Basophils and mast cells at the centre of the immunological response.
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Basophils↗
Biomedical subjects
C M van Drunen
Publications and source records attributed to C M van Drunen.
Reduced nasal IL-10 and enhanced TNFalpha responses during rhinovirus and RSV-induced upper respiratory tract infection in atopic and non-atopic infants.
Rhinovirus and respiratory syncytial virus (RSV) are the most prevalent inducers of upper respiratory tract infections (URTI) in infants and may stimulate immune maturation. To estimate the amount of immune stimulation, nasal immune responses were examined during rhinovirus and RSV-induced URTI in infants. Nasal brush samples were taken from infants (2-26 months; 57% atopic family) with rhinovirus-induced URTI (N=20), with RSV-induced URTI (N=7), and with rhinovirus-induced rhinitis (N=11), from children with asymptomatic rhinovirus infection (N=7) and from eight non-infected children. Numbers of nasal brush cells positive for Th1-, Th2-, regulatory and proinflammatory cytokines were measured by immunohistochemistry or by measuring protein levels using a cytometric bead array analysis. During rhinovirus and RSV-induced URTI, fewer regulatory cytokine IL-10 positive cells were found compared to non-infected children. This fall was accompanied by an increase in levels of the Th1 cytokine TNFalpha. IL-10 responses were inversely related to TNFalpha responses. No enhanced responses were observed for IFNgamma, IL-12 and IL-18. Cytokine responses were comparable in children with rhinovirus-induced URTI and in children with rhinitis, while responses in asymptomatic rhinovirus-infected children were located between those for symptomatic and asymptomatic rhinovirus-infected children. Cytokine responses did not depend on the age of the child or atopy in the family. In conclusion, reduced nasal IL-10 responses during URTI in infants could facilitate the induction of a TNFalpha response. TNFalpha in turn could replace the immature production of IL-12, IL-18 and IFNgamma during URTI to induce an effective clearance of the viral infection and which could stimulate the maturation of Th1 cytokine production in infancy.
Child, Preschool↗
Age- and infection-related maturation of the nasal immune response in 0-2-year-old children.
BACKGROUND: The hygiene hypothesis suggests that exposure to micro-organisms influences development of the immune system in children. METHODS: In this study, we examined nasal immune responses in the first 2 years of life in relation to age of children and the number of viral infections they have experienced. Nasal brushes were taken during rhinovirus- (n = 20) or respiratory syncytial virus (RSV)-induced (n = 7) upper respiratory tract infections (URTI), and of controls (n = 40). RESULTS: The number of macrophages were higher during URTI and increased with age. The number of T lymphocytes increased with age in controls and were higher during URTI at all ages. We found an age-related decrease in the number of interleukin (IL)-4- and IL-10-positive cells in controls, while the number of IL-12-positive cells remained unchanged. Changes in T lymphocyte and IL-4 cell number were stronger related to the age of the child than to the number of respiratory infections, while the opposite was true for macrophages. CONCLUSIONS: In infants, we found an infection- and age-related increase respectively for nasal macrophages and T lymphocytes during URTI. Furthermore, the number of IL-4- and IL-10-positive cells decreased with age. Whether this maturation reflects a natural age-related maturation, the degree of exposure to respiratory infections, or possibly both, could not be resolved and needs further study.
Age Distribution↗
Evaluation of an intranasal house dust mite provocation model as a tool in clinical research.
BACKGROUND: As a result of the all-year-round exposure to house dust mite (HDM), perennial rhinitis patients never have a clear symptom-free period. In this study, we investigated whether, despite these symptoms, we can still use nasal HDM provocations to study perennial allergic rhinitis and the effects of treatment. METHODS: In a parallel-group study, after 1 week treatment with either fluticasone propionate aqueous nasal spray (FPANS) or placebo, 20 patients, allergic to HDM, registered symptoms (nasal obstruction, rhinorrhoea, sneezing, pruritus and eye symptoms) using three different scoring methods [Lebel, categorical and visual analogue scale (VAS)] and peak nasal inspiratory flow (PNIF) after HDM provocations. Provocations were performed with 1000 biological units/ml and 24 h later with 10,000 biological units/ml of HDM. Before and after the provocations, nasal mucosa biopsies were taken for immunohistochemical staining to determine the number of eosinophils. RESULTS: House dust mite provocations resulted in an increase in symptoms and a decrease in PNIF. Even at high-dose provocation, the FPANS group registered significantly lower symptoms than the placebo group for nasal blockage, sneezing, eye symptoms, and PNIF in both early and late phases. FPANS also suppressed rhinorrhoea during the late phase and the influx of eosinophils in the lamina propria. CONCLUSION: Despite the high background of symptoms, allergic responses can be induced in this perennial rhinitis model. The VAS score seems most suited to detect these changes and the suppression of symptoms by 7 days of FPANS treatment. Epithelial eosinophilia at baseline was correlated positively with the severity of the reaction after the first provocation.
Administration, Intranasal↗
Desloratadine reduces systemic allergic inflammation following nasal provocation in allergic rhinitis and asthma patients.
