Increased breath naphthalene in children with asthma and wheeze of the All Age Asthma Cohort (ALLIANCE).

Exhaled breath contains numerous volatile organic compounds (VOCs) known to be related to lung disease like asthma. Its collection is non-invasive, simple to perform and therefore an attractive method for the use even in young children. We analysed breath in children of the multicenter All Age Asthma Cohort (ALLIANCE) to evaluate if 'breathomics' have the potential to phenotype patients with asthma and wheeze, and to identify extrinsic risk factors for underlying disease mechanisms. A breath sample was collected from 142 children (asthma: 51, pre-school wheezers: 55, healthy controls: 36) and analysed using gas chromatography-mass spectrometry (GC/MS). Children were diagnosed according to Global Initiative for Asthma guidelines and comprehensively examined each year over up to seven years. Forty children repeated the breath collection after 24 or 48 months. Most breath VOCs differing between groups reflect the exposome of the children. We observed lower levels of lifestyle-related VOCs and higher levels of the environmental pollutants, especially naphthalene, in children with asthma or wheeze. Naphthalene was also higher in symptomatic patients and in wheezers with recent inhaled corticosteroid use. No relationships with lung function or TH2 inflammation were detected. Increased levels of naphthalene in asthmatics and wheezers and the relationship to disease severity could indicate a role of environmental or indoor air pollution for the development or progress of asthma. Breath VOCs might help to elucidate the role of the exposome for the development of asthma. The study was registered at ClinicalTrials.gov (NCT02496468).

The immunoinformatics of cancer immunotherapy.

We review here the developments in the field of immunoinformatics and their present and potential applications to the immunotherapeutic treatment of cancer. Antigen presentation plays a central role in the immune response, and as a result in immunotherapeutic methods such as adoptive T-cell transfer and antitumor vaccination. We therefore extensively review the current technologies of antigen presentation prediction, including the next generation predictors, which combine proteasomal processing, transporter associated with antigen processing and major histocompatibility complex (MHC)-binding prediction. Minor histocompatibility antigens are also relevant targets for immunotherapy, and we review the current systems available, SNEP and SiPep. Here, antigen presentation plays a key role, but additional types of data are also incorporated, such as single nucleotide polymorphism data and tissue/cell-type expression data. Current systems are not capable of handling the concept of immunodominance, which is critical to immunotherapy, but efforts have been made to model general aspects of the immune system. Although tough challenges lie ahead, when measuring the field of immunoinformatics on its contributions thus far, one can expect fruitful developments in the future.

The nature of recombination in HLA-B*4207.

The identification of the novel human leukocyte antigen (HLA)-B*4207 allele, which was found in a blood donor of Caucasian origin, is described. The sequence of the new allele differs from HLA-B*4201 in three nucleotide substitutions in exon 2, resulting in three consecutive amino acid (AA) exchanges at position 69, 70, and 71. AA positions 69 and 70 affect the peptide-binding site of the HLA molecule in the formation of pockets A, B, and C. Therefore, it is likely that the peptide-binding motif of HLA-B*4207 differs from the HLA-B*4201 motif. HLA-B*4207 exhibits a high level of structural homology to HLA-B*08 alleles as well as to HLA-B*4201. Rating of the AA variations of these alleles according to the AA distance matrix score gives the lowest overall matching score between the HLA-B*4207 and the HLA-B*0801 alleles, indicating a high functional similarity. To further address this, homology modeling was performed using B8 as the closest structural template. The portion of the molecule that is accessible to the T-cell receptor and antibodies is identical between B*4207 and B*0801. Under consideration of allele frequencies, close inspection of these sequences shows that the new allele is most likely a result of a recombination involving B*0702 and B*0801. Unfortunately, patient consent could not be obtained for retrospective serological typing to definitively determine whether B*4207 reacts in the B8 serological group.

Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: comparison with other steroid metabolizing enzymes.

Effects of phytoestrogens on human health have been reported for decades. These include not only beneficial action in cancer prevention but also endocrine disruption in males. Since then many molecular mechanisms underlying these effects have been identified. Targets of phytoestrogens comprise steroid receptors, steroid metabolising enzymes, elements of signal transduction and apoptosis pathways, and even the DNA processing machinery. Understanding the specific versus pleiotropic effects of selected phytoestrogens will be crucial for their biomedical application. This review will concentrate on the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases from a comparative perspective with other steroid metabolizing enzymes.

Peptide-binding characteristics of the novel allele DRB1*0112 are probably identical to DRB1*0101.

