bluuebunny/crossref_metadata_2025 huggingface dataset:
P67 Hand eczema, skin exposure and glove use in dental technicians
Authors: Meding Birgitta , Hosseiny S , Wrangsjö K , Andersson E , Hagberg S , Wass K , Torén K , Brisman J
Published: August 2010

Aims:  To estimate the incidence of hand eczema in dental technicians and to elucidate occupational risk factors.

Methods:  This was a retrospective cohort study among former dental technician students (n = 2139). Controls comprised population controls (n = 2288) with similar distribution of age and sex as the dental technicians. A mail questionnaire asked for occurrence of hand eczema including age of onset, occupational exposure and use of protective gloves. The response rate was 58%(1210 dental technicians and 1316 controls answered the questionnaire).

Results:  In dental technicians the incidence of hand eczema was 7.1 cases/1000 person years in males and 10.8 in females during acrylate‐exposed time. In controls the incidence of hand eczema was 3.1 in males and 3.7 in females. The relative risk (RR) for males was 2.3 (95% CI 1.6–3.4) and for females 2.9 (95% CI 2.1–4.0). 48% of the dental technicians and 30% of the controls reported more than 10 hand‐washings/day (p < 0.001). 80% of the dental technicians reported skin exposure to uncured acrylates, 14% daily. 87% had skin contact with grinding dust from acrylates, 42% daily. 38% used protective gloves when handling uncured acrylates, males 29%, females 48%(p < 0.001). 58% did not know for how long the normally used gloves protected the skin against uncured acrylates.

Conclusions:  The risk of hand eczema is more than doubled in dental technicians. The work involves frequent and unprotected exposure to acrylates and wet work. Education regarding skin protection is important.
FS01.1 The use of azo dyes in the European Union – legislation review
Authors: Azenha Angelo
Published: August 2010

The aim of this paper is to review the European legislation concerning the use of textile dyes.

It is known that different dyes of different classes can be sensitizing and cause allergic contact dermatitis for both textile workers and consumers.

The European Union has created restraining directives regarding the manufacture and distribution of potentially dangerous products, such as aromatic amines. A specific scientific committee of EU stated that the carcinogenic capacity of some azo dyes is related to chemical releasing of aromatic amines (groups MAK III; A1; A2).

In this paper, focus will be centred on the latest European Union Directives in this matter and the prohibition of some azo dyes in textile industry.
FS01.3 Disperse (yes), orange (yes), 3 (no): what do we test in textile dye dermatitis?
Authors: Le Coz Christophe J , Jelen G , Goossens A , Vigan M , Ducombs G , Bircher A , Giordano‐Labadie F , Pons‐Guiraud A , Milpied‐Homsi  , Castelain M , Tennstedt D , Bourrain J‐L , Bernard G
Published: August 2010

Introduction:  Patients sensitized to para‐phenylenediamine (PPD) have a high degree of patch test reactivity to Disperse Orange 3 (DO3), and a lesser one to Disperse Red 1 and Red 17. Two successive patients positive to PPD, Disperse Red 1 and 17, negative to DO3 were real eye‐openers for our considerations about purity of our current allergen DO3.

Materials and methods:  We realized comparative thin‐layer chromatography (TLC), with DO3 from Chemotechnique®(DO3‐Chem) and Trolab®(both extracted from petrolatum), and “pure” DO3 from two chemical providers. TLC clearly indicated that DO3‐Chem was not DO3. HPLC analysis with pure DO3 from Chemotechnique® and comparison of structures by NMR with samples of DO3, revealed that DO3‐Chem was Disperse Orange 31 (DO31). In addition, signals through the GERDA network allowed the collection of test materials and observations. Among other members, only 2 used DO3‐Chem (from 2 different batches) that was DO31 too, according to TLC Results: According to their data, they observed no or a lower reactivity to DO3 than expected (4 patients DO3‐Chem + among 23 PPD+ e.g.). Finally, the error was proved to be due to the provider of the dye to Chemotechnique®, who likely deleted the 1 of Disperse Orange 31 on his packaging.

Discussion:  Chemical structure of DO31 indicates a possible in vivo hydrolysis into nitroaniline and a second compound, a substituted PPD derivative that clearly does not frequently react in PPD positive patients. Like drugs, patch tests are submitted to post‐commercialization controls. In addition to allergens providers who should enhance their quality controls, dermato‐allergologists have to be vigilant, and must active networks when they observe a rare bird.
FS01.8 Substance‐specific modulation of TNF‐Alpha and IL‐1Beta in human monocytes by para‐Phenylenediamine and not by its autoxidation product Bandrowski’s Base
Authors: Hamidizadeh Nasrin , Breuer S , Merk HF , Bloemeke B
Published: August 2010

