Infections and disease

Scientific publications - Infections and disease


Yordanov, M., Dimitrova, P., Patkar, S., Saso, L., Ivanovska, N.

"Inhibition of Candida albicans extracellular enzyme activity by selected natural substances and their application in Candida infection"

Canadian Journal of Microbiology, 54 (6), pp. 435-440. (2008)

Extracellular enzymes secreted by Candida albicans are claimed to be virulence factors responsible for penetration of the yeast into host cells. Substances able to inhibit lipolytic and proteinase activities of the fungus might be of therapeutic use in some pathologic conditions caused by C. albicans. In the present work, we have tested the influence of the flavonoid compounds apigenin and kaempferol, the indole alkaloid ibogaine, and the protoberberine alkaloid berberine on the in vitro enzyme activity of C. albicans. The substances showed complex suppressive effects concerning the processes of adherence to epithelial cells, secreted aspartyl proteinase activity, and the rate of cell wall protein glycosylation. Apigenin and kaempferol were administered in systemic C. albicans infection, demonstrating an increased number of survivors by kaempferol. The application of apigenin, kaempferol, ibogaine, and berberine in cutaneous infection suppressed the symptoms and accelerated elimination of the yeast from the site of inoculation. © 2008 NRC Canada.

Bagger-Skjøt, L., Sandvang, D., Frimodt-Møller, N., Lester, C.H., Olsen, K.E.P., Porsbo, L.J., Monnet, D.L., Hammerum, A.M.

"Association between antimicrobial resistance and virulence genes in Escherichia coli obtained from blood and faeces"

Scandinavian Journal of Infectious Diseases, 39 (8), pp. 724-727. (2007)

Escherichia coli isolates obtained from faeces (n = 85) and blood (n = 123) were susceptibility tested against 17 antimicrobial agents and the presence of 9 virulence genes was determined by PCR. Positive associations between several antimicrobial resistances and 2 VF genes (iutA and traT) were found among blood isolates, sometimes among faecal isolates.

Matharoo-Ball, B., Hughes, C., Lancashire, L., Tooth, D., Ball, G., Creaser, C., Elgasim, M., Rees, R., Layfield, R., Atiomo, W.

"Characterization of biomarkers in Polycystic Ovary Syndrome (PCOS) using multiple distinct proteomic platforms"

Journal of Proteome Research, 6 (8), pp. 3321-3328. (2007)

A variety of prefractionation methods (including a novel reversed-phase solid-phase-extraction (RP-SPE) combined with SDS-PAGE and proteomic based approaches (e.g., 2-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry combined with Artificial Neural Network (ANN) bioinformatic tools) were used to investigate the protein/peptide signatures in patients with Polycystic Ovary Syndrome (PCOS). Four potential PCOS biomarkers were identified (complement C4α3c and C4γ and haptoglobin α and β chains). © 2007 American Chemical Society.

Jakobsen, L., Sandvang, D., Jensen, V.F., Seyfarth, A.M., Frimodt-møller, N., Hammerum, A.M.

"Gentamicin susceptibility in Escherichia coli related to the genetic background: Problems with breakpoints"

Clinical Microbiology and Infection, 13 (8), pp. 830-832. (2007)

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GENR) genes aac(3)-II, aac(3)-IV or ant(2″)- I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2″)- I had an MIC distribution of 8-64mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512mg/L, suggesting a relationship between the distribution of MICs and the specific GENR mechanism. The MIC distribution, regardless of the GENR mechanism, was 8 - >512mg/L, which supports the clinical breakpoint of MIC>4mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (≥16mg/L). © 2007 The Authors Journal Compilation © 2007 European Society of Clinical Microbiology and Infectious Diseases.

Sharma, H.S., Lundstedt, T., Flärdh, M., Skottner, A., Wiklund, L.

"Neuroprotective effects of melanocortins in CNS injury"

Current Pharmaceutical Design, 13 (19), pp. 1929-1941. (2007)

New compounds having affinity to various melanocortin receptors have recently been identified as possible neuroprotective agents. This review is focused on the role of neuroprotective effects of melanocortins in CNS injury and repair mechanisms. Using selective non-peptidic compounds with varying affinity to melanocortin receptors, our laboratory has shown their anti-edematous effects in the spinal cord injury. This effect of the compounds is related with their ability to attenuate blood-spinal cord barrier permeability. The functional significance and possible therapeutic strategies of these-compounds in CNS injury are discussed. © 2007 Bentham Science Publishers Ltd.