So far 11 different amino acid variants of the HLA-DRB1*01 family have been reported. We here describe the identification of a new HLA-DRB1*01 allele in a healthy female Caucasian. The allele was detected by sequencing-based typing during high-resolution typing of a potential unrelated donor from the North German Bone Marrow Registry (NKR). Compared with DRB1*010101, to which it is closest, the new variant is characterized by a new replacement mutation (G-->T) at nucleotide position 202 of exon 2, resulting in the amino acid substitution Arg-->Leu at position 72. Because this amino acid position is not involved in peptide binding or T-cell interaction, it is likely to represent a permissive mismatch to the more common HLA-DRB1*0101 allele.

Identification and characterization of 17 beta-hydroxysteroid dehydrogenases in the zebrafish, Danio rerio.

The 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs) are key enzymes in the final steps of steroid hormone synthesis. 17beta-HSD type 1 (HSD17B1) catalyzes the reduction of estrone to estradiol, while type 3 (HSD17B3) performs the conversion of androstenedione to testosterone. Here we present a functional genomics study of putative candidates of these enzymes in the zebrafish. By an in silico screen of zebrafish EST databases we identified three candidate homologs for both HSD17B1 and HSD17B3. Phylogenetic analysis, unique expression patterns (RT-PCR) during embryogenesis and adulthood, as well as activity measurements revealed that one of the HSD17B1 candidates is the zebrafish homolog, while the other two are paralogous photoreceptor-associated retinol dehydrogenases. All three HSD17B3 candidate genes showed nearly identical, ubiquitous expressions in embryogenesis and adult tissues and were identified to be paralogs of HSD17B12 and a yet uncharacterized putative steroid dehydrogenase. Phylogenetic analysis shows that HSD17B3 and HSD17B12 are descendants from a common ancestor.

HistoCheck: rating of HLA class I and II mismatches by an internet-based software tool.

HLA polymorphism is a major barrier for hematopoietic stem cell and solid organ transplantation. To estimate the allogeneic potential between HLA-mismatched stem cell donor/recipient pairs, we recently proposed a matching score (dissimilarity index) that is based on the structural data of HLA class I molecules, and on the functional similarity of amino acids (AA). This first approach revealed new features about presumptive subtype allogenicities within the HLA-A*23 and A*24 groups. We have now developed an internet-based software tool ("HistoCheck") that is capable to assess the allogenicity (matching score) between any pair of clinically relevant HLA class I, and also class II, alleles. Newly described HLA sequences will be regularly integrated into the database according to the nomenclature for factors of the HLA system updates. The software is intended to be a first step for estimating the allogenicity of HLA mismatches in peculiar clinical settings, as long as there are no reliable in vitro or clinical studies available. The algorithm can later be modified according to functional data, for example, peptide-binding specificities. With the extension of the sequence similarity concept to all clinically relevant HLA class I and II loci, HistoCheck may contribute to prevent HLA mismatching being a matter of chance.

Characterization of 17beta-hydroxysteroid dehydrogenase type 7 in reproductive tissues of the marmoset monkey.

In contrast to the known rodent enzymes, the physiological significance of 17beta-hydroxysteroid dehydrogenase type 7 (17HSD7) and its presumed function in reproductive biology is not well understood in primates. As a first step, we recently cloned the complete coding regions of human and marmoset monkey (Callithrix jacchus) 17HSD7 (cj17HSD7). In the present work the complete cDNA of marmoset 17HSD1 (cj17HSD1), including the proximal promoter region, and a partial sequence of marmoset aromatase (cjARO) were sequenced in order to compare the expression of these estradiol synthesizing enzymes with that of 17HSD7 in a primate model and to identify tissues where 17HSD7 might participate in the pathway of estradiol synthesis. The gene structures of cj17HSD1 and cj17HSD7 were determined and proved to be very similar to the human orthologues. Northern hybridization showed that cjARO mRNA seems to be coexpressed preferably with cj17HSD1 in placenta, whereas in other tissues it is expressed in parallel only with cj17HSD7. Especially in corpora lutea, the cj17HSD7 transcript is detectable throughout the luteal phase of the ovarian cycle and increases during pregnancy, in parallel with the transcript of aromatase. Results were confirmed by immunoblots and immunohistochemistry using new polyclonal antisera directed against cj17HSD7 and cjARO protein. The enzymatic conversion of estrone to estradiol was assessed in marmoset corpora lutea. The pattern of coexpression with aromatase supports the hypothesis that luteal 17HSD7 complements placental 17HSD1, ensuring continued estradiol synthesis throughout pregnancy in primates.

Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa.

Fungal conventional kinesins are unusually fast microtubule motor proteins. To compare the functional organization of fungal and animal conventional kinesins, a set of C-terminal deletion mutants of the Neurospora crassa conventional kinesin, NcKin, was investigated for its biochemical and biophysical properties. While the shortest, monomeric construct comprising the catalytic core and the neck-linker (NcKin343) displays very high steady-state ATPase (k(cat) = 260/s), constructs including both the full neck and adjacent hinge domains (NcKin400, NcKin433 and NcKin480) show wild-type behaviour: they are dimeric, show fast gliding and slower ATP turnover rates (k(cat) = 60-84/s), and are chemically processive. Unexpectedly, a construct (NcKin378, corresponding to Drosophila KHC381) that includes just the entire coiled-coil neck is a monomer. Its ATPase activity is slow (k(cat) = 27/s), and chemical processivity is abolished. Together with a structural analysis of synthetic neck peptides, our data demonstrate that the NcKin neck domain behaves differently from that of animal conventional kinesins and may be tuned to drive fast, processive motility.

Therapeutic alteration of insulin-dependent diabetes mellitus progression by T cell tolerance to glutamic acid decarboxylase 65 peptides in vitro and in vivo.

We have reported previously that nonobese diabetic (NOD) fetal pancreas organ cultures lose the ability to produce insulin when maintained in contact with NOD fetal thymus organ cultures (FTOC). Initial studies indicated that exposure to glutamic acid decarboxylase (GAD65) peptides in utero resulted in delay or transient protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice. We also found that exposure of young adult NOD mice to the same peptides could result in acceleration of the disease. To more closely examine the effects of early and late exposure to diabetogenic Ags on T cells, we applied peptides derived from GAD65 (GAD AA 246-266, 509-528, and 524-543), to our "in vitro IDDM" (ivIDDM) model. T cells derived from NOD FTOC primed during the latter stages of organ culture, when mature T cell phenotypes are present, had the ability to proliferate to GAD peptides. ivIDDM was exacerbated under these conditions, suggesting that GAD responsiveness correlates with the ivIDDM phenotype, and parallels the acceleration of IDDM we had seen in young adult NOD mice. When GAD peptides were present during the initiation of FTOC, GAD proliferative responses were inhibited, and ivIDDM was reduced. This result suggests that tolerance to GAD peptides may reduce the production of diabetogenic T cells or their capacity to respond, as suggested by the in utero therapies studied in NOD mice.

Fetal thymi from diabetes-prone but not diabetes-resistant BB/Wor rats fail to generate mature ART2+ T-cells in organ culture.

Diabetes-prone (BBDP) BB rats develop spontaneous autoimmune diabetes mellitus. They are lymphopenic and severely deficient in ART2+ T-cells. Diabetes-resistant BB (BBDR) rats do not develop spontaneous diabetes and have normal numbers of ART2+ T-cells. T-cell lymphopenia in BBDP rats results from hematopoietic stem cell defects leading to abnormal intrathymic T-cell maturation. To study this process, we established rat fetal thymic organ cultures (FTOC). Like mouse FTOC, cultures of BBDR rat thymi yielded approximately 10(5) cells per lobe. The majority of cells were CD8+ART2+ T-cells. In contrast, BBDP rat FTOC yielded 60% fewer cells (approximately 0.3 x 10(5)/lobe), a smaller percentage of CD8+ and TcRalphabeta+ T-cells, and almost no detectable ART2+ T-cells. ART2 mRNA was detectable in BBDR but not BBDP FTOC. In contrast, expression of mRNAs encoding bcl-2 and a panel of cytokines was comparable in BBDP and BBDR FTOC. Addition of anti-ICAM-1 (CD54) antibody reduced T-cell number in BBDR rat FTOC by approximately 70%, but addition of IL-7 or IL-1beta had no effect. The data demonstrate that BBDP thymocytes fail to generate mature ART2+ T-cells in rat FTOC, a system that can now be used to study the mechanism of this process.

Regional differences in phorbol 12-myristate 13-acetate stimulation of PDGF production in the normal canine aorta.