Para‐phenylenediamine (PPD), an arylamine dye, is a strong allergen causing allergic contact dermatitis. Cytokines such as TNF‐α and IL‐1beta are key mediators in the initiation of this reaction. Both cytokines are predominantly produced by stimulated monocytes and macroghages. We investigated the responses of PPD and Bandrowski's base (BB), an autoxidation product of PPD in human monocytes. We isolated monocytes from healthy volunteers and incubated them with the allergens. TNF‐α and IL‐1beta mRNA expression and protein levels were estimated after 45 min, 2 h, 4 h and 24 h after allergen contact. IL‐1beta and TNF‐alpha were measured in cell culture supernatants by ELISA (n = 7) and mRNA expression was determined by real‐time RT‐PCR. We found that PPD reduced TNF‐α protein secretion by 20–69.9%(n = 6). Further, IL‐1beta levels were decreased by 44–98%. The same tendency was found studying IL‐1beta and TNF‐α mRNA steady state levels (n = 3; 1 h incubation). These effects were substance‐specific and not found for PPD derivatives nor for the autoxidation product BB. These findings suggest that PPD may specifically modify immune responses by directly infering with the cellular proinflammatory cytokine network.

This study was supported by the EU.
FS02.7 Rates of persistent itchy nodules after fourth dose of DT vaccines
Authors: Netterlid Eva , Bruze M , Hindsén M , Isaksson M , Olin P
Published: August 2010

Background:  Studies in Gothenburg, Sweden, reported unexpectedly high rates, 0.35–1.66%, of late occurring itching nodules at the site of injection of aluminium containing vaccines. Among 455 children with itching nodules after vaccination, 352 had a positive epicutaneous test to aluminium. When switching to a new booster diphtheria‐tetanus vaccine in Sweden these findings warranted a comparative study of local itching nodules that had remained at least 2 months after injection, and of aluminium contact allergy.

Methods:  A prospective cluster randomised study was done in 25,232 10‐year‐olds. Participating schools in each municipality were randomised in a 1/1 ratio to use the old diphtheria‐tetanus toxoid (DT) vaccines, Duplex® or the new vaccine, diTeBooster®. Parental reports 6 months after vaccination were obtained for 22,365 (88%) pupils in 851 schools. Patch testing with aluminium chloride‐hexahydrate was performed in 9 children with an itching nodule, 6 children only pruritus and 17 children without any itching nodule after the fourth dose of DT vaccine.

Results:  We identified 3–6 children per 10000 with a local itching nodule persisting for at least 2 months. There were no significant differences between the vaccine groups. Contact allergy to aluminium was not detected.

Conclusion:  Our findings support the use of the vaccine presently available in the Swedish vaccination program. Continued surveillance of persistent itching nodules after vaccination with different aluminium containing DT and diphtheria‐tetanus‐pertussis toxoid (DTP) vaccines is however warranted.
Adenosine Receptor A2a is Differentially Expressed in CD4+ T Lymphocytes of Asthmatic and Healthy Individuals
Authors: Holopainen P. , Karagiannidis C. , Rückert B. , Hense G. , Schmidt‐Weber C. , Blaser K.
Published: August 2010

The endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti‐inflammatory effects. Adenosine acts via four adenosine receptors of which the A2a receptor is the predominant form in T cells. Adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. Instead, the role of anti‐inflammatory mechanisms of adenosine on T cells in asthma is unclear.

Aim:  To study the A2a receptor expression in peripheral blood CD4+ T cells in asthmatic and healthy individuals using flow cytometric and quantitative real‐time PCR methods.

Results:  Unstimulated CD4+ cells of asthmatic patients expressed significantly lower levels (P < 0.001) of A2a receptor in protein level (mean percentage of cells positive ± SEM: 76.8 ± 1.2, n = 6) compared to healthy individuals (90.4% ± 1.9, n = 4). Double staining for CD69 expression showed that stimulation of CD4+ cells decreased A2a expression in both groups but indicated that the detected lower levels of A2a in unstimulated cells of asthmatics was not due to preactivation in these patients. Surprisingly, A2a mRNA expression in unstimulated CD4+ cells was significantly higher (P < 0.05) in asthmatics (n = 28) compared to healthy controls (n = 7). The expression did not correlate with serum total IgE levels.

Conclusions:  Asthmatic individuals express less A2a adenosine receptor on their peripheral CD4+ T cells. The higher mRNA levels instead may point to a negative feedback regulation in the receptor expression. The role of possibly decreased adenosine‐mediated anti‐inflammatory effects in asthma pathogenesis require further studies on this T‐cell mediated disease.
A Novel Adjuvant Allergen Complex, CBP‐Fel d 1, Induces Upregulation of CD86 and Cytokine Release in Human Dendritic Cells
Authors: Neimert‐Andersson T. , Ekman G. J. , Grönlund H. , Buentke E. , Scheynius A. , Van Hage‐Hamsten M. , Gafvelin G.
Published: August 2010

Allergen‐specific immunotherapy (SIT) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). Drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. An alternative carrier is 2 μm carbohydrate‐based particles (CBPs). In mouse, allergen‐coupled CBPs have been demonstrated to skew the allergen‐specific immune response towards a Th1‐like activity (Grönlund et al. Immunology, 2002). We here coupled the recombinant major cat allergen Fel d 1 to CBPs (CBP‐Fel d 1) by cyanogen‐bromide activation, resulting in covalent binding. The effect of CBP‐Fel d 1 on monocyte‐derived dendritic cells (MDDCs) from healthy human blood donors was studied. We found that the majority of the CD1a+ MDDCs were capable of taking up FITC‐labelled CBP‐Fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. Furthermore, incubation with CBP‐Fel d 1 resulted in an upregulation of the costimulatory molecule CD86 on the MDDCs, which was not observed with Fel d 1 or CBPs alone. Finally, CBP‐Fel d 1 induced a fivefold increase in the release of the pro‐inflammatory cytokine tumour necrosis factor (TNF)‐α and a fourfold increase in the release of the chemokine interleukin‐8 from MDDCs. Taken together, the effects CBPs possess make them interesting as novel allergen carriers for SIT.
Recominant Expression and Immunological Characterization of House Dust Mite Allergen Der P 1
Authors: Draborg H. , Roggen E. L. , Soni N. K. , Patkar S. , Friis E. P. , Lyngstrand S. T. , Christensen L. L. H. , Batori V. , Danielsen S. , Ernst S.
Published: August 2010

The cysteine protease Der p1 from dust mite of the genus Dermatophagoides pteronyssinus is a major type I allergen. About 80% of house dust mite (HDM) allergic individuals are reactive to this protease in standard assays for detection of IgE. A curative treatment for atopic allergy is immunotherapy (IT) with HDM extracts which are complex mixtures occasionally resulting in anaphylactic reactions. Novozymes focuses on developing a recombinant variant of Der p1 which exhibit lowered risk of IgE‐mediated allergic reactions, while maintaining its ability to trigger proper Th‐cell responses. This may provide a safer alternative for specific IT of HDM allergy. A secreted recombinant form of pro‐Der p 1 expressed by Saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. The N‐glycosylation site of Der p1 was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of 35 kDa. Protein purification procedure was developed to obtain nearly pure Der p1 protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor E64. The deglycosylated recombinant pro‐Der p 1 revealed immunologic similarity to the native Der p 1 molecule when compared in basophile histamine release, IgE‐binding assays and T‐cell proliferation assays. By in silico epitope mapping of a modelled 3‐dimensional structure of Der p1, five putative IgG and IgE epitopes were predicted. By protein engineering, the predicted epitopes were removed one by one in Der p1 and screening for hypoallergenic variants was performed.
Lysine‐Dependent Binding of OspE to the C‐terminus of Factor H Mediates Complement Resistance in Borrelia burgdorferi
Authors: Alitalo A. , Meri T. , Lankinen H. , Cheng Z.‐Z. , Jokiranta S. , Seppälä I. , Lahdenne P. , Brooks C. , Hefty P. S. , Akins D. R. , Meri S.
Published: August 2010

Serum resistance of Borrelia burgdorferi strains belonging to the B. afzelii and B. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor H. We recently reported that factor H binding by B. burgdorferi is due to inducible expression of several approximately 20 kDa plasmid‐encoded, surface‐exposed lipoproteins related to OspE (e.g. ErpA, ErpP and P21). In addition, a second class of factor H‐binding proteins of approximately 27–35 kDa has been described. The OspE‐related lipoproteins are dramatically induced by B. burgdorferi during transmission from its tick vector into the mammalian host. The induction of OspE‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. The goal of the present study was to define the factor H‐binding regions of OspE‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (Biacore). The combined studies revealed that the C‐terminal regions of both human and mouse factor H (SCRs 18–20) specifically bind to OspE‐related lipoproteins. We also found FHR‐1, whose C‐terminal SCRs 3–5 are homologous to SCRs 18–20 of factor H, to bind to OspE. Peptide mapping revealed five putative regions (designated I‐V) in OspE that could directly interact with factor H. Deleting the C‐terminal 15 amino acid residues from region V of P21 abolished its ability to bind factor H. At the same time, however, synthetic peptides corresponding to the C‐termini of OspE, P21 and ErpP did not inhibit factor H binding to OspE. Thus, the C‐terminal‐binding region V appears to be necessary but not sufficient for factor H binding. When a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor H‐binding regions were mutated to alanines, we observed that lysines in the factor H‐binding regions of OspE were required for factor H binding. The combined data have revealed that key lysine residues in OspE‐related lipoproteins and ionic interactions are crucial for factor H interactions. Furthermore, binding of OspE to the C‐termini of both mouse and human factor H suggests that Borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. In Borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the OspE sequences as well as in the expression of factor H‐binding proteins may account for their susceptibility to serum lysis.
Role of YadA, Ail and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3
Authors: Biedzka‐Sarek M. , Venho R. , Skurnik M.
Published: August 2010

Complement attack is one of the host strategies leading to elimination of pathogens. Yersinia enterocolitica, resistant to human serum, expresses several factors that may contribute to this resistance, the outer membrane proteins YadA and Ail, and lipopolysaccharide (LPS). To study relative roles of these factors for the survival of Y. enterocolitica serotype, O:3 (Ye O:3) in nonimmune human serum, we constructed 23 mutant strains of Ye O:3 expressing different combinations of YadA, Ail, LPS O‐antigen and LPS outer core. Survival of bacteria was analysed in normal serum (with functional classical and alternative complement activation pathways) and EGTA‐Mg‐treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single serum resistance factor needed for long‐term survival was YadA. Ail was important for short‐term survival and thus delayed the bacterial killing. On the contrary, the LPS O‐antigen and outer core when in combination with YadA or Ail, or both, had a minor and often negative effect on serum resistance.
Interferon‐α mRNA in Splenic CD11b+ Marginal Zone Macrophages of C4‐Deficient Mice
Authors: Finke D. , Hoerster R. , Brockmann C. , Kropf K. , Hennig H. , Zawatzky R. , Goerg S.
Published: August 2010

The absence of early complement components (C1, C4 and C2 but not C3) is a predisposing factor for systemic lupus erythematosus (SLE). Recently, we demonstrated that, in C4‐deficient (C4 def.) mice, IgM‐containing immune complexes (IgM‐IC) are filtered by the splenic barrier of marginal zone macrophages (MZM), resulting in an increased immune response against antigens within these IgM‐IC, but this could not be observed in wildtype or C3 def. mice. We hypothesized that splenic CD11b+ MZM play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mRNA was isolated, and real‐time PCR was performed with specific primers for murine IFN‐γ (IFN‐γ), interleukin‐12 (IL‐12) and IFN‐α (IFN‐α). We observe a moderate increase of IL‐12 and IFN‐γ mRNA in CD11b+ cells of C4 def. mice compared to wildtype cells. Surprisingly, the concentration of IFN‐α mRNA is six times higher in C4 def. mice. Preliminary results suggest that mRNA in CD11b+ cells of C3 def. mice is even lower than that in wt. Six hours following i.v. application of 20 µg of a murine monoclonal IgM anti‐dsDNA antibody, production of IL‐12, IFN‐γ and IFN‐α mRNA is increased in CD11b+ cells of both C4 def. and wt mice. Several references described increased levels of INF‐α in patients with SLE. Dendritic cells are discussed as a major source of IFN‐α. Our observation that C4‐deficient, SLE‐susceptible mice demonstrate an increased spontaneous IFN‐α production by splenic CD11b+ marginal zone macrophages could be an early sign and a trigger for the development of SLE. This is supported by the fact that the absence of C3 is not a predisposing factor for SLE and our observation that C3 def. animals display low levels of IFN‐α mRNA.
Mannan‐Binding Lectin Inhibits Humoural Responses
Authors: Ruseva M. , Gajdeva M. , Takahashi K. , Ezekowitz A. , Thiel S. , Jensenius J. C.
Published: August 2010

Chronic hepatitis B virus (HBV) infection affects about 200–400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. Control over the HBV infection is achieved mainly by vaccination with Hepatitis B surface antigen (HBsAg). HBsAg contains N‐linked glycosylation side and is recognized by both MBL‐A and MBL‐C in a Ca‐dependent manner. HbsAg–MBL complexes activate complement and may thus affect humoural immunity. To investigate the role of MBL in humoural responses to HBsAg, we immununized mice that lack both MBL‐A and MBL‐C proteins with soluble HBsAg. It has been shown that deficiencies in other complement components like C1q, C4 and C3 result in decreased antibody responses. However, MBL double KO animals mounted dramatically increased humoural responses. After priming, MBL double KOs mounted HbsAg‐specific IgM responses, which were threefold higher than WT controls. After boosting the HBsAg, total IgG was 10‐fold higher in MBL KO than in WT control animals. Similar to the response to HbsAg, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in MBL double KO animals, suggesting that MBL plays an important role in a negative feedback regulation of adaptive immunity. Reconstitution experiments with rMBL partially rescued the KO phenotype. We propose that the clearance of glycoprotein antigens in MBL KO is handled differently from the WT, resulting in better stimulation of humoural responses. Alternatively, glycoprotein‐Ag‐MBL‐rich complexes inhibit B‐cell responsiveness via putative MBL receptors.
Human Isolates of Lactic Acid Bacteria Differentially Affect Maturation and Cytokine Production by Human Dendritic Cells
Authors: Hjerrild L. , Christensen H. R. , Frøkiær H.
Published: August 2010

The intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut‐associated immune system. Evidence indicates that lactic acid bacteria (LAB), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. Established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. The objective of this study is to evaluate the immunomodulating properties of a range of LAB of human origin. As dendritic cells (DCs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro‐generated immature DCs serve as a suitable model for studying the immunomodulating effects of lab. Human immature DCs were generated in vitro from monocytes and exposed to lethally UV‐irradiated LAB. The effect of various species of LAB on DCs in direct contact was evaluated. Furthermore, the maturation pattern of DCs separated from the bacteria by an epithelial cell layer (CaCo‐2 cells), which should mimic the intestinal environment, was studied. Cytokine secretion (IL‐12, IL‐10 and TNF‐α) and upregulation of maturation surface markers on DCs (CD83 and CD86) was measured. Different LAB induced diverse cytokine responses. Some strains were strong IL‐12 and TNF‐α inducers and others weak. All strains induced IL‐10. Different LAB also differentially modulated expression of CD83 and CD86 on DCs. Although some variation in the response to LAB of DCs generated from different blood donors was observed, general differences in the effect of the various LAB was revealed. Experiments with the DC CaCo‐2 coculture system are ongoing. Different species of LAB differentially affect DC maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response.
Lactobacilli Modulate Proliferation and Cytokine Production of Human Peripheral Blood Natural Killer Cells In Vitro
Authors: Fink L. N. , Christensen H. R. , Frøkiær H.
Published: August 2010

Natural killer (NK) cells are cells of the nonspecific immune system lysing altered self‐cells. A noncytolytic subset of NK cells may serve a regulatory role by secreting cytokines. Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. Here, we investigated how human gut flora‐derived lactobacilli affect NK cells in vitro, by measuring proliferation and IFN‐γ production of human NK cells upon bacterial stimulation. CD3–CD56+NK cells were isolated from buffy coats by negative isolation using non‐NK lineage‐specific antibodies and magnetic beads. NK cells were incubated with 10μg/ml UV‐inactivated bacteria or 10μg/ml phytohemagglutinin (PHA) for 4 days. Proliferation was assessed by incorporation of radioactive thymidine into NK‐cell DNA. The IFN‐γ concentration was measured by ELISA. Incubation of NK cells with a Lactobacillus acidophilus strain increased the proliferation of the NK cells and induced IFN‐γ production, both to levels comparable to PHA stimulation. The proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various strains of lactobacilli have the capacity to activate NK cells in vitro, in a monocyte‐dependent or ‐independent way. Hence, the encounter of NK cells with lactic acid bacteria will affect NK‐cell activation. Such activation of NK cells may potentially skew an on‐going or subsequent immune response towards a Th1 response.
Proteome Analysis Based on Human Recombinant Antibody Microarrays
Authors: Ingvarsson J. , Lindstedt M. , Borrebaeck C. A. K. , Wingren C.
Published: August 2010

Protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform high‐throughput global proteome analysis. A chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. We aim to develop protein microarrays based on human recombinant scFv antibody fragments for global proteome analysis. The concept of comparing proteomic maps of healthy versus diseased samples will allow disease‐specific proteins to be detected. In fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species‐independent manner, as long as a proteome can be isolated. However, the complexity of proteomes, containing several thousands of different proteins, is a problem. Here, we have designed antibody microarrays targeting the water‐soluble fraction of a proteome. To this end, an anticytokine antibody array was developed and human dendritic cells (±activation) was used as model system. The results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. Furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. Due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one‐step procedure (based on size) prior to the labelling step. In more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut‐offs. The results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable.
The Human‐Inducible Costimulator Ligand is Polymorphic
Authors: Jacobsen K. , Ohm‐Laursen L. , Barington T.
Published: August 2010

Inducible costimulator ligand (ICOSL) is a costimulatory molecule related to B7.1 (CD80) and B7.2 (CD86). B cells, monocytes, dendritic cells and endothelial cells express ICOSL. Inducible costimulator (ICOS) interacts with ICOSL, and this interaction leads to signals involved in isotype switching and the development of immunological memory. Hitherto, no polymorphisms of this gene have been described. The aim of this study was to reveal variation of the ICOSL gene in normal individuals. All eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. Eight single nucleotide polymorphisms (SNPs) and two length polymorphisms were found. One of the SNPs was found in the coding regions of the gene. The base involved was located in exon 3 and caused a conservative amino acid change from valine (GTT) to isoleucine (ATT). Three individuals were heterozygous G/A for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype G/G. Exon 3 encodes the immunoglobulin variable (IgV)‐like domain of the molecule which is situated outside the cell. This means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for ICOS. The results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. Especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. Besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional.
Monitoring Patients Treated with Type 1 Interferons: Antiviral versus MxA Induction Assays
Authors: Sønder S. U. S. , Hedegaard C. J. , Bendtzen K.
Published: August 2010

Interferon‐α/β (IFN‐α/β) is increasingly used as antiviral and immunomodulatory therapies. Unfortunately, bioavailability varies with IFN species and mode of administration, and all IFN species are potentially immunogenic. Assays for antiviral activity (IFN) and antiviral neutralization (antibodies, NAb) have been used for some time to monitor patients on IFN biologicals. These assays require laborious titrations making them unsuitable for large‐scale clinical use. Our laboratories have therefore modified the antiviral assays for IFN bioactivity and Nab, so that they are suitable for large‐scale screening in specialized laboratories. The read‐out is survival of a subcloned A549 cell line in the presence of an otherwise lethal amount of virus. Thus, survival increases in the presence of type 1 IFN and decreases in the presence of NAb against the IFN added to the cells. MxA is induced by type 1 IFN and can be used for measuring the Nab activity. In another assay, the MxA level in the A549 cell line is measured. In an attempt to find a new and better reporter gene for type 1 IFN than MxA and genes specific for either IFN‐α or ‐β, a micro array screen was carried using the U133A chip from Affymetrix. The expression of 22,000 genes can be studied simultaneous with this technology. The results will be presented at the conference.
Capacity of AE to Modulate Nitric Oxide Production Depended on Intercellular Contact
Authors: Mijatovic S. , Maksimovic‐Ivanic D. , Miljkovic D. J. , Harhaji L. J. , Trajkovic V.
Published: August 2010

Aloe emodin (AE) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. This anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. High amounts of nitric oxide (NO) released by activated macrophages induce tumour cell death. Therefore, we explored the capacity of AE to modulate NO‐mediated antitumour response in vitro. Interestingly, while AE markedly suppressed NO release from macrophages alone, it significantly potentiated NO production in cocultures of macrophages and C6 cells, after 48 h of cultivation. Accordingly, the viability of C6 cells cocultivated with macrophages was reduced in the presence of AE. Moreover, the observed AE‐imposed potentiation of NO production in macrophages was closely related to macrophage culture cell density. According to these data, we proposed that NO modulator capacity of AE strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by AE.
Inhibitors of Apoptosis as Targets for Spontaneous T‐cell Responses in Cancer Patients: Potential Universal Antigens in Therapeutic Vaccinations Against Cancer
Authors: Andersen M. H. , Reker S. , Kvistgaard P. , Svane I. M. , Becker J. C. , Straten P. T.
Published: August 2010

Immunotherapy represents an attractive fourth‐modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. To this end, a large number of peptide antigens derived from TAA have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. In some cases the response rates have been impressive and no adverse autoimmunity have been observed. A major strategic difficulty associated with these trials relates to the choice of best‐suited peptide antigens. The vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen‐loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. In this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. Obviously, the development of loss‐variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. Ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss‐variant tumour cells. In this respect, several inhibitors of apoptosis proteins (IAPs) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. We have characterized spontaneous T‐cell reactivity against IAP‐derived peptides in cancer patients. From the IAP survivin, we have characterized peptides restricted to the Class I molecules HLA‐A1, A2, A3, A11, B7 and B35. Furthermore, we have demonstrated that survivin‐specific T cells infiltrate metastatic lesions and that isolated survivin‐specific CTLs are capable of killing HLA‐matched tumour cells. Survivin‐derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other IAPs are targets for spontaneous T‐cell reactivity in cancer patients.
Clonally Expanded CD8+ T cells in Allogeneic Bone Marrow Transplantation
Authors: Kollgaard T. M. , Reker S. , Petersen S. L. , Masmas T. N. , Vindelov L. L. , Straten P. T.
Published: August 2010

Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with haematologic malignancies. Several lines of evidence demonstrate that donor T cells are involved in the antitumour effects observed after BMT. Thus, patients receiving T‐cell‐depleted BMT have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated BMT, and patients experiencing graft‐versus‐host disease (GVHD) have a lower risk of disease relapse than patients who do not experience GVHD. Although the importance of donor T cells for the curative action of BMT has been established, the exact mechanisms and molecules involved in this graft‐versus‐tumour effect remain largely unknown. In a recently initiated project, we have conducted a longitudinal study of T‐cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. Peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (MNCs) were isolated and cryopreserved. CD8+ T cells were isolated from the MNCs by use of immunomagnetic beads or FACS and analysed for the presence of clonally expanded cells by T‐cell receptor clonotype mapping based on RT‐PCR and denaturing gradient gel electrophoresis (DGGE). Using this gel‐based methodology, clonally expanded T cells were monitored after transplant and compared to the clinical data of the patients. The preliminary results demonstrates the presence of clonally expanded CD8+ T cells at all time points analysed. Furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. The appearance of newly emerged clonotypes which coincided with clinical GVHD could indicate a role for these T cells in the pathogenesis of GVHD.
Expression of Human Collectins in Colorectal Carcinoma
Authors: Siassi M. , Hohenberger W. , Croner R.
Published: August 2010

Introduction:  The human collectins, mannan‐binding lectin (MBL), surfactant protein‐A (SP‐A) and surfactant‐protein‐D (SP‐D) play a central role in the innate immune system. Immunological responses to malignant transformation of epithelial cells gained increasing interest recently. A former study could demonstrate binding of MBL to certain colorectal carcinoma (CRC) cell lines in vitro. We therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas.

Materials and methods:  Colon samples from 20 CRC patients and 10 normal mucosa samples were collected immediately after surgery. The tissue was microdissected and RNA isolated (Qiagen, Rneasy‐Kit). Gene expression profiles were analysed using Gene‐chips (Affymetrix, HG‐U133). We analysed the data for the expression of MBL, its associated serine proteases mannan‐binding lectin‐associated serine protease 1/2 (MASP 1/2), SP‐A and SP‐D. The signal intensity of the genes of interest was compared using the Mann–Whitney U‐test.

Results:  The expression of human collectins in normal human colon mucosa was generally low. Only the expression of SP‐A and MASP‐2 reached the noise threshold of 250 signals. These genes were significantly downregulated in CRC specimens. The expression of the other proteins showed no difference in normal mucosa and CRC.

Conclusion:  As demonstrated before, the expression of human collectins in normal colon was low in this study. Only SP‐A showed a significant expression in normal mucosa which was downregulated in CRC. As the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. Our results suggest that there is no major role for the human collectins in colorectal cancer.
Anti‐Inflammatory Liver X Receptors and Related Molecules in Multiple Sclerosis Patients from Sardinia and Sweden
Authors: Huang Y.‐M. , Liu X. , Steffensen K. , Sanna A. , Arru G. , Sominanda A. , Sotgiu S. , Rosati G. , Gustafsson J.‐Å. , Link H.
Published: August 2010

The nuclear receptor heterodimers of liver X receptors (LXRs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Multiple sclerosis (MS), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. Sweden belongs to the countries with a high MS incidence. In Italy, incidence is lower, with an exception for Sardinia where the incidence is even higher than that in Sweden. Subjects from Sardinia are ethnically more homogeneous and differ from Swedes, also regarding genetic background and environment. We studied LXRs and their related molecules of blood mononuclear cells (MNCs) from female patients with untreated relapsing‐remitting MS from Sassari, Sardinia and Stockholm, Sweden. Sex‐ and age‐matched healthy controls (HCs) were from both areas. mRNA expression was evaluated by real‐time PCR. LXR‐α was lower (P < 0.05) in MS (mean ± SEM: 3.1 ± 0.2; n = 37) compared to HC (3.6 ± 0.1; n = 37). LXR‐α was lower in MS from Stockholm (2.6 ± 0.2; n = 22) compared to corresponding HC (3.4 ± 0.1; n = 22; P < 0.01) and compared to MS (3.8 ± 0.2; n = 15; P < 0.001) and HC (4 ± 0.2; n = 15; P < 0.001) from Sardinia. MS patients from Stockholm, but not from Sassari, also expressed lower (P < 0.05) LXR‐β (−4.1 ± 0.4) compared to corresponding HC (−2.9 ± 0.3). MS from Stockholm was associated with higher ABCA‐1 (6.1 ± 0.4 versus 5.0 ± 0.3; P < 0.05) and higher estrogen receptor‐β‐Cx (2.4 ± 0.4 versus 0.8 ± 0.4; P < 0.01) compared to corresponding HC. The HC from Sassari had higher androgen receptor (2.9 ± 0.2) compared to MS from Sassari (1.4 ± 0.3; P < 0.01), MS (1.3 ± 0.4; P < 0.01) and HC from Stockholm (1.2 ± 0.3; P < 0.01). MS from Sassari had lower cyclooxygenase‐1 compared to corresponding HC (5.1 ± 0.4 versus 6.6 ± 0.3; P < 0.01) and lower prostaglandin‐E (−0.03 ± 0.5) compared to the HC (1.4 ± 0.5; P < 0.05) and MS (2.7 ± 0.4; P < 0.05) and HC from Stockholm (1.9 ± 0.4, P < 0.001). Our findings identify LXRs and their related molecules as being involved in MS from Stockholm but not from Sassari, while sex hormone receptors seem to be involved in MS in Sassari.
Peripheral Blood T‐Cell Responses to Keratin Peptides that Share Sequences with M Proteins are Largely Restricted to Skin‐Homing CD8+ T Cells
Authors: Johnston A. , Gudjónsson J. E. , Sigmundsdóttir H. , Löve T. H. , Valdimarsson H.
Published: August 2010

The association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the T cells that are known to infiltrate dermis and epidermis of psoriatic skin. Streptococcal M protein shares an extensive sequence homology with human epidermal keratins. Keratins 16 (K16) and 17 (K17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. There is increasing evidence that CD8+ T cells play an important effector role in psoriasis and M protein‐primed T cells may recognize these shared epitopes in skin via molecular mimicry. To identify candidate epitopes, peptides with sequences from K17 were selected on the basis of predicted binding to HLA‐Cw6 and sequence similarities with M6 protein. Matched peptides from the sequence of M6 protein and a set of peptides with poor predicted binding were also selected. Cw6+ individuals with psoriasis and Cw6+ healthy controls, having a family history of psoriasis, were recruited. PBMCs were incubated with the peptide antigens. T‐cell activation in the CD4+, CD8+ and later the skin‐homing cutaneous lymphocyte‐associated antigen (CLA)‐expressing subset of CD8+ T cells was evaluated by CD69 expression and intracellular IFN‐γ accumulation using flow cytometry. We demonstrate that Cw6+ psoriasis patients had significant CD8+ T‐cell IFN‐γ responses to peptides from K17 and M6 protein selected on the basis of sequence homology and predicted HLA‐Cw*0602 binding. These responses were about 10 times more frequent in the skin‐homing cutaneous lymphocyte‐associated antigen‐expressing (CLA+) subset of CD8+ T cells. CD4+ T cells showed only borderline responses. CD8+ T cells from Cw6 + nonpsoriatic individuals responded to some M6 peptides but very rarely to K17 peptides, and this also applied to the CLA+CD8+ subset. These findings indicate that psoriatic individuals have CD8+ T cells that recognize keratin self‐antigens and that epitopes shared by streptococcal M protein and human keratin may be targets for the CD8+ T cells that infiltrate psoriatic skin lesions.
Cutaneous Larva Migrans
Authors: Allan A. , Oriba H.
Published: August 2010

Cutaneous larva migrans is the most common tropically acquired dermatosis caused by larvae of various nematodes, which penetrate the skin and subsequently migrate, causing serpiginous lesions and pruritus. The diagnosis is usually made clinically. While a biopsy taken just ahead of the leading edge of a lesion is felt to enhance the yield of a positive biopsy, the larvae are migratory and thus rarely identified on histologic sections. We present a case of cutaneous larva migrans in which the larval forms are well demonstrated both in a horizontal and longitudinal orientation. A 75‐year old man presented with intractable pruritus and multiple erythematous plaques on his back, poorly responsive to topical steroids. The diagnosis was made on biopsy. Associated with a perivascular lymphocytic infiltrate with eosinophils, a cross section of a larval form was identified within the epidermis. In a second biopsy, the entire organism was present within a sebaceous gland. This case clearly demonstrates the rarely identified larval forms of cutaneous larva migrans.
Tissue Eosinophilia as an Indicator of Drug‐Induced Leukocytoclastic Vasculitis: A Clinicopathologic Investigation
Authors: Bahrami S. , Malone J. , Webb K. , Callen J.P.
Published: August 2010

Cutaneous leukocytoclastic vasculitis (LCV) may be idiopathic, drug‐induced, related to infection, or may occur in patients with autoimmune disorders. Our objective was to determine whether tissue eosinophilia is a reliable indicator of a drug‐induced etiology in the setting of LCV. Thorough chart investigation of 63 patients with LCV was performed with division of patients into drug‐induced and non‐drug‐induced groups. Corresponding histopathologic material was reviewed by a dermatopathologist blinded to the etiologic associations.Â An eosinophil score was tabulated by averaging the number of eosinophils in 10 high‐power fields (400x) and dividing by the density of inflammation. Statistical analysis was performed using the Wilcoxin rank sums test. Mean eosinophil scores in the drug‐induced (n = 16) and non‐drug‐induced (n = 47) groups were 5.20 and 1.05 respectively (p = 0.0126). Vascular fibrin deposition and epidermal changes were present in both groups. Clinical evidence of systemic vasculitis was present in 6%(1/16) of the drug‐induced group vs. 38%(18/47) of the non‐drug‐induced cases. This study establishes tissue eosinophilia as a reliable indicator of drug induction in the setting of LCV. Furthermore, this information may be useful for guiding management decisions, especially in settings of unclear etiology.
Herpetic Folliculitis is Usually a Consequence of Varicella Zoster Virus Infection
Authors: Blair J , Kutzner H , McCalmont TH
Published: August 2010

Skin biopsies of 8 patients diagnosed with herpetic folliculitis by light microscopy were retrieved from the files of the UCSF Dermatopathology Service. Clinical and microscopical features were reviewed and tabulated, and PCR analysis was employed to seek DNA sequences specific for herpes simplex virus (HSV) and varicella zoster virus (VZV). The study group included 4 women and 4 men, ages 15 to 54. Five patients (62%) were immunosuppressed, with underlying conditions including HIV infection, leukemia, rheumatoid arthritis, and lupus erythematosus with polyarteritis nodosa. Microscopically, herpetic cytopathic changes involved the isthmus in 7/8 cases (87%), and involved the sebaceous apparatus in 4/8 cases (50%). Herpetic viropathic changes were not found within eccrine epithelium. A moderate to dense perifollicular infiltrate, comprised mostly of lymphocytes, was evident in 7/8 cases (87%). After PCR expansion of genetic material extracted from the original paraffin blocks, VZV‐specific DNA sequences were detected in 8/8 cases. We conclude that herpetic folliculitis is a consequence of VZV infection. Because follicular herpetic infection is often accompanied by a dense perifollicular lymphoid infiltrate, the microscopical presentation can simulate inflammatory skin diseases such as lupus erythematosus. Level sections may be required for a specific diagnosis to be made.