No patient receiving vitamin D(2) experienced hypercalcaemia. Patients receiving 2.5 mg vitamin D(2) experienced a greater mean increase in serum 25(OH)D at 1 week post-dose than healthy adults receiving 2.5 mg vitamin D(2). CONCLUSION: A single oral dose of 2.5 mg vitamin D(2) corrects hypovitaminosis D at 1 week but not at 8 weeks post-dose in TB patients.

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

Novel molecular aspects of pituitary adenomas.

Ghrelin stimulates while somatostatin inhibits GH release and they thus serve as functional antagonists. We have compared their effects on cell proliferation. Ghrelin stimulates while somatostatin inhibits cell proliferation in most tissues and cell lines. Here we show that ghrelin and desoctanoyl ghrelin stimulate cell proliferation in rat pituitary cell line (GH3), and these effects could be inhibited with mitogen-activated protein kinase (MAPK), tyrosine kinase and protein kinase C inhibitors. Somatostatin and its analogs negatively regulate the growth of pituitary cells, and we now show that they inhibit MAPK activation. We hypothesised that one of the mechanisms involved in the somatostatin effect is a stimulation of cell cycle inhibitor p27, as pituitary adenomas have decreased p27 peptide content. Both octreotide and a new somatostatin analog SOM230 treatment resulted in an upregulation of p27 protein levels in human somatotrophinoma cells. In summary, we suggest that ghrelin and somatostatin have opposite effects on somatotroph cells not just at the level of GH release but also in terms of cell proliferation. Ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway. Our results also suggest that the antiproliferative effect of somatostatin analogs octreotide and SOM230 involve the up-regulation of p27 and down-regulation of the MAPK pathway in human somatotrophinomas.

Dynamical studies of peptide motifs in the Plasmodium falciparum circumsporozoite surface protein by restrained and unrestrained MD simulations.

The immunodominant region on the circumsporozoite surface (CS) protein of the malaria parasite Plasmodium falciparum contains 37 repeated copies of a asparagine-alanine-asparagine-proline (NANP) motif NMR studies of linear synthetic peptides containing one, two or three repeat units provided evidence for nascent type I beta-turns within the NPNA cadence in aqueous solution. The beta-turns could be stabilised upon substituting proline for alpha-methylproline (p(Me)) in the dodecamer (NP(Me)NA)3, without loss of the ability to elicit antibodies cross-reactive with P. falciparum sporozoites. In this work, four 4 ns MD simulations of the dodecapeptide Acetyl-(NP(Me)NA)3, in water, using NOE distance restraints, using 3J-coupling constant restraints, using both these restraints and without restraints, were carried out to determine the conformations of this peptide in aqueous solution. An unrestrained MD simulation of the unmethylated Ac-(NPNA)3 peptide in water was also carried out to investigate the effect of the additional methyl groups on the structure and dynamics of the peptide. The application of NOE distance restraints and 3J-coupling constant restraints leads to contradictory results, probably due to different averaging time scales inherent to the measurement of these data, which exceed the 100 ps averaging applied in the simulations. The additional methyl groups lead to more compact structures, which display enhanced local fluctuations. The central tetrapeptide adopts a type I beta-turn, while the outer motifs display more conformational variability. The three motifs in the methylated dodecamer peptide, however, adopt frequently in the distance restrained MD simulation a compact structure such that the outer motifs appear to form a hydrophobic core by stacking of their two proline rings. This arrangement also suggests how a peptide containing multiple tandemly linked copies of a stable beta-turn NPNA motif might adopt a folded stem-like structure, which conceivably may be of biological relevance in the native CS protein.

Molecular dynamics simulation using weak-coupling NOE distance restraining.

Application of the weak-coupling scheme to restrain the configurations of a molecular system to a set of NOE distance restraints is investigated using two test systems: (i) a 15-atom chain molecule with one distance restraint; and (ii) a protein molecule with hundreds of NOE distance restraints. Atom-atom distance restraining by the weak-coupling technique is possible, but this method does not produce as good results as the penalty function method normally used to maintain NOE distance restraints.

