Session I: Insects and Animals (including man)

Antibiotic peptides of insects

Jules A. HOFFMANN , Institut de Biologie Moléculaire et Cellulaire, CNRS, 15 Rue René Descartes, Strasbourg, France. jhoff@ibmc-u.strasbg.fr

Insects are particularly resistant to microbial infections. The host defense relies essentially on three facets: (i) phagocytosis of invading microorganisms; (ii) activation of proteolytic cascades leading to localized blood coagulation, to formation of melanin and to opsonisation; (iii) synthesis of a battery of small-sized, cationic peptides with large activity spectra against bacteria and/or fungi. Drosophila has become a favourite model system for the study of the host defense of insects and the presentation will focus on the induction and the role of the antimicrobial peptides in this species. The structures of the inducible antibacterial and antifungal peptides will be presented and the control of expression of their genes will be analysed, namely through the use of Drosophila genetics. The presentation will stress the parallels between the induction of antimicrobial genes by bacterial challenge in Drosophila and the cytokine-induced expression of immuneresponse genes in mammals. A central theme will be the marked conservation of innate defenses between insects and mammals, which points to a common ancestry of these systems.  

Development of animal antibiotics into drugs

Michael A. Zasloff, M.D., Ph.D., Magainin Pharmaceuticals Inc., 5110 Campus Dr. Plymouth Meeting, PA, USA. mzasloff@magainin.com

Gifts from Frogs and Sharks: Components of the Innate Immune System of Vertebrates Under Development as Anti-infective and Anti-cancer Drugs. Over the past ten years, a great number of substances with antimicrobial and anti-cancer activity have been discovered in animals. In most cases, they have been found to function as the effector arm of the innate immune system. I will discuss the development of two such molecules, each of which has entered advanced stages of human clinical trials. The first is pexiganan, a 22 amino acid antimicrobial peptide developed for the treatment of infected diabetic ulcers. The second is squalamine, a cationic steroid, originally discovered as a broad-spectrum antimicrobial, but later shown to exhibit antiangiogenic activity in vivo. Lastly, I will speculate on the possibility of creating therapeutics that can provide protective benefit to man and other animals through stimulation of the arm of innate immunity involved in protection of our epithelial barrier defenses.

Defensins and Defensinoid Peptides

Michael E. Selsted, Department of Pathology, College of Medicine. University of California, Irvine, California, U.S.A. meselste@uci.edu

Mammalian defensins are disulfide-rich antimicrobial peptides expressed in granulocytes and the epithelium of many tissues. Defensins include a and b subfamilies which, though encoded by distinct gene families, are conformationally quite similar. Leukocyte defensins are packaged in the azurophil granules of neutrophils, and are delivered to the phagosome following phagocytosis. Conversely, epithelial a-defensins that are produced by Paneth cells in crypts of the small intestine are constitutively secreted into the bowel lumen, though lumenal levels may be increased by cholinergic stimuli. Studies on defensin microbicidal action have disclosed that the peptides produce lesions in model membranes in at least two ways. One relies on the formation of large transient disruptions that allow for the efflux of macromolecules; the second involves the formation of stable pores formed by multimers of defensin dimers. A common mechanistic feature of both a and b defensins is the antagonism of their in vitro microbicidal action by increasing ionic strength. It has been hypothesized that salt sensitivity of airway b-defensins underlies the susceptibility of cystic fibrosis patients to pulmonary infections. This salt sensitivity contrasts with microbicidal activities of other disulfide-containing peptides, such as protegrins, that appear unaffected by ionic strength. The isolation, structure, and microbicidal properties of a novel broad spectrum antimicrobial peptide that is similarly unaffected by ionic strength will be presented.

Cathelicidin Peptides:  Variations on an Antimicrobial Theme

Margherita Zanetti, Dip. Scienze e Tecnologie Biomediche - University of Udine, Piazzale Kolbe, Udine, Italy. zanetti@icgeb.trieste.it