OBJECTIVE: Platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen implicated in the development of intimal hyperplasia and atherosclerosis. A regional variation in canine aortic production of PDGF (greater in the distal than in the proximal aorta) was demonstrated previously in organ culture. The response of aortic segments in organ culture, as well as of aortic endothelial cells and smooth muscle cells, to stimulators of PDGF secretion-phorbol 12-myristate 13-acetate (PMA) and thrombin-was assessed to elucidate whether these regional variations were due to intrinsic differences in the abilities of cells to produce PDGF. METHODS: Proximal and distal aortic segments were removed from 10 dogs and placed in organ culture, then treated with PMA or thrombin for 72 hours. PDGF in the conditioned media was measured by radioreceptor assay. RESULTS: PDGF production in the distal, unstimulated aorta was 2.5-fold higher than that in the proximal aorta (P <.05). Treatment of the proximal aorta with 10 nmol/L and 100 nmol/L PMA increased PDGF production twofold and threefold, respectively, whereas no increase with PMA treatment was seen in the distal aorta. After thrombin treatment, no increase in PDGF production was noted in the proximal aorta and only a minimal increase was noted in the distal aorta. Endothelial cells and smooth muscle cells (n = 6) were cultured from four aortic segments (ascending thoracic, descending thoracic, abdominal, and infrarenal) and treated with PMA. PDGF production by unstimulated endothelial cells from the infrarenal aorta was 2.5-fold higher (P <.01) than that from the ascending thoracic aorta. With PMA treatment, PDGF secretion increased in endothelial cells from all segments, the greatest percentage increase being observed in the proximal segments. Thrombin also increased PDGF release from endothelial cells, but with no regional variation. Unstimulated smooth muscle cells did not exhibit regional variation in PDGF production and did not increase PDGF secretion after treatment with PMA or thrombin. CONCLUSIONS: These findings suggest that endothelial cells in the aorta may have a differential capacity to produce PDGF in response to stimulants, reflecting intrinsic differences in endothelial cells from the proximal aorta versus the distal aorta, and this may account in part for the propensity of the distal aorta to develop atherosclerosis.

Feeding oleamide to lactating Jersey cows. 2. Effects on nutrient digestibility, plasma fatty acids, and hormones.

Six lactating Jersey cows were used in a 6 x 6 Latin square with 14-d periods to evaluate different ratios of canola oil and oleamide on nutrient digestibility, plasma fatty acids, and plasma hormones. The control diet contained no added fat. All other diets contained 3.5% added fat consisting of 0, 25, 50, 75, and 100% as oleamide and the remainder as canola oil. Data were collected during the final 4 d of each period. Dry matter intake was reduced by the addition of canola oil to the diet, and further reduced by replacing canola oil with oleamide. Milk yield was not affected by diet but increasing oleamide proportion in the fat supplement caused linear increases in cis-C18:1 and linear decreases in C4 to C16 fatty acids in milk. Adding canola oil reduced total tract digestibilities of fiber and fatty acids, but had no effect on the digestibilities of dry matter or protein. Replacing canola oil with oleamide increased protein digestibility linearly, and increased digestibility of fiber (quartic relationship) and fatty acids (quadratic relationship). Oleic acid concentration in plasma increased by adding canola oil to the diet, and was further increased by replacing canola oil with oleamide. Diet had no effect on plasma concentrations of insulin or IGF-I. Oleamide fed to Jersey cows in this study was highly digestible and had no deleterious effects on total tract digestility of fiber or protein. Increasing oleic acid concentration in plasma lipids while maintaining a constant level of added fat in the ration had no effect on circulating concentrations of insulin or IGF-I in Jerseys.

NOD fetal thymus organ culture: an in vitro model for the development of T cells involved in IDDM.

This paper introduces a model which incorporates fetal thymus organ culture (FTOC) from NOD mice to replicate thymic development of diabetogenic T cells. NOD fetal pancreas organ culture (FPOC) co-cultured with 13-16 day NOD FTOC for an additional 14-21 days produced less insulin than FPOC cultured alone. Insulin production from the FTOC of non-diabetic strains C57BL/6 and BALB/c was not inhibited by co-culture with FTOC from their syngeneic counterparts. Sections of the NOD co-cultures showed peri-islet infiltration with lymphocytes. Insulin reduction by FTOC/FP co-culture was prevented by co-culture of the NOD FT with FT from immunologically incompetent C.B-17 SCID/SCID mice. Co-culture of NOD FP with NOD FT prior to the development of T cells prevented generation of diabetogenic FTOC. Thus, early exposure of NOD T cell precursors to the thymic stromal elements of C.B-17 SCID/SCID FT or to islet antigens can negatively select for diabetogenic T cells or activate immuno-regulatory cells that can suppress diabetogenic T cell activity. The addition of blocking F(ab')2 fragments of anti-CD3epsilon monoclonal antibody to NOD FTOC/FP co-cultures prevented insulin reduction, implicating a role for TcR-mediated recognition in this "in vitro IDDM" model. The addition of activating whole anti-CD3epsilon caused the complete ablation of insulin production in FTOC/FP co-cultures from all strains tested. Transfer of unprimed syngeneic FTOC cells to prediabetic NOD mice prevented the onset of IDDM while transfer of islet-cell primed FTOC/FP cells slightly increased disease incidence. These data suggest that while diabetogenic T cells are present in the FT, they are normally suppressed, even after organ culture. However, these cells can induce the destruction of islet cells, in vitro and in vivo, if they are appropriately activated with pancreatic tissue.