Bagger-Skjøt, L., Nielsen, E.M., Sandvang, D., Ethelberg, S., Monnet, D.L., Hammerum, A.M.

"Less frequent Salmonella serovars as a reservoir of antimicrobial resistance [11]"

Journal of Antimicrobial Chemotherapy, 59 (4), pp. 814-815. (2007)


Lobedanz, S., Bokma, E., Symmons, M.F., Koronakis, E., Hughes, C., Koronakis, V.

"A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps"

Proceedings of the National Academy of Sciences of the United States of America, 104 (11), pp. 4612-4617. (2007)

Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower α-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal α-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state. © 2007 by The National Academy of Sciences of the USA.

Tobiasen, C., Aahman, J., Ravnholt, K.S., Bjerrum, M.J., Grell, M.N., Giese, H.

"Nonribosomal peptide synthetase (NPS) genes in Fusarium graminearum, F. culmorum and F. pseudograminearium and identification of NPS2 as the producer of ferricrocin"

Current Genetics, 51 (1), pp. 43-58. (2007)

Fungi have the potential to produce a wide range of secondary metabolites including polyketides and small peptides produced by nonribosomal peptide synthetases (NPS). Fusarium graminearum is a mycotoxin producing pathogen of cereals and knowledge of the infection process is essential for the development of disease control. Bioinformatics provide a means to identify genes encoding NPSs, the products of which may act as fungal virulence factors. The F. graminearum genome sequence was analysed and similarity searches and application of prediction server service identified 15 putative NPS genes. NPS1 and PS2, were found to be related to genes involved in NPS hydroxamate siderophore biosynthesis and chemical analysis of a F. graminearum NPS2 deletion mutant showed that this gene encodes the NPS responsible for the biosynthesis of ferricrocin. The expression of the NPS genes was analysed in Fusarium culmorum. NPS1 and NPS19 differed from the remainder of the genes, as they were only expressed during infection of barley roots and not under the different culture conditions tested. Strains of F. graminearum, F. culmorum and Fusarium pseudograminearum were examined for the presence and expression of the 15 identified NPS genes. With the exception of NPS18, that is absent in F. pseudograminearum, all the NPS genes are represented in the diffferent species. Lack of transcripts from some genes and the presence of frameshift and stop codons in four of the NPS genes in the sequenced F. graminearum strain suggest that some are pseudogenes. © Springer-Verlag 2006.

Wang, Y.-S., Pi, L.-Y., Chen, X., Chakrabarty, P.K., Jiang, J., De Leon, A.L., Liu, G.-Z., Li, A., Benny, U., Oard, J., Ronald, P.C., Song, W.-Y.

"Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance"

Plant Cell, 18 (12), pp. 3635-3646. (2006)

XA21 is a receptor-like kinase protein in rice (Oryza sativa) that confers gene-for-gene resistance to specific races of the causal agent of bacterial blight disease, Xanthomonas oryzae pv oryzae. We identified XA21 binding protein 3 (XB3), an E3 ubiquitin ligase, as a substrate for the XA21 Ser and Thr kinase. The interaction between XB3 and the kinase domain of XA21 has been shown in yeast and in vitro, and the physical association between XB3 and XA21 in vivo has also been confirmed by coimmunoprecipitation assays. XB3 contains an ankyrin repeat domain and a RING finger motif that is sufficient for its interaction with the kinase domain of XA21 and for its E3 ubiquitin ligase activity, respectively. Transgenic plants with reduced expression of the Xb3 gene are compromised in resistance to the avirulent race of X. oryzae pv oryzae. Furthermore, reduced levels of Xb3 lead to decreased levels of the XA21 protein. These results indicate that Xb3 is necessary for full accumulation of the XA21 protein and for Xa2f-mediated resistance. © 2006 American Society of Plant Biologists.

Bendifallah, N., Rasmussen, F.W., Zachar, V., Ebbesen, P., Nielsen, P.E., Koppelhus, U.

"Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)"

Bioconjugate Chemistry, 17 (3), pp. 750-758. (2006)

Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan- amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 μM. (D-Arg) 9-PNA showed optimal efficacy at 5 μM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data. © 2006 American Chemical Society.

Rasmussen, F.W., Bendifallah, N., Zachar, V., Shiraishi, T., Fink, T., Ebbesen, P., Nielsen, P.E., Koppelhus, U.

"Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers"

Oligonucleotides, 16 (1), pp. 43-57. (2006)

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transferable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way. © Mary Ann Liebert, Inc.

Sharma, H.S., Skottner, A., Lundstedt, T., Flärdh, M., Wiklund, L.

"Neuroprotective effects of melanocortins in experimental spinal cord injury. An experimental study in the rat using topical application of compounds with varying affinity to melanocortin receptors"

Journal of Neural Transmission, 113 (4), pp. 463-476. (2006)

The possibility that local administration of low molecular weight non-peptide compounds with varying affinities at melanocortin receptors in the spinal cord will influence pathophysiological outcome of spinal cord injury (SCI) was examined in a rat model. Five new Melacure compounds ME10092, ME10354, ME10393, ME10431 and ME10501 were used in this investigation. Each compound was dissolved in saline and tested at 3 different doses, i.e. 1 μg, 5 μg and 10 μg total dose in 10 μl applied topically 5 min after SCI. The animals were allowed to survive 5 h and trauma induced edema formation, breakdown of the blood-spinal cord barrier (BSCB) and cell injuries were examined and compared with untreated injured rats. A focal SCI inflicted by an incision into the right dorsal horn of the T10-11 segments resulted in marked edema formation, breakdown of the BSCB to Evans blue albumin and caused profound nerve cell injury in the T9 and the T12 segments. Topical application of ME10501 (a compound with high affinity at melanocortin, MC-4 receptors) in high doses (10 μg) resulted in most marked neuroprotection in the perifocal spinal cord (T9 and T12) segments. On the other hand, only a mild or no effect on spinal cord pathology was observed in the traumatized animals that received ME10092, ME10354, ME10393 and ME10431 at 3 different doses. These observations suggest that non-peptide compounds with varying affinity to melanocortin receptors are able to influence the pathophysiology of SCI. Furthermore, compounds acting at melanocortin, MCR4 receptors are capable to induce neuroprotection in spinal cord following trauma. © Springer-Verlag 2006.

Jepsen, J.R., Laursen, L.H., Hagert, C.-G., Kreiner, S., Larsen, A.I.

"Diagnostic accuracy of the neurological upper limb examination II: Relation to symptoms of patterns of findings"

BMC Neurology, 6, art. no. 10, . (2006)

Background: In a sample of patients in clinical occupational medicine we have demonstrated that an upper limb neurological examination can reliably identify patterns of findings suggesting upper limb focal neuropathies. This further study aimed at approaching the diagnostic accuracy of the examination. Methods: 82 limbs were semi-quantitatively assessed by two blinded examiners (strength in 14 individual muscles, sensibility in 7 homonymous territories, and mechanosensitivity at 10 locations along nerves). Based on the topography of nerves and their muscular and sensory innervation we defined 10 neurological patterns each suggesting a localized nerve affliction. Information on complaints (pain, weakness and/or numbness/tingling) collected by others served as a reference for comparison. The relation between the presence of pattern(s) and complaints was assessed by κ-statistics. Sensitivity, specificity, and positive/negative predictive values were calculated, and pretest odds were compared to post-test probability. Results: The two examiners identified pattern(s) suggesting focal neuropathy in 34/36 out of 38 symptomatic limbs, respectively (κ = 0.70/ 0.75), with agreement in 28 limbs. Out of 44 non-symptomatic limbs the examiners agreed on absence of any pattern in 38 limbs. With concordance between the examiners with regard to the presence or absence of any pattern, the sensitivity, specificity, positive and negative predictive values were 0.73, 0.86, 0.93 and 0.90, respectively. While the pre-test odds for a limb to be symptomatic amounted to 0.46 the post-test probability was 0.81. For each examiner the post-test probability was 0.87 and 0.88, respectively. Conclusion: The improved diagnostic confidence is an indication of one aspect of construct validity of the physical examination. For determination of clinical feasibility of the examination further studies are required, most importantly 1) studies of validity by means of comparison with additional references and 2) studies of the potential benefit that can be attained from its use. © 2006 Jepsen et al; licensee BioMed Central Ltd.

Jepsen, J.R., Laursen, L.H., Hagert, C.-G., Kreiner, S., Larsen, A.I.