Cathelicidin-derived peptides are a group of structurally heterogeneous antimicrobial peptides that are thought to contribute to host defense and which include a-helical, Cys-rich, Pro- and Arg-rich, and Trp-rich sequences. These peptides are synthesized at the carboxyl-terminus of 15-18 kDa precursors (cathelicidins) that contain a conserved ‘cathelin’ amino-terminal domain belonging to the superfamily of cystatins. Members of the cathelicidin gene family are widely distributed in mammals. Only one is present in humans, while other mammalian species have a varied number of congeners with different C-terminal domains. They are primarily expressed in myeloid cells and are stored in the secretory granules of neutrophils in unprocessed forms containing the cathelin domain which inhibits the antimicrobial activity of the C-terminal peptide. Processing of the proforms by elastase upon neutrophil degranulation liberates the microbicidal peptides in cow and pig, and a similar activation mechanism has also been suggested for the human congener. By contrast, horse cathelicidins are released as unprocessed forms under these experimental conditions and mature peptides have only been detected in addition to the proforms, in neutrophil-rich tracheobronchial secretions of horses affected by chronic obstructive pulmonary disease. Cathelicidins also show species-distinctive features with respect to their expression in tissues. In addition to bone marrow cells, the human congener is diffusely expressed throughout epithelia, including lung epithelia, and is induced in inflammed skin, whereas expression of bovine cathelicidins seems to be mainly restricted to lymphoid organs as indicated by in situ hybridization and immunocytochemistry of various tissues. Furthermore, in situ hybridization localizes high levels of cathelicidin mRNA to neutrophils infiltrating the lower airways in bovine pneumonia, suggesting inducible expression of bovine congeners in neutrophils. Important inter-species differences can also be observed when considering the activity of the mature peptides. These show in general broad spectrum antimicrobial properties and, for some, endotoxin binding activity has also been demonstrated. However, a Pro- and Arg-rich peptide of this family has been shown to affect cellular processes in human cells by selectively binding SH3 domains of cytosolic proteins. Thus, cathelicidins show a surprising set of variations on a common antimicrobial theme, with respect to processing and tissue distribution, structural variety and multiple functions of the antimicrobial peptides.

Poster No.1

Novel synthetic peptides with antimicrobial and antiendotoxin properties

Abraham PR, Appelmelk BJ¹ and Van Deventer JH
Department of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam; ²Department of Medical Microbiology, Vrije Universiteit, Amsterdam, The Netherlands. p.r.abraham@amc.uva

The recent emergence of multidrug-resistant bacterial pathogens has intensified efforts to provide a new class of antibiotics to combat the high mortality associated with systemic infection. Bacterial membrane components or endotoxins such as lipopolysaccharides (LPS) or lipoteichoic acids (LTA) activate the cellular host-defense system to produce chemical mediators of inflammation responsible for clinical symptoms characteristic of severe infection. A rational approach in the development of new therapeutic modalities is directed against neutralization of liberated endotoxin to block pathological inflammatory processes. With rational design techniques, we have devised a novel class of linear cationic peptide antibiotics designated bactericidal peptides (BP) of 19 amino acid residues in length with idealized amphipathic a-helical structures. A central feature incorporated in the design was a tandem array of minimal LPS-binding motifs derived from sequence features of a number of naturally occurring antimicrobial peptides and proteins, to generate model peptides with extended antibiotic spectra, improved endotoxin-neutralizing properties and low toxicity. Characterization of a typical model peptide BP2, revealed broad-spectrum bactericidal activity towards clinical strains of antibiotic-resistant Gram-negative and Gram-positive organisms (MIC = 0.4 to 3.5 mM), synergism with conventional antibiotics against antibiotic-resistant strains, bactericidal activity at micromolar concentrations against multi-resistant clinical isolates in whole blood and negligible hemolytic activity at several-fold excess of antimicrobial concentrations. BP2 efficiently inhibited LPS- and LTA-mediated cytokine release in whole blood at concentrations in the micromolar range and bound LPS (K_D = 5nM) and LTA (K_D = lOnM) with high apparent affinities. The antibiotic and antiendotoxin properties of BP2 were confirmed in experimental animal models of lethal endotoxemia, peritonitis and pneumonia. With a limited number of sequence analogues of BP2 we have shown that multiple motifs are essential for antibiotic and endotoxin-neutralizing properties and that LPS-or LTA-binding is not predictive for antimicrobial activity or synonymous with endotoxin-neutralization.

Poster No. 2

Impaired Innate Immunity in the Newborn: Newborn Neutrophils are Deficient in Bactericidal/Permeability-Increasing Protein (BPI)

Ofer Levy*, Sarah Martin***, Eric Eichenwald^Ù, Tomas Ganz⁺, Erika Valore⁺, Stephen F. Carroll^#, Kelly Lee^#, Donald Goldmann* *, & Grace M. Thorne * * *

From the Divisions of Medicine* & Infectious Diseases** and General Clinical Research Center*** , Children’s Hospital and Harvard Medical School, Boston, Massachusetts; Division of Newborn Medicine^Ù Brigham & Woman’s Hospital, Boston, Massachusetts; ⁺Dept. of Medicine, University of California at Los Angeles, California; & ⁺Dept. Preclinical Research, Xoma Corp. Berkeley, California. levy-o@a1.tch.harvard.edu

Objective: The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. In particular, little is known about the oxygen-independent killing mechanisms of newborn neutrophils. The activity of adult neutrophils against Gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary granules of the 55 kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial surface lipopolysaccharides and kills Gram-negative bacteria. In light of BPI’s importance to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns.
Design: The cellular content of BPI was determined by Western blotting of neutrophils derived from fullterm newborn cord blood (n = 21, mean gestational age 38.6 weeks) and from adult peripheral blood (n = 22, mean age 29 years). Neutrophil content of other primary granule markers was also assessed: myeloperoxidase (MPO) by Western blotting and defensin peptides by acid-urea PAGE.
Results: The neutrophils of newborns contain at least 3-4 fold less BPI per cell than adult neutrophils (67+/-13 ng per 10⁶ cells vs 234 +/- 27 ng per 10⁶ cells; p < 0.001). The relative BPI-deficiency of newborn neutrophils was apparently not due to perinatal stress-related degranulation of intracellular BPI stores since newborn and adult neutrophils contained nearly identical amounts of MPO and of defensin peptides, both of which, like BPI, are also localized to neutrophil primary granules.
Conclusion: These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against Gram-negative bacteria. This deficiency could contribute to the increased incidence of Gram-negative sepsis among newborns relative to healthy adults.