BACKGROUND: Preclinical studies have demonstrated that some second-generation antihistamines have anti-inflammatory effects. It is not known whether these effects are also demonstrable in vivo. In this study we investigated the effect of treatment with desloratadine (DL) on systemic inflammation and on nasal and bronchial mucosal inflammation after nasal allergen provocation (NP) in subjects with grass-pollen-allergic rhinitis and asthma. METHODS: Twenty-six subjects with grass-pollen-allergic rhinitis and asthma were randomly allocated to 8 days of treatment with DL (n = 13) or placebo (n = 13) outside the grass pollen season. On day 7 they underwent nasal provocation with grass pollen allergen. Nasal and bronchial biopsies were taken for immunohistochemical evaluation, and blood samples were analysed. Rhinitis and asthma symptoms, peak nasal inspiratory flow and peak expiratory flow, were also measured at specified times. RESULTS: The number of circulating eosinophils decreased during DL treatment, and there was a reduced increase in circulating eosinophils after NP in these subjects. There was also a significant reduction in early bronchial clinical response. There was no significant lessening in the severity of the nasal symptoms. Nasal and bronchial mucosal inflammation parameters did not alter under DL treatment. CONCLUSION: These data suggest that treatment with DL reduces systemic eosinophilia and prevents the increase in circulating eosinophils after NP. DL also significantly reduces the early bronchial clinical response to NP. However, airway mucosal inflammation is not altered by 1 week of treatment.
Adult↗
dSIR2 and dHDAC6: two novel, inhibitor-resistant deacetylases in Drosophila melanogaster.
We have identified new members of the histone deacetylase enzyme family in Drosophila melanogaster. dHDAC6 is a class II deacetylase with two active sites, and dSIR2 is an NAD-dependent histone deacetylase. These proteins, together with two class I histone deacetylases, dHDAC1 and dHDAC3, have been expressed and characterized as epitope-tagged recombinant proteins in Schneider SL2 cells. All these proteins have in vitro deacetylase activity and are able to deacetylate core histone H4 at all four acetylatable lysine residues (5, 8, 12, and 16). Recombinant dHDAC6 and dSIR2 are both insensitive to TSA and HC toxin and resistant, relative to dHDAC1 and dHDAC3, to inhibition by sodium butyrate. Indirect immunofluorescence microscopy of stably transfected SL2 lines reveals that dHDAC1 and dSIR2 are nuclear, dHDAC6 is cytosolic, and dHDAC3 is detectable in both cytosol and nucleus. dHDAC6 and dSIR2 elute from Superose 6 columns with apparent molecular weights of 90 and 200 kDa, respectively. In contrast, dHDAC1 and dHDAC3elute at 800 and 700 kDa, respectively, suggesting that they are components of multiprotein complexes. Consistent with this, recombinant dHDAC1 coimmunoprecipitates with components of the Drosophila NuRD complex and dHDAC3 with an as yet unknown 45-kDa protein.
Amino Acid Sequence↗
SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.
SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.
Amino Acid Motifs↗
A bipartite sequence element associated with matrix/scaffold attachment regions.
We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.
Animals↗
Involvement of a nuclear matrix association region in the regulation of the SPRR2A keratinocyte terminal differentiation marker.
The small proline-rich protein genes ( SPRRs ) code for precursors of the cornified cell envelope, and are specifically expressed during keratinocyte terminal differentiation. The single intron of SPRR2A enhanced the activity of the SPRR2A promoter in transient transfection assays. This enhancement was position dependent, and did not function in combination with a heterologous promoter, indicating that the intron does not contain a classical enhancer, and that the enhancement was not due to the splicing reaction per se. Mild DNAse-I digestion of nuclei showed the SPRR2 genes to be tightly associated with the nuclear matrix, in contrast to the other cornified envelope precursor genes mapping to the same chromosomal location (epidermal differentiation complex). In vitro binding studies indicated that both the proximal promoter and the intron of SPRR2A are required for optimal association of this gene with nuclear matrices. Neither nuclear matrix association nor the relative transcriptional enhancement by the intron changed during keratinocyte differentiation. Apparently, the association of the SPRR2A gene with the nuclear matrix results in a general, differentiation-independent enhancement of gene expression.
3T3 Cells↗
Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis thaliana.
The Arabidopsis thaliana genome is currently being sequenced, eventually leading towards the unravelling of all potential genes. We wanted to gain more insight into the way this genome might be organized at the ultrastructural level. To this extent we identified matrix attachment regions demarking potential chromatin domains, in a 16 kb region around the plastocyanin gene. The region was cloned and sequenced revealing six genes in addition to the plastocyanin gene. Using an heterologous in vitro nuclear matrix binding assay, to search for evolutionary conserved matrix attachment regions (MARs), we identified three such MARs. These three MARs divide the region into two small chromatin domains of 5 kb, each containing two genes. Comparison of the sequence of the three MARs revealed a degenerated 21 bp sequence that is shared between these MARs and that is not found elsewhere in the region. A similar sequence element is also present in four other MARs of Arabidopsis.Therefore, this sequence may constitute a landmark for the position of MARs in the genome of this plant. In a genomic sequence database of Arabidopsis the 21 bp element is found approximately once every 10 kb. The compactness of the Arabidopsis genome could account for the high incidence of MARs and MRSs we observed.