Parametrisation of time-averaged distance restraints in MD simulations.

Time-averaged restraints in molecular dynamics simulations offer a means to account for the averaging that is implicit in NMR spectroscopic data. We present a systematic investigation of the parameters which characterise time-averaged distance restraints. Using previously published data for a small protein, chymotrypsin inhibitor 2, we identify conditions which can lead to undesirable heating or which grossly distort the dynamics of the system.

A reassessment of the structure of chymotrypsin inhibitor 2 (CI-2) using time-averaged NMR restraints.

Chymotrypsin inhibitor 2 (CI-2) is one of the growing family of proteins for which well-defined solution and crystal structures have been published and for which small, but distinct differences between these were found. It presents an ideal case to address the question of whether a structural difference is physically real or due to the simplifying approximations with respect to averaging that are used in the conventional methods for structure refinement. NOE distance and 3J coupling constant restrained molecular dynamics simulations were performed using conventional and time-averaged restraints, both in vacuo and in aqueous solution, and the trajectories were compared with structural properties of published structures. The time-averaged restrained molecular dynamics simulations sampled more conformations at various times and visited states consistent with both previously published solution and crystal structures. It was found that the difference between these structures is due to the refinement methodology used. Application of time-averaged restraints in structure refinement yields a physically different picture of the molecular mobility.

Variable radiomorphologic data of high altitude pulmonary edema. Features from 60 patients.

The purpose of the study was to collect radiomorphologic data of a large population of subjects with high altitude pulmonary edema. A blinded retrospective analysis of 60 patients severe enough to warrant hospital admission is reported. Immediately after rescue to low altitude, the severity of HAPE was graded using a quadrant-based scoring system (0-4 each quadrant). Its distribution and the morphologic features were noted. HAPE was more severe in the base, and specifically, the right lower quadrant, as compared to the other quadrants. It was often located both centrally and peripherally (60 percent) and in 92 percent was characterized by air space disease of homogeneous (n = 40) rather than patchy distribution (n = 15). In recurrent HAPE (n = 13), radiomorphologic data were as variable as among different HAPE patients. We conclude that HAPE does not have one common radiomorphologic condition. Based on the literature, earlier experience, and follow-up observations, we hypothesize that it may start patchy and peripheral, supporting the concept of uneven vasoconstriction with overperfusion and/or permeability leak. Later on, such as in the severe cases studied, it becomes homogeneous. Recurrent episodes generally do not show an identical distribution of HAPE, suggesting that structural abnormalities are not involved in the pathogenesis of HAPE.

[Altitude edema in the Swiss Alps. Observations on the incidence and clinical course in 50 patients 1980-1984]. / Das Höhenödem in den Schweizer Alpen. Beobachtungen über Inzidenz, Klinik und Verlauf bei 50 Patienten der Jahre 1980-1984.

In the Swiss Alps 50 patients with high altitude edema (high altitude pulmonary edema and/or high altitude cerebral edema) had to be rescued by helicopter during the period 1980-1984. The development, clinical picture and clinical course have been analyzed retrospectively. The patients were 49 men and one woman, generally in good health and well trained. They had ascended from the low-lands to an altitude above 2500 m and subsequently climbed higher. The climbers developed symptoms of acute mountain sickness on the second to third day of high altitude exposure and had to be evacuated by air on the fourth to fifth day. 70% of the cases occurred in the Valais Alps and the rest in the region of the Bernese Alps and the Bernina. The highest incidence of high altitude edema was observed in the Capanna Margherita (4559 m), where one of 588 climbers who stayed overnight had to be air-rescued. This ratio was about one in 4000 mountaineers at the Finsteraarhorn hut (3050 m) and the Monte Rosa hut (2795 m). Evacuation by air was the most successful therapeutic measure and resulted in immediate amelioration of clinical symptoms in 16 patients. 34 climbers had to be admitted to local hospitals due to severe high altitude pulmonary and/or cerebral edema. The pulmonary edema was bilateral in two thirds of these patients and unilateral in one third. Arterial blood gases showed moderate to severe limitation of oxygen diffusion capacity. All patients recovered completely within a few days.