"Diagnostic accuracy of the neurological upper limb examination I: Inter-rater reproducibility of selected findings and patterns"

BMC Neurology, 6, art. no. 8, . (2006)

Background: We have previously assessed the reproducibility of manual testing of the strength in 14 individual upper limb muscles in patients with or without upper limb complaints. This investigation aimed at additionally studying sensory disturbances, the mechanosensitivity of nerve trunks, and the occurrence of physical findings in patterns which may potentially reflect a peripheral neuropathy. The reproducibility of this part of the neurological examination has never been reported. Methods: Two blinded examiners performed a semi-quantitative assessment of 82 upper limbs (strength in 14 individual muscles, sensibility in 7 homonymous territories, and mechanosensitivity of nerves at 10 locations). Based on the topography of nerves and their muscular and cutaneous innervation we defined 10 neurological patterns each suggesting a focal neuropathy. The individual findings and patterns identified by the two examiners were compared. Results: Strength, sensibility to touch, pain and vibration, and mechanosensitivity were predominantly assessed with moderate to very good reproducibility (median κ-values 0.54, 0.69, 0.48, 0.58, and 0.53, respectively). The reproducibility of the defined patterns was fair to excellent (median correlation coefficient = 0.75) and the overall identification of limbs with/without pattern(s) was good (κ = 0.75). Conclusion: This first part of a study on diagnostic accuracy of a selective neurological examination has demonstrated a promising inter-rater reproducibility of individual neurological items and patterns. Generalization and clinical feasibility require further documentation: 1) Reproducibility in cohorts of other composition, 2) validity with comparison to currently applied standards, and 3) potential benefits that can be attained by the examination. © 2006 Jepsen et al; licensee BioMed Central Ltd.

Yordanov, M., Dimitrova, P., Patkar, S., Falcocchio, S., Xoxi, E., Saso, L., Ivanovska, N.

"Ibogaine reduces organ colonization in murine systemic and gastrointestinal Candida albicans infections"

Journal of Medical Microbiology, 54 (7), pp. 647-653. (2005)

In the present study the effect of the indole alkaloid ibogaine on the in vitro lipolytic activity and adherence to epithelial cells of Candida albicans was investigated. The substance was administered intraperitoneally at a dose of 5 mg kg-1 day-1 in mice with disseminated and gastrointestinal C. albicans infections. Ibogaine significantly decreased the rate of mortality and the number of C. albicans c.f.u. recovered from the kidney, liver and spleen. Ibogaine interfered with the early stages of both disseminated and gastrointestinal C. albicans infections but did not reduce the number of C. albicans c.f.u. in the organs at the late phase of infections. The development of a specific immune response was not influenced by ibogaine, since the delayed-type hypersensitivity reaction to C. albicans and the production of interferon (IFN)-γ were similar in control and ibogaine-treated mice. The combined use of amphotericin B plus ibogaine in the treatment of mice with gastrointestinal infection reduced organ colonization more strongly than each substance alone. © 2005 SGM.

Young, P.J., Newman, A., Jensen, K.T., Burger, L.R., Pintel, D.J., Lorson, C.L.

"Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies"

Journal of General Virology, 86 (4), pp. 1009-1014. (2005)

The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A92L cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation. © 2005 SGM.

Hansen, E.H., Schäfer, T., Molin, S., Gram, L.

"Effect of environmental and physiological factors on the antibacterial activity of Curvularia haloperoxidase system against Escherichia coli"

Journal of Applied Microbiology, 98 (3), pp. 581-588. (2005)

Aims: The aim of this study was to investigate the influence of environmental and physiological factors on the susceptibility of Escherichia coli to the Curvularia haloperoxidase system. Methods and Results: The Curvularia haloperoxidase system is a novel enzyme system that produces reactive oxygen species which have an antimicrobial effect. Escherichia coli MG1655 was exposed to the Curvularia haloperoxidase system under different temperatures and NaCl concentrations and after exposure to different stress factors. Temperature clearly affected enzymatic activity with increasing antibacterial effect at increasing temperature. The presence of NaCl interfered with the enzyme system and in the presence of 1% NaCl, no antibacterial effect could be observed at pH 7. Cells grown at pH 8.0 were in one experiment more resistant than cells grown at pH 6.5, whereas cells grown in the presence of 2% NaCl were more susceptible to the Curvularia haloperoxidase system. Conclusions: Environmental and physiological factors can affect the antibacterial activity of the Curvularia haloperoxidase system. Significance and Impact of the Study: The study demonstrates a systematic approach in assessing the effect of environmental and physiological factors on microbial susceptibility to biocides. Such information is crucial for prediction of application as well as potential side-effects. © 2004 The Society for Applied Microbiology.