Amino Acid Sequence↗
Transposase A binding sites in the attachment sites of bacteriophage Mu that are essential for the activity of the enhancer and A binding sites that promote transposition towards Fpro-lac.
In this paper we determine which of the A binding sites in the attachment sites of phage Mu are required for the stimulatory activity of the transpositional enhancer (IAS). For this purpose the transposition frequencies of mini-Mu's with different truncated attachment sites to an Ftet target were measured both in the presence and the absence of the IAS. The results show that in our in vivo assay the L3 and R3 sites are dispensable for functioning of the IAS. An additional deletion of L2 or R2 however abolishes the stimulating activity of the enhancer suggesting an interaction between A molecules bound to these sites and the IAS. The residual transposition activity of a IAS-containing mini Mu in which R2 (and R3) are deleted is much lower than the activity of the comparable construct without the IAS. This means that in the absence of R2 the IAS is inhibiting transposition. Such an inhibition is not observed when L2 (and L3) are deleted. This suggests that the IAS interacts with the attachment sites in an ordered fashion, first with attL and then with attR. Furthermore we show that mini-Mu transposition is enhanced when Fpro-lac is used as a target instead of Ftet. We show that this elevated transposition is dependent on the Mu A binding sites L2,L3 and R2. These sequences could possibly mediate an interaction between the mini-Mu plasmid and sequences present on Fpro-lac.
Bacteriophage mu↗
Inhibition of bacteriophage Mu transposition by Mu repressor and Fis.
In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3-80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site lead to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4 bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.
Bacteriophage mu↗
Enhanced induction of SV40 replication from transformed rat cells by fusion with UV-irradiated normal and repair-deficient human fibroblasts.
Fusion of SV40-transformed rat (BRKSV) cells which do not spontaneously produce infectious virus, with permissive monkey cells resulted in a low level of production of infectious virus in the heterokaryons. UV-Irradiation of the BRKSV cells prior to fusion did not result in increased virus production, but irradiation of the monkey cells prior to fusion did result in enhanced induction (EI) of SV40, as compared to control experiments in which neither cell type was irradiated. This indicated that rat cells lack the ability to initiate replication of integrated SV40 upon UV-irradiation and do not contain "permissiveness" factors that are required to support SV40 replication. In contrast, monkey cells do contain such permissiveness factors which seem to be temporally enhanced by UV-irradiation, and thus may be responsible for the EI phenomenon. Expression of EI was dose-dependent and reached maximum values approximately 24 h after UV-irradiation. The kinetics of EI resembled that of EI previously established for SV40 induction in semi-permissive cells, and of enhanced reactivation (ER) and enhanced mutagenesis (EM) of SV40 in monkey cells. Similar kinetics of EI were obtained when human diploid fibroblasts were used for fusion with BRKSV cells. Similar levels of EI were found with normal human cells and repair-deficient xeroderma pigmentosum (XP) cells of complementation groups A and C, and XP variant cells. This suggests that expression of EI is not related to excision repair. Since EI is also normally expressed in XP cells which display an abnormal ER of HSV and in XP variant cells which show a delayed EM of HSV, we conclude that EI may occur independently of ER and EM. Finally it was shown that treatment of human cells with N-ethyl-N-nitrosourea results in similar induction of EI as irradiation with UV-light, and that addition of TPA in fusion experiments has no effect on EI.
Animals↗
Enhanced induction of SV40 replication from transformed mammalian cells by fusion with UV-irradiated untransformed cells.
DNA-damaging agents such as ultraviolet (UV) light are known to cause stimulation of virus replication in SV40-transformed hamster and human cells. The dose-response curves of UV-induced SV40 replication in transformed hamster cells resemble that obtained for UV-enhanced reactivation (ER) and UV-enhanced mutagenesis (EM) of SV40 or herpes viruses in mammalian cells. We have investigated whether UV-enhanced production of SV40 from transformed hamster (THK) and human (NB-E) cells belongs to the same category of conditional responses as ER and EM. To answer this question we have made use of the phenomenon that fusion of the SV40-transformed cells with monkey cells that are permissive to SV40 results in a considerable increase in the production of SV40 virus. When THK or NB-E cells were fused with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased and reached a maximum value of 2--3-fold when fusion was delayed for 24-48 h after irradiation. The kinetics of enhanced SV40 induction resembled that of ER and EM, suggesting that the UV-stimulated part of the induction represents one of the pleiotropic responses that are transiently induced in mammalian cells by DNA-damaging agents. Evidence is presented, showing that this transient effect can be induced only in cells that are permissive to SV40 replication. This suggests that the enhanced induction observed after fusion with irradiated monkey cells may be attributed to a transient increase in the activity of "permissiveness' factors. No enhanced induction was found when the THK or NB-E cells were fused with irradiated rodent cells, that are not or only slightly permissive to SV40 replication.
Animals↗