Poster No. 3

Expression of Natural Antibiotics in Human Gingival Epithelium.

A.Weinberg¹, S. Krisanaprakornkit ² , B.A. Dale ². (¹Dept Periodontics, Case Western Reserve University., Cleveland, OH; ² Dept. Oral Biology, University of Washington, Seattle, WA, USA, e-mail:axw47@po.cwru.edu.

Epithelial derived antimicrobial peptides (EAPs) are a new class of antibiotics recently discovered in various human mucosal epithelia, and believed to act as a first line of innate host defense. We reasoned that since the human oral cavity is constantly challenged with fungi, viruses and over 300 species of bacteria, the gingiva is a source of EAP expression. The goals of this study were (1) to determine b-defensin (hBD-1, hBD-2) and cathelin class (LL-37) mRNA expression in normal human gingival epithelial cells (NHGECs) and tissues, (2) to test bacterial cell wall fractions for EAP regulation, and (3) to localize expression of hBD-1 peptide in human gingival tissue. Quiescent NHGECs and NHGECs challenged with cell walls of Porphyromonas gingivalis (Pg; pathogen associated with periodontal disease; not found in healthy sites) or Fusobacterium nucleatum (Fn; commensal found in healthy and diseased sites), E coli LPS, Pg LPS, or PMA were analyzed by RT-PCR using primers specific for hBD­-1, hBD-2, LL-37, and controls. Results indicated that hBD-1 and LL-37 mRNAs were constitutively expressed in all the situations studied, while hBD-2 was transcriptionally upregulated by all stimulants except Pg. Moreover, hBD-2 mRNA was not appreciably induced, beyond serum induction, by E. coli LPS or Pg LPS. Interestingly, acidurea gel/Western and dot blot assays of the NHGECs, using antibody to hBD-1 (T. Ganz, UCLA), detected hBD-1 peptide expression only in Fn cell wall challenged cells. Thus, hBD-1 is post transcriptionally upregulated by Fn, but not Pg.While there was no difference in mRNA expression of hBD-1 -and hBD-2 between normal and inflamed sites from the same subject (n=4,mRNA expression of these EAPs was found to vary between subjects (n). Immunohistochemical analysis of gingival tissue localized expression of hBD-1 in the upper third of sulcular and oral epithelium, but not in junctional epithelium; consistent with expression in differentiated epithelium. Our findings indicate that the human gingival epithelial cell compartment is a source for hBD-1, hBD-2, and LL-37, and suggest a role of these peptides in gingival health and disease. Supported by NIH NIDCR P50 DE08229 and DE10329.

Poster No. 4

Innate Immunity In The Human Airway: Expression Of Beta Defensins, CD 14 And Toll-Like Receptors In Tracheal Epithelium And Alveolar Macrophages.

Diamond G ¹, Becker M.², Verghese M.², Randell S.², Agagan C.¹, Santomauro E.¹ and Kisich, K.^3
1Dept. Anatomy, Cell Biology and Injury Sciences, UMDNJ, Newark, NJ. ²Cystic Fibrosis Center, Univ. North Carolina, Chapel Hill, NC.³National Jewish Medical Center, Denver, CO. gdiamond@umdnj.edu

As part of a study to define the role of the airway in innate immunity, we examined respiratory epithelial cells and alveolar macrophages (AM) for the expression of antimicrobial peptide genes and pathogen associated molecular pattern receptors. We observed by RT-PCR that well differentiated airliquid interface cultures of human tracheobronchial epithelial cells (TEC) express human beta-defensins (hBD) 1 and 2. Northern blot analysis indicated that expression of hBD2 is inducible upon stimulation with lipopolysaccharide (LPS) as well as with inflammatory mediators, while hBD1 is uninducible. Northern and western blot analysis also indicated that TEC express membrane bound CD14. Using RTPCR, we further detected mRNA encoding human toll-like receptors (hTLR) 1,2,3 and 5 in TEC. Northern blot analysis indicated that while hTLR2 mRNA levels were induced upon stimulation with LPS, CD14 mRNA levels remained constant. These results suggest that the respiratory epithelium functions as an innate immune response tissue. We also examined alveolar macrophages isolated from freshly lavaged patients for the presence of these transcripts. Similar to what has been published for peripheral blood derived (PB) monocytes/macrophages, we were only able to detