bluuebunny/crossref_metadata_2025 huggingface dataset:
Probiotic Bacteria Induce Regulatory Cytokine Production via Dendritic Cells
Authors: Hvas C. L. , Kelsen J. , Agnholt J. , Höllsberg P. , Dahlerup J. F.
Published: August 2010

Probiotic bacteria, e.g. Lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. We examined cytokine production and phenotypic change after in vitro stimulation of T cells from healthy volunteers using different probiotic strains.

Methods:  T cells were cultured from colonic biopsies from eight healthy volunteers (Agnholt and Kaltoft, Exp Clin Immunogenet 2001;18:213–25), and dendritic cells were matured from their peripheral blood mononuclear cells. T‐cell cultures were stimulated with autologous bacterial sonicate or strains of Lactobacillus spp., with and without the addition of dendritic cells. Cytokine levels (TNF‐α, IFN‐γ, IL‐10 and GM‐CSF) and phenotype (CD3, CD4, CD25 and CD69) were measured on day 4.

Results:  Lactobacillus spp. induced higher productions of TNF‐α and IL‐10 than did autologous bacteria. In presence of dendritic cells, the production of all cytokines increased. However, the increases of IFN‐γ and TNF‐α were more pronounced in wells with autologous bacteria than in wells with Lactobacillus spp. The addition of dendritic cells upregulated CD25 expression without simultaneous upregulation of CD69. The upregulation was pronounced after stimulation with Lactobacillus rhamnosus GG compared with autologous bacteria and other lactobacilli.

Discussion:  In presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. Lactobacillus rhamnosus GG induced a regulatory phenotype (cd25+), in part mediated by dendritic cells. Future studies will address whether this shift to a CD25+ phenotype represents a differentiation into competent regulatory T cells. In a clinical context, such cells might be used for treatment of inflammatory diseases.
The Immunomodulatory Effect of Heat Shock Protein 70: Immunization with a DNA Construct Based on the Malarial Antigen Fused with a Fragment of HSP 70 Primes for a Th‐1 Type of Response
Authors: Rahman Q. K. , Wikman M. , Vasconcelos N.‐M. , Berzins K. , Ståhl S. , Fernández C.
Published: August 2010

Finding an appropriate adjuvant for human vaccination is crucial. Heat shock proteins (HSPs) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. However, there is a potential risk of autoimmunity when using the complete molecules, because HSPs are evolutionary conserved. To overcome this, we first evaluated the adjuvant effect against two different antigens of a less‐conserved fraction of Plasmodium falciparum HSP70 (Pf70C) and compared it to the whole HSP70 molecule from Trypanosoma cruzi (TcHSP70). We found that Pf70C exhibited similar adjuvant properties as the whole molecule. We later evaluated the adjuvant potential of Pf70C against the malarial antigen EB200 in a chimeric DNA construct. No appreciable levels of EB200‐specific antibodies were detected in mice immunized only with the DNA constructs. However, DNA primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong Th‐1 antibody response. In contrast, no priming effect was observed for ex vivo IFN‐γ production but stimulation with the HSP‐chimeric fusion protein induced a stronger secretion of IFN‐γin vitro than other proteins used. These results indicate that the use of HSPs is promising in the design of new vaccines.
High‐Throughput Proteomics on Antibody‐based Microarrays: the Importance of Probe and Surface Design
Authors: Steinhauer C. , Wingren C. , Borrebaeck C. A. K.
Published: August 2010

In analogy to DNA microarrays, protein microarrays offer a new distinct possibility to perform sensitive high‐throughput global proteome analysis. However, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. The analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. We have recently generated a human recombinant single‐chain Fv antibody library, genetically constructed around one framework, the nCoDeR‐library, containing 2 × 1010 clones. Single framework antibody fragments (sinFabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). However, the choice of framework is critical. We have shown that the selected nCoDeR framework displayed excellent functional on‐chip stability and arrayed dehydrated probes retained their activity for several months. Furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. An in‐house‐designed substrate, macroporous silicon coated with nitrocellulose (MAP3‐NC7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. We have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. Using a novel affinity tag, the double‐(his)6‐tag, we increased the binding efficiency of sinFab‐molecules to Ni2+‐coated solid supports, thereby allowing nonpurified probes to be directly applied.
Plasma TNF‐Binding Capacity and Soluble TNF Receptors in Patients with Juvenile Idiopathic Arthritis
Authors: Bjoernhart B. , Svenningsen P. , Gudbrandsdottir S. , Zak M. , Nielsen S. , Bentzen K. , Müller K.
Published: August 2010

Background:  Unbalanced production of proinflammatory cytokines may be related to disease progression in rheumatoid arthritis and juvenile idiopathic arthritis (JIA). Within the TNF system, the two agonists, TNF‐α and TNF‐β, also called lymphotoxin‐α (LT), are bound by soluble TNF receptors (sTNFR‐I and ‐II) that act as natural inhibitors of TNF‐induced inflammation. We investigated the plasma levels of sTNFR‐I in parallel with LT‐binding capacity (LTBC) in patients with JIA.

Methods:  The levels of sTNFR‐I were measured by ELISA (R&D). LTBC was determined by spiking diluted plasma samples with recombinant LT. Detectable LT was measured by an in‐house ELISA measuring unbound LT only. LTBC was expressed in arbitrary units (AUs) as the percentage value of bound LT to added LT.

Result:  In contrast to previous findings of elevated sTNFR levels in patients with various chronic inflammatory diseases, we found slightly reduced sTNFR‐I levels in JIA patients (n = 123) compared with age‐matched healthy controls (n = 37): 1077 pg/ml (819–2280) versus 1185 pg/ml (625–2303) [median (range)], P = 0015. However, the sTNFR‐I levels correlated positively with the number of active joints, physicians' global assessment and CRP. In contrast, patient LTBC values were elevated compared to healthy controls: 44 AU (36–52) versus 31 AU (13–41), P < 0.0001.

Conclusion:  Despite overall slightly reduced plasma levels of sTNFR‐I, the capacity to bind TNF was increased in plasma samples from JIA patients. Studies to identify the TNF‐binding substances in plasma are in progress.
Monitoring Patients Treated with Type1 Interferons: Potential Reporter Genes in Patient Leucocytes
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. Myxovirus A (MxA) is a resistance GTP‐binding protein that is specifically induced by treatment with type 1 IFNs. For example, IFN‐β‐induced MxA in blood leucocytes has been used as a biomarker in IFN‐β‐treated patients with multiple sclerosis. However, the degree of specificity of MxA in this regard is unclear, and measurements of MxA protein and/or mRNA are not yet suitable for routine clinical use. In an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for IFN‐α and ‐β), microarray screenings with U133A GeneChips (Affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line A549. We studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant IFN‐α and IFN‐β and other antiviral and immunomodulatory cytokines. The results will be presented at the conference.
Production of Functionally Active Recombinant HLA Molecules Representing the Supertypes B62 and B58 and the Generation of Corresponding Peptide‐Binding Assays and Tetramers
Authors: Røder G. , Lamberth K. , Buus S.
Published: August 2010

Rationale:  Major histocompatibility complex class I (MHC I) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic CD8+ T cells. The goal of the human MHC project is to predict the binding strength of any given peptide/MHC complex. This prediction allows the design of peptide‐based vaccines. The prediction requires representative binding data from MHC alleles from all the nine HLA supertypes. Here, we describe the genetic construction, protein production and purification as well as the establishment‐binding assays for two recombinant MHC supertype alleles, HLA‐B*1501 and HLA‐B*5801.

Methods:  Using the Quikchange Multisite Directed Mutagenesis Kit (Stratagene), codon‐optimized genes encoding HLA‐B*1501 and HLA‐B*5801 are created. The two MHC I molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. The binding (KD) of natural T‐cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (RIAs) and enzyme‐linked immunosorbent assays (ELISAs). The MHC molecules are biotinylated during expression.

Results:  The expression of MHC I resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of KD for various peptide ligands ranging from strong binders (KD < 50 nM) to low binders (KD > 5 µM). Tetramerization is visualized by SDS‐PAGE.

Conclusion:  An effective method for the production of highly pure MHC I molecules has been applied to HLA‐B*1501 and HLA‐B*5801, and RIA and ELISA binding assays for those alleles have been established, showing the binding of various peptide ligands to the MHC I molecules.
‘Query‐by Committee’— An Efficient Method to Select Information‐Rich Data for the Development of Peptide—HLA‐Binding Predictors
Authors: Lamberth K. , Nielsen M. , Lundegaard C. , Worning P. , Laurmøller S. L. , Lund O. , Brunak S. , Buus S.
Published: August 2010

Rationale:  We have previously demonstrated that bioinformatics tools such as artificial neural networks (ANNs) are capable of performing pathogen‐, genome‐ and HLA‐wide predictions of peptide–HLA interactions. These tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. A crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide–HLA interaction). This is particularly important when it is difficult and expensive to obtain data. Herein, we demonstrate the importance in selecting information‐rich data and we develop a computational method, query‐by‐committee, which can perform a global identification of such information‐rich data in an unbiased and automated manner. Furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools.

Methods:  A large panel of binding affinities of peptides binding to HLA A*0204 was measured by a radioimmunoassay (RIA). This data was used to develop multiple first generation ANNs, which formed a virtual committee. This committee was used to screen (or ‘queried’) for peptides, where the ANNs agreed (‘low‐QBC’), or disagreed (‘high‐QBC’), on their HLA‐binding potential. Seventeen low‐QBC peptides and 17 high‐QBC peptides were synthesized and tested. The high‐ or low‐QBC data were added to the original data, and new high‐ or low‐QBC second generation ANNs were developed, respectively. This procedure was repeated 40 times.

Results:  The high‐QBC‐enriched ANN performed significantly better than the low‐QBC‐enriched ANN in 37 of the 40 tests.

Conclusion:  These results demonstrate that high‐QBC‐enriched networks perform better than low‐QBC‐enriched networks in selecting informative data for developing peptide–MHC‐binding predictors. This improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high‐QBC experiment and in the low‐QBC experiment. Finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly.
Interleukin‐18 Inhibition of Oral Carcinoma Cell Proliferation
Authors: Nilkaeo A. , Bhuvanath S.
Published: August 2010

Interleukin‐18 (IL‐18), a pro‐inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. Its primary function in stimulation of IFN‐γ production and stimulation of NK‐cell‐cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. In oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. However, direct effects of this cytokine on oral cancer cells have not been elucidated. In this project, we investigated IL‐18 effect on an oral carcinoma (KB) cell line. With RT‐PCR technique, KB‐cell line was found to express IL‐18 receptors (IL‐18Rα and IL‐18Rβ), indicating that this oral carcinoma line is a target for IL‐18 study. We showed that recombinant human IL‐18 inhibited KB‐cell proliferation by 17% at concentration of 100 ng/ml (P < 0.05), whereas LDH release by these cells in treatment group and control groups was comparable, indicating that IL‐18 suppression of cell proliferation was not mediated by the induction of cell death. To further address this hypothesis, we found that IL‐18 treatment did not induce apoptotic cell death, as studied by DNA laddering and TUNEL assays. In addition, expression pattern of cell death‐controlling genes (bcl‐2 and bax) was not altered by this cytokine. Findings in these studies indicated that suppression of KB‐cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. The data presented in this project could provide an insight of how cancer cell directly responds to IL‐18, as this cytokine is an important regulator of anticancer mechanisms.
BCL‐6 Prevents Mammary Epithelial Apoptosis, Promotes Cell Survival and is Expressed in Breast Carcinoma
Authors: Alenzi F. Q. B.
Published: August 2010

Background:  Proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. The hypothesis examined in this study is that the transcription factor BCL‐6 is critically important not only for regulating B‐cell growth and development but also for mammary epithelial apoptosis.

Methodology:  Twenty breast cancer cases and 31 healthy controls were used to investigate whether BCL‐6 protein in involved in breast cancer (grade III). Full length BCL‐6 cDNA was retrovirally transduced into EpH‐4 cell line. We then used flow cytometry of BrdUrd‐stained cells to investigate the cell‐cycle duration of the control and transduced cell lines. TUNEL was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. Finally, immunohistochemistry staining was performed to detect BCL‐6 in breast cancer (III).

Results:  Restoration of BCL‐6 into EpH‐4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. The results also indicated that the cell‐cycle time of BCL‐6‐transduced EpH‐4 cells is prolonged by about 3 h, presumably as a result of the action of BCL‐6 at the BCL‐6 at the G1/S transition. We found differences in the frequencies of viable and apoptotic cells in cultures of the parent EpH‐4 cells, control‐transduced EpH‐4 cells and BCL‐6‐transduced EpH‐4 cells. Consistently, we demonstrated that BCL‐6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours.

Conclusion:  Together, these results suggest that BCL‐6 is likely to be involved in mammary gland development and carcinogenesis.
Inflammatory Cytokine Modulation of Cancer Cell Proliferation
Authors: Bhuvanath S. , Nilkaeo A.
Published: August 2010

Inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. They also play an important role in anticancer immunity. For example, they can promote cell‐mediated immunity against cancer cells. With their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of DNA vaccine or adjuvant or therapeutic cytokines. Direct effect of these cytokines on cancer cell, however, is still unclear. In this project, we investigated whether IL‐1( and IL‐18 can modulate cancer cell proliferation. We employed a simple nonradioactive proliferation (MTT) assay and detection of lactate dehydrogenase (LDH) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (MCF‐7), oral carcinoma cell line (KB), colon cancer cell line (Caco‐2) and choriocarcinoma cell line (Jar). Cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. Findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.
Preoperative Mannan‐Binding Lectin Pathway and Prognosis in Colorectal Cancer
Authors: Ytting H. , Christensen I. J. , Thiel S. , Jensenius J. C. , Nielsen H. J.
Published: August 2010

Background:  Deficiency of the mannan‐binding lectin (MBL) pathway of innate immunity leads to increased susceptibility to infections. In patients with colorectal cancer, postoperative infection is associated with poor prognosis. The aim of the present study was to evaluate (1) the relation between the MBL pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of MBL as acute phase reactant compared to CRP.

Methods:  Preoperative MBL concentration, MBL/MBL‐associated serine protease (MASP) activity and CRP were determined in serum from 611 patients and 150 healthy controls. The patients were observed for 8 years. Postoperative infections, recurrence and survival were recorded.

Results:  The MBL pathway components were increased in the patients (P < 0.0001) compared to healthy controls. Low MBL levels were predictive of pneumonia (P = 0.01), and pneumonia (n = 87) was associated with poor survival (P = 0.003, HR = 1.5, 95% CI 1.1–1.9). MBL and MBL/MASP activity could not predict postoperative overall infections. MBL showed no correlation (spearman's ρ = 0.02, 95% CI −0.06–0.10) with CRP.

Conclusions:  Low preoperative MBL levels are predictive of pneumonia, which is associated with poorer survival. MBL concentration and MBL/MASP activity was not predictive of other postoperative infections or long‐term prognosis. MBL apparently is not a surrogate measure of CRP.
Multiple Sclerosis: IFN‐β Induces CD123+BDCA2– Dendritic Cells that Produce IL‐6 and IL‐10 and have No Enhanced Type I Interferon Production
Authors: Huang Y. M. , Adikari S. , Båve U. , Sanna A. , Alm G.
Published: August 2010

IFN‐β, an approved drug for multiple sclerosis (MS), acts on dendritic cell (DC) by suppressing their production of IL‐12p40 and increasing IL‐10. This results in Th2‐biased immune responses. The nature of IFN‐β‐modulated DC remains elusive. Previously, we observed that IFN‐β dose dependently induces expression of CD123, i.e. a classical marker for plasmacytoid DC, on human blood monocyte‐derived myeloid DC. Such IFN‐β‐modulated DC produce predominantly IL‐10 but are IL‐12 deficient, with potent Th2 promotion. In the present study, we further characterize IFN‐β‐modulated DC by using recently identified blood DC antigens (BDCA) and investigate their ability to produce Type I IFN in response to virus stimulation. We show that IFN‐β induces development of CD123+ DC from human blood monocytes, which coexpress BDCA4+ but are negative for BDCA2–, a specific marker for plasmacytoid DC. Such IFN‐β‐modulated DC produce large amounts of IL‐6 and IL‐10, but no IL‐12p40 and have no enhanced IFN‐β and IFN‐β production. The findings indicate that IFN‐β‐modulated DC represent a myeloid DC subset with diminished CD11c, BDCA‐1 and CD1a expression, having potent Th2‐promoting function but lacking antiviral capacity.
Germinal Centres in Primary Sjögren's Syndrome Indicate a Certain Clinical Immunological Phenotype
Authors: Jonsson M. V. , Brun J. G. , Skarstein K. , Jonsson R.
Published: August 2010

Ectopic germinal centers (GCs) can be detected in the salivary glands of approximately 1/5 of patients with Sjögren's syndrome (SS) and appear in both primary and secondary SS. Previously, ectopic GC have been associated with increased local autoantibody production. The aim of this study was to determine whether GC in primary Sjögren's syndrome (pSS) defines a distinct seroimmunological phenotype. Retrospectively, a material of 130 haematoxylin and eosin‐stained paraffin‐embedded tissue sections of minor salivary gland tissue from patients with pSS was morphologically screened for the presence of ectopic GC. GC‐like lesions were detected in 33/130 (25%) of the pSS patients. Seventy‐two pSS patients lacking these structures (GC‐) were randomly selected for comparison. Focus score was significantly increased in the GC+ patients compared to the GC– patients (P = 0.035). In the GC+ group, 54.5% of the patients presented with anti‐Ro/SSA compared to 43.7% in the GC– group. Anti‐La/SSB was detected in 31.3% of the GC+ patients compared to 25.7% of the GC– patients. Sixty‐one percentage of GC+ patients presented with increased levels of IgG, a nonsignificant difference when compared to 39.4% in the GC– patients (P = 0.089). Levels of RF, ANA, ENA, IgM and IgA were similar in both patient groups, as were ESR and CRP. In conclusion, patients with ectopic GC have a higher focus score and more often present with autoantibodies and increased levels of IgG compared to pSS patients with regular focal infiltration (GC–). Our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies.
The Role of Immune Complexes Consisting of Myelin Basic Protein (MBP), Anti‐MBP Antibodies and Complement in Promoting CD4+ T‐cell Responses to MBP in Health and Multiple Sclerosis
Authors: Hedegaard C. J. , Bendtzen K. , Nielsen C. H.
Published: August 2010

Multiple sclerosis (MS) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. A candidate autoantigen, myelin basic protein (MBP), has especially attracted attention. The presence of anti‐MBP antibodies is a predictor of definite MS, but their role in the pathogenesis remains obscure. T cells have long been known to play a pivotal role in the pathogenesis of MS. Recently, an important role for B cells as autoantigen‐presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. The uptake of MBP by B cells and the presentation of MBP‐derive peptides to T helper (Th) cells by B cells may be promoted by the formation of complement (C) activating immune complexes (ICs) between MBP and natural autoantibodies in healthy individuals and disease‐associated anti‐MBP antibodies in MS patients, respectively. We have investigated the formation of MBP‐containing IC, the binding of MBP to B cells, the MBP‐elicited induction of Th‐cell and B‐cell proliferation and the cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors grown in the presence of intact or C‐inactivated serum from healthy donors or patients with MS. While MBP did not induce measurable proliferation of B cells nor CD4+ T cells, we observed the production of TNF‐α, IFN‐γ and IL‐10 by PBMC in response to incubation with MBP in the presence of sera from healthy controls as well as sera from MS patients. By contrast, no production of IL‐2, IL‐4 and IL‐5 was detected. We are currently investigating the capability of MS sera to promote the formation of MBP‐containing IC and thereby enhance the cytokine responses, by virtue of elevated anti‐MBP contents.
Delayed Elimination of the LCM Virus from Acid Sphingomyelinase‐Deficient Mice due to Reduced Expansion of Virus‐Specific CD8+ T Lymphocytes
Authors: Utermöhlen O. , Karow U. , Baschuk N. , Herz J. , Loegters T. T. , Krönke M.
Published: August 2010

The phagolysosomally localized acid sphingomyelinase (ASMase) activated by proinflammatory cytokines such as TNF and IFN‐γ generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin D. These characteristics of ASMase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. We show here that ASMase–/– mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (LCM) virus as rapidly as littermate wildtype mice. Investigation of the immune response revealed a reduced expansion of CD8+ T cells. The secretion of IFN‐γ in response to contact with target cells as well as the cytolytic activity of virus‐specific CD8+ T cells was severely impaired. Additionally, both phases of the LCM virus‐specific DTH response, mediated by CD8+ and CD4+ T cells consecutively, were diminished in ASMase–/– mice. However, the secondary memory response of virus‐specific CTL was not altered, and the virus was effectively controlled for at least 3 months by ASMase–/– mice. In conclusion, the results of this study suggest an involvement of the ASMase in the activation, expansion or maturation of virus‐specific CD8+ T cells during the acute infection of mice with the LCM virus.
Novel Markers for Alternative Activation of Macrophages: Macrophage Galactose‐Type C‐Type Lectins 1 and 2
Authors: Van Den Bergh R. , Hassanzadeh G. H. , Brys L. , Dahal B. K. , Brandt J. , Grooten J. , Brombacher F. , Vanham G. , Noël W. , Bogaert P. , Boonefaes T. , Kindt A. , De Baetselier P. , Raes G.
Published: August 2010

In parallel with the Th1/Th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. While the best‐studied, classically activated macrophage is induced by type I stimuli such as IFN‐γ, a type II cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. However, molecular markers associated with these type II cytokine‐dependent, alternatively activated macrophages remain scarce. Besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of FIZZ1 and Ym. We now report that expression of the two members of the mouse macrophage galactose‐type C‐type lectin gene family, termed mMGL1 and mMGL2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei or the Helminth Taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. We also demonstrate that, in vitro, interleukin‐4 and interleukin‐13 upregulate mMGL1 and mMGL2 expression and that, in vivo, induction of mMGL1 and mMGL2 is dependent on interleukin‐4 receptor signalling. Moreover, we show that regulation of MGL expression is similar in human monocytes and monocyte‐derived macrophages. Hence, macrophage galactose‐type C‐type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in Th2 conditions.
Mapping of the Ex Vivo Cellular Immune Response Against the Complete Human Parvovirus B19 Genome During Acute Infection
Authors: Isa A. , Norbeck O. , Pöhlmann C. , Tolfvenstam T.
Published: August 2010

Background:  Human parvovirus B19 (B19) is a ubiquitous pathogen, normally causing a mild self‐limiting disease, but also capable of causing both significant pathology and long‐term persistence. The small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection.

Methods:  Five patients with acute primary B19 infection were included in the study and followed consecutively for up to 200 weeks. Cellular immune responses were mapped by IFNγ enzyme‐linked immunospot to overlapping peptides spanning the whole B19 genome.

Results:  In all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. Responses peaked at levels of 850–1850 SFC/million PBMCs, roughly corresponding to 0.3–0.6% B19‐specific CD8+ cells circulating in peripheral blood at 10–80 weeks post‐infection. The responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same CD8 epitopes present in the pools throughout the follow‐up period. The majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases.

Conclusion:  The cellular immune responses to acute B19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post‐infection. The initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection.
Malaria and Nutritional Status in Children Living on the Coast of Kenya
Authors: Nyakeriga A. M. , Troye‐Blomberg M. , Chemtai A. K. , Marsh K. , Williams T. N.
Published: August 2010

The relationship between malnutrition and malaria is controversial. On one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. We investigated the association between Plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of Kenya. The study involved longitudinal follow‐up for clinical malaria episodes and anthropometric measurements at four cross‐sectional surveys. We used Poisson regression analysis to investigate the association between malaria and nutritional status. Compared to baseline (children with a WAZ or HAZ score of ≥−2), the crude incidence rate ratios (IRRs) for malaria in children with low HAZ or WAZ scores (<−2) during the period prior to assessment were 1.17 (95% CI 0.91–1.50; 0 = 0.21) and 0.94 (0.71–1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of PEM. However, we found that age was acting as an effect modifier in the association between malaria and malnutrition. The IRR for malaria in children 0–2 years old who were subsequently characterized as wasted was 1.65 (1.10–2.20; P = 0.01), and a significant overall relationship between malaria and low‐HAZ was found on regression analysis when adjusting for the interaction with age (IRR 1.89; 1.01–3.53; P < 0.05). Although children living on the coast of Kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life.
Presence of Helicobacter pylori Antibodies in Haemodialysis Patients
Authors: Astrinidou‐Vakaloudi A. , Xytsas S. , Diamanti I. , . Ioannidis H , Pangidis P.
Published: August 2010

Aim:  Renal dysfunction may influence the colonization of gastric mucosa by urea‐splitting bacteria such as Helicobacter pylori, by increasing urea concentrations in the gastric juice. Our aim was to investigate the prevalence of H. pylori in patients with end‐stage renal disease (ESRD), receiving long‐term haemodialysis treatment.

Methods:  This study included 40 sera from patients with ESRD (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. Using ELISA technique, we investigated the presence of IgG and IgA antibodies against H. pylori as well as IgG CagA (antibodies specific for CagA(+) strains of H. pylori). Sera from 40 healthy blood donors were used as a control group.

Results:  H. pylori IgG antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for IgA. IgG CagA antibodies were present in 13 out of 40 (32.5%). Prevalence of H. pylori IgG, IgA and CagA IgG antibodies in the control group was 33, 7 and 15%, respectively.

Conclusions:  Although international data suggest that prevalence of H. pylori infection is the same in ESRD patients as in healthy individuals, in our study that seems not to be the case. The higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of H. pylori infection in this group of patients.
Mucosally Targeted Prime‐Boost Vaccination Approaches for Tuberculosis Based on the TLR2/4 Ligand OprI Adjuvant
Authors: Gartner T. , Baeten M. , De Baetselier P. , Huygen K. , Revets H.
Published: August 2010

Immunity against tuberculosis (TB), caused by Mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell‐mediated immune responses involving both CD4+ and CD8+ T cells and the ability to respond with Th1‐type cytokines, particularly IFN‐γ. Recent studies suggested that BCG, the only licensed vaccine against M. tuberculosis, may fail to induce T‐cell responses in the lung mucosa and may therefore not protect against pulmonary TB. A decrease in TB mortality may be achieved by enhancing immunity in the lung. The present study evaluated the induction of antigen‐specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein I (OprI) from Pseudomonas aeruginosa. OprI has shown to be a Toll‐like receptor 2/4 agonist that, when given subcutaneously, induces Type‐1 immune responses against heterologous antigens. Here, a fusion of OprI to Ag85A of Mtb (OprI‐Ag85A) was used as a subunit vaccine in homologous prime‐boost immunizations. In addition, OprI‐Ag85A was combined with an Ag85A‐encoding DNA vaccine (Ag85A DNA) or with BCG in heterologous prime‐boost vaccinations. Intranasal and parenteral delivery with OprI‐Ag85A elicited comparable T‐cell responses in the spleen; in addition, i.n. delivery elicited specific T‐cell responses in the lung lymph nodes (LLNs). Intramuscular delivery of Ag85A DNA induced significant systemic Th1 immune responses. Intranasal boosting with OprI‐Ag85A enhanced this response and in addition induced an antigen‐specific IFN‐γ response in LLN. OprI may therefore be an efficient adjuvant for mucosal boosting. We continue to evaluate the protection induced by OprI‐based prime‐boost vaccinations against pulmonary TB. Results on the immunogenicity and protection against intravenous Mtb H37Rv infection will be presented.
Differential Requirements for Toll‐Like Receptor Signalling for Induction of Chemokine Expression by Herpes Simplex Virus and Sendai Virus
Authors: Melchjorsen J. , Bowie A. G. , Matikainen S. , Paludan S. R.
Published: August 2010

Toll‐like receptors (TLRs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress‐associated molecules. TLR–ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. In this study, we have examined the requirement for different TLR adaptor molecules in virus‐induced chemokine expression and are currently trying to identify the TLR involved. We have found that both a herpesvirus [herpes simplex virus (HSV)] and a paramyxovirus (Sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. For both viruses, this is independent of the TLR adaptor molecules TRIF and Mal. However, overexpression of the Vaccinia virus‐encoded inhibitor of TLR‐signalling A52R or dominant‐negative MyD88 totally inhibited HSV‐induced RANTES expression but only partially prevented Sendai virus from inducing this chemokine. This suggests that HSV‐induced RANTES expression occurs via a TLR pathways, whereas Sendai virus utilizes both TLR‐dependent and ‐independent pathways to stimulate expression of RANTES. We are currently trying to identify the TLRs involved. Data from these studies will also be presented at the meeting.
Supplementation of Vitamin C to Weaner Diets Increases IgM Concentration and Improves the Biological Activity of Vitamin E in Alveolar Macrophages
Authors: Lauridsen C. , Jensen S. K.
Published: August 2010

Vitamins E and C have been found to increase the cellular and humeral immunity of pigs. Vitamin E deficiency has also been found to predispose pigs to different diseases, E. coli infection is one among them. After weaning, the vitamin E status of pigs often decreases to a critical low level. In this experiment, we studied whether vitamin C supplementation would be a possible feeding strategy to optimize the immune status of weaners. The interaction between vitamin E and C is interesting due to the reported sparing action on vitamin E or synergism between these to vitamins. Piglets were weaned at day 28 of age from sows fed increasing dietary vitamin E during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg STAY‐C per kg. Blood sampling was obtained weekly from day 28 and until day 49 of age. On the same days, one piglet per dietary treatment was killed and alveolar macrophages (AM) were harvested. Vitamin C supplementation increased the concentration of IgM in serum of piglets throughout the weaning period. Although the vitamin E concentration in AM decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin E. However, vitamin C supplementation tended to increase the total AM concentration of vitamin E after weaning and increased the proportion of the biologically most active isomer of vitamin E [RRR‐(α‐tocopherol)] in the AM. The eicosanoid synthesis by AM was not influenced by the vitamin C supplementation, but the synthesis of leukotriene B4 was decreased 2 weeks after weaning compared to other days of AM harvesting. In conclusion, dietary vitamin C supplementation improved the immune responses of piglets after weaning.
Do High and Low Tumour Necrosis Factor‐α Responders Exist in Dairy Cows?
Authors: Røntved C. M. , Dernfalk J. , Ingvartsen K. L.
Published: August 2010

A whole blood stimulation assay with Escherichia coli (O111:B4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor‐α (TNF‐α) ex vivo. Initially, a time‐ and dose‐dependent study was carried out to find the optimal stimulation conditions for the TNF‐α response. The TNF‐α response peaked between 3 and 4 h at 38.5 °C. A dose in the range of 5–10 g of E. coli lipopolysaccharide (LPS)/ml whole blood was found to give the maximum TNF‐α response. Thirty‐eight Danish–Holstein dairy cows were investigated for their TNF‐α responsiveness ex vivo in the periparturient period. Heparin‐stabilized blood samples were collected seven times over a period of 4 months (weeks −3, −1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of E. coli LPS. Indeed, fluctuations in the TNF‐α responsiveness occurred over time. Moreover, the mean TNF‐α responsiveness of 38 cows was found to be significantly increased (P < 0.001) in the weeks close to calving. However, in the more stabile physiological periods, some cows had a consistently low TNF‐α response, whereas others had high a TNF‐α response. We are currently investigating whether high and low TNF‐α responders to E. coli LPS also exist in dairy cows in vivo. Moreover, the importance of TNF‐α responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental E. coli infections in the udder is being investigated.
Release of sVEGF and sVEGFR1 from White Blood Cells and Platelets During Surgery and Stimulation with Bacterial Antigens
Authors: Svendsen M. N. , Werther K. , Bisgaard T. , Christensen I. J. , Nielsen H. J.
Published: August 2010

Introduction:  The influence of surgery on release of soluble vascular endothelial growth factor (sVEGF) and the soluble vascular endothelial growth factor inhibitory receptor 1 (sVEGFR1) is unknown. We studied the effect of major and minor surgery on potential variations in sVEGF and sVEGFR1 concentrations in vivo and on bacterial antigen‐induced release of sVEGF and sVEGFR1 from whole blood in vitro.

Methods:  Sixty‐one patients with abdominal diseases undergoing five different surgical procedures were included. Blood samples were drawn from anaesthetized patients before and after the operation. White blood cells and platelets were counted, and plasma sVEGF and sVEGFR1 was determined by an ELISA method. Whole blood from each blood sample was stimulated in vitro with bacteria‐derived antigens (LPS or protein‐A) and sVEGF and sVEGFR1 levels were subsequently determined in the supernatants. Stimulation with isotonic saline served as control assay.

Results:  Neither sVEGF or sVEGFR1 in plasma changed during surgery. In vitro stimulation of blood samples with bacteria‐derived antigens resulted in a significant increase in sVEGF (P < 0.0001) and a less pronounced but still significant increase in sVEGFR1. Release of sVEGF due to stimulation was significantly higher after the operation (nonsignificant), whereas sVEGFR1 release remained largely unchanged after surgery. Correlation between bacterial antigen‐induced release of sVEGF and neutrophile cell count was highly significant (P < 0.0001). There was no correlation between sVEGF and platelet cell count, and bacterial antigen‐induced sVEGFR1 release did not correlate with counts of neutrophils and platelets.

Conclusions:  Plasma sVEGF and sVEGFR1 concentrations did not change during surgery. In vitro bacterial stimulation led to increased release of sVEGF and sVEGFR1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils.
Natural Killer Cell Functions and Subsets After In Vitro Stimulation with IL‐2 and IL‐12, with Special Emphasis on Intracellular IFN‐γ and NK‐Cell Cytotoxicity
Authors: Nyboe R. , Rix T. , Krog J. , Tønnesen E. , Hokland M.
Published: August 2010

Materials and methods:  Isolated cryopreserved human peripheral blood mononuclear cells (PBMCs) were stimulated with IL‐2 and IL‐12. This stimulation has previously been shown to activate NK cells. Cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. This method was compared to the current standard 51Cr release assay. Cells were treated with BFA to accumulate IFN‐γ, stained for surface markers, permeabilized and stained for intracellular IFN‐γ. Flow cytometry was then performed to measure intracellular IFN‐γ production in PBMC, especially in NK cells.

Results:  We have demonstrated that stimulation with IL‐2 and IL‐12 is effective in increasing the number of IFN‐γ‐positive cells. There is a distinct difference between the CD3‐CD56dim and the CD3‐CD56bright subsets, with a much greater proportion of IFN‐γ‐positive cells in the CD3‐CD56bright subset. The effects of stimulation with IL‐2 and IL‐12 on cytotoxicity will be presented, as will the relation between IFN‐γ production and cytotoxicity. In addition, we will present results of these assays applied to porcine cells.

Discussion:  In combination, these tests will address NK cell function by combining cytotoxicity with IFN‐γ production in NK cell subsets. The results will demonstrate whether this could serve as a useful tool in describing NK‐cell function, which could be of value in clinical and experimental settings.
Culture of Regulatory T‐Cell Lines from Bronchial Mucosa
Authors: Rømer J. L. , Kelsen J. , Dahl R. , Hoffmann H. J.
Published: August 2010

T lymphocytes play a major role in many immune responses. In the last decade, special focus has been on the function of Th1 and Th2 effector cells. Now the importance of regulatory CD4+CD25+ T cells in maintenance of the immunological homeostasis emerges. Sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. The typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly CD4+ T cells of Th1 phenotype. We have cultured T cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (IL‐2) and IL‐4 and demonstrate spontaneously arising CD4+ CD25+ populations and high concentrations of IL‐10 in these cultures. The main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines IL‐6 and TNF‐α in cultures of sarcoid origin.
Axillary Metastasis of Malignant Melanoma Mimicking a Breast Carcinoma
Authors: Andea A. , Satter E. , McEvoy J.R. , Williams K. , Mikhitarian K. , Metcalf J.
Published: August 2010

We describe here the case of a female patient with an otherwise classic invasive melanoma of the upper extremity, demonstrating the typical immunohistochemical profile, who presented three years later with massive ipsilateral axillary node metastases. The metastatic tumor had morphologic features similar to the primary melanoma, however at immunohistochemical level the tumor cells not only gained expression of pan‐cytokeratin (AE1/AE3) but also lost expression of all standard melanoma markers (S100, Melan‐A, HMB‐45, MIT‐1, and tyrosinase) thus mimicking a poorly differentiated mammary carcinoma. Additional stains reveal expression of Vimentin, NSE, c‐kit, CD10 and negativity for CK7, CK20, BR2, ER/PR, mucin, and LCA. To rule out a breast carcinoma, real‐time RT‐PCR was performed on a lymph node using six breast cancer‐associated genes (>99% sensitivity), which was also negative. Clinical work‐up of the patient failed to reveal abnormal masses in the breast or elsewhere. The clinical history, histological appearance, immunohistochemical profile (including negative CK7 and positive NSE, c‐kit and CD10) as well as a negative RT‐PCR renders almost null the possibility of a breast carcinoma and supports a diagnostic of melanoma in this case. Cases like ours may be easily misclassified unless a concerted approach is used to reach the correct diagnosis.