Peripheral blood from human immunodeficiency virus type 1-infected patients displays diminished T cell generation capacity.

An organ culture chimera system was used to assess the effect of human immunodeficiency virus type 1 (HIV-1) infection on the T cell-generation capacity of precursors derived from human peripheral blood. Peripheral blood mononuclear cells from HIV-1-infected patients and uninfected controls were placed on fetal thymus lobes of NOD/LtSz-scid/scid mice. Blood from the HIV-1-infected patients consistently produced fewer CD4 and CD8 cells compared with blood from controls (P < .01). Addition of zidovudine to the cultures did not alter this profile. Limit dilution experiments suggested that there were fewer functional precursors in the infected patients. These results were not dependent on the patient's level of peripheral CD4 cells; even samples from patients with normal CD4 cell counts were unable to generate T cells in organ cultures. The results are consistent with a loss in the capacity of HIV-1-infected patients to produce functional T cell progenitors in their peripheral blood.

Conformational studies of mono- and bicyclic parathyroid hormone-related protein-derived agonists.

Parathyroid hormone-related protein (PTHrP) is expressed in a wide variety of cells where it acts as an autocrine and/or paracrine factor involved in regulation of cellular growth, differentiation, and embryonic development. It may also play a physiological endocrine role in calcium transport across the placenta or during lactation. The N-terminal portion, PTHrP-(1-34), retains all the calciotropic parathyroid hormone-like activity and is a lead structure for the design of novel, bone anabolic agents for the treatment of bone disorders such as osteoporosis. To characterize the putative bioactive conformation, we have carried out a detailed structural analysis of a series of three conformationally constrained PTHrP-(1-34)-based mono- and bicyclic lactam-containing biologically active analogs: (III) The conformational properties were studied by circular dichroisim, nuclear magnetic resonance spectroscopy, distance geometry calculations, and molecular dynamic simulations in water/trifluoroethanol (TFE) mixtures. The helical content in water of both monocyclic analogs I and II is approximately 22%; that of the bicyclic analog III is approximately 40%. In 30% TFE, all analogs reached a maximal helical content of 80%, corresponding to 26 or 27 residues out of 34 in a helical conformation. High-resolution structures obtained with 50:50 TFE/water revealed that all three analogs display two helical domains and a hinge region around Gly12-Lys13. The highly potent mono- and bicyclic agonists I and III display a second hinge around Arg19-Arg20 which is shifted to Ser14-Asp17 in the weakly potent monocyclic agonist II. We suggest that the presence and localization of discrete hinges in the sequence together with the high propensity for helicity of the C-terminal sequence and the enhancement of helical nucleation at the N-terminal sequence are essential for generating a PTH/PTHrP receptor-compatible bioactive conformation.

Carbonyl cyanide phenylhydrazones as probes of the anionic activator site of the human erythrocyte glutathione adduct transport ATPase.

We have previously shown that the ATPase activity associated with the erythrocyte glutathione adduct transporter is also stimulated by 2,4-dinitrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, both well-known anionic and lipophilic uncouplers of oxidative phosphorylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazones as activators of the ATPase. All of the compounds tested stimulated the ATPase to similar extents, based on Vmax values. The K0.5 for stimulation of the ATPase depended on the electron-withdrawing characteristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slope of this relationship, with lower K0.5 values for electron-withdrawing substituents, suggests that an anionic residue in the active site partially discourages binding of this class of activators. ortho-Substituted carbonylcyanide phenylhydrazones do not follow this relationship, but show lower apparent affinities than expected from their pKa values. This finding suggests that steric effects in that region of the binding site negatively influence the affinity.