Ferrer, M., Soliveri, J., Plou, F.J., López-Cortés, N., Reyes-Duarte, D., Christensen, M., Copa-Patiño, J.L., Ballesteros, A.

"Synthesis of sugar esters in solvent mixtures by lipases from Thermomyces lanuginosus and Candida antarctica B, and their antimicrobial properties"

Enzyme and Microbial Technology, 36 (4), pp. 391-398. (2005)

The lipases from Thermomyces lanuginosus (immobilized by granulation with silica) and Candida antarctica B (adsorbed on Lewatit, "Novozym 435") were comparatively assayed for the synthesis of sugar esters by transesterification of sugars with fatty acid vinyl esters in 2-methyl-2-butanol:dimethylsulfoxide mixtures. We found that lipase from C. antarctica B is particularly useful for the preparation of 6,6′-di- acylsucrose, whereas T. lanuginosus lipase catalyzes selectively the synthesis of 6-O-acylsucrose. The granulated T. lanuginosus lipase retained more than 80% of its initial activity after 20 cycles of 6 h. Both lipases were similarly effective for the regioselective synthesis of 6′-O-palmitoylmaltose and 6-O-lauroylglucose. The effect of the synthesized sugar esters on the growth in liquid medium of various microorganisms (Gram-positive, Gram-negative and yeasts) was evaluated. 6-O-lauroylsucrose and 6′-O-lauroylmaltose inhibited the growth of Bacillus sp. at a concentration of 0.8 mg/ml, and of Lactobacillus plantarum at 4 mg/ml. Sucrose dilaurates and 6-O-lauroylglucose did not show antimicrobial activity, probably due to their low aqueous solubility. As regards the inhibition of yeasts, none of the tested carbohydrate esters inhibited significantly the growth of Zygosaccharomyces rouxii and Pichia jadinii. © 2004 Elsevier Inc. All rights reserved.

Jakobsen, C.G., Bodtger, U., Poulsen, L.K., Roggen, E.L.

"Vaccination for birch pollen allergy: Comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance"

Clinical and Experimental Allergy, 35 (2), pp. 193-198. (2005)

Background: Allergen-specific immunotherapy (SIT) is associated with increased levels of allergen-specific IgG in serum. However, it is not clear to what extent qualitative changes in the allergen binding capacity of IgG may be induced as well. Objective: The purpose of this study was to investigate the influences of SIT on antibody affinity. Methods: The binding affinity of purified serum IgG1, IgG4 and IgE to the major allergen in birch (Betula verrucosa) pollen, Bet v 1, was analysed by surface plasmon resonance. The antibodies were obtained from 10 birch pollen-allergic patients receiving SIT and from 10 patients with no SIT. Results: The patients having received SIT have a significant higher titre of anti-Bet v 1 antibodies in their blood, but the affinity to Bet v 1 of allergen-specific IgE, IgG 1 and IgG4 does not differ between the two groups. For IgG1 and IgG4, correlations between less allergic symptoms and affinity of the antibodies were observed both in the SIT group and to a smaller extent in the non-SIT group. Conclusion: SIT has no effect on antibody affinity of allergen-specific IgE, IgG1 or IgG4. Allergic patients with high-affinity IgG1 and IgG4 antibodies report less symptoms than patients with low-affinity antibodies. © 2005 Blackwell Publishing Ltd.

Schiött, Å., Lindstedt, M., Johansson-Lindbom, B., Roggen, E., Borrebaeck, C.A.K.

"CD27- CD4+ memory T cells define a differentiated memory population at both the functional and transcriptional levels"

Immunology, 113 (3), pp. 363-370. (2004)

The memory T-cell population is a heterogeneous population, including both effector cells, which exert a direct secondary immune response, and resting or intermediate cells, which serve as a reservoir and exert a possible regulatory role. To further dissect the T-cell memory population residing in the CD4 +CD45RO+ T-cell pool, we studied the functional properties of memory populations identified by the CD27 marker. This marker clearly divides the memory population into two groups. One group consists of effector cells lacking CD27 and displaying a high antigen recall response. The other group consists of an intermediate memory population, displaying CD27. This latter group lacks an antigen recall response and requires costimulation for T-cell receptor triggering. To evaluate the function of the CD27+ memory pool, we analysed the transcriptional profile, using high-density microarray technology. These gene data strongly support the different functional profiles of CD27+ and CD27+ memory populations, in terms of protein expression and the capacity to respond to antigen.

Da Silva, F.G., Shen, Y., Dardick, C., Burdman, S., Yadav, R.C., De Leon, A.L., Ronald, P.C.

"Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response"

Molecular Plant-Microbe Interactions, 17 (6), pp. 593-601. (2004)

Innate immunity to microorganisms relies on the specific sensing of pathogen-associated molecules by host recognition receptors. Whereas studies in animals have largely focused on the recognition of extracellular pathogen-associated molecules by the TLR (toll-like receptor) superfamily, few studies have been carried out in plants, and it is not understood how these molecules are secreted or modified. The rice Xa21 gene encodes a receptor-like kinase that provides immunity against strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae carrying AvrXa21 activity. We identified four X. oryzae pv. oryzae genes that are required for AvrXa21 activity. raxA, raxB, and raxC encode proteins with similarity to a membrane fusion protein, an ATP-binding cassette transporter, and an outer membrane protein, respectively, of bacterial type I secretion systems. The fourth gene, raxST, encodes a sulfotransferase-like protein. Sequence analysis of three naturally occurring X. oryzae pv. oryzae strains no longer recognized by Xa21 revealed alterations in the raxST and raxA genes. The raxC gene complemented an Escherichia coli tolC mutant for secretion of a double glycine-leader peptide confirming the function of raxC in type I secretion. These results indicate that bacterial type I secretion is necessary for Xa21-mediated recognition and immunity and further suggest that type I secretion and modification of pathogen-associated molecules play an important role in triggering the innate immune response in rice.

Rung, J.H., Draborg, H.H., Jørgensen, K., Nielsen, T.H.

"Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase"

Physiologia Plantarum, 121 (2), pp. 204-214. (2004)

The role of fructose-2,6-bisphosphate (Fru-2,6-P2) in regulation of carbon metabolism was investigated in transgenic potato plants (Solanum tuberosum Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC,6- bisphosphatase (F26BPase, EC in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P2 was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic 14CO2 metabolism, showed that in plant lines with reduced Fru-2,6-P2 level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P2 only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-14C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-14C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P2 is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P2 on tuber metabolism was limited.

Nilsson, L., Ng, Y.Y., Christiansen, J.N., Jørgensen, B.L., Grótinum, D., Gram, L.

"The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold-smoked salmon systems"

Journal of Applied Microbiology, 96 (1), pp. 133-143. (2004)

Aims: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. Methods and Results: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac- mutant was higher than that of the wild-type at 5 and 37°C but not at 25 or 30°C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac- and bac +, respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac- caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3.5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5°C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. Conclusions: The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes Significance and Impact of the Study: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.

Stonebraker, J.S., Amand, R.E., Bauman, M.V., Nagle, A.J., Larson, P.J.

"Modelling haemophilia epidemiology and treatment modalities to estimate the unconstrained factor VIII demand"

Haemophilia, 10 (1), pp. 18-26. (2004)

The article presents a new method for estimating the unconstrained factor VIII (FVIII) demand based on the principles of decision analysis. Epidemiology and treatment modalities were integrated into a model for unconstrained FVIII demand. Assumptions for each variable with impact on the unconstrained FVIII demand were defined and probability estimates for these variables were obtained from the literature and medical experts. The sensitivity of the unconstrained FVIII demand to each of the variables was determined, and the variables with the greatest impact were modelled probabilistically. The probability-weighted average for the unconstrained FVIII demand model was 6.9 units per capita with a 90% uncertainty interval of 2.7-13.6 units per capita. When compared with FVIII usage in countries, only Luxembourg's use of FVIII (7.7 units per capita) exceeded the probability-weighted average for the modelled unconstrained FVIII demand. As better information becomes available, revision of model variables is easily accomplished allowing for a more accurate and dynamic forecast of demand over time. More accurate modelling of the 'true' demand longitudinally should help prevent shortages of FVIII concentrates such as those that have occurred in the past. In addition, a more accurate forecast of FVIII demand will allow national health care policy makers to better allocate financial and other resources. Sufficient and consistent supply of FVIII concentrates and appropriate financing of haemophilia care will allow the clinical benefits of more aggressive treatment regimens such as prophylaxis to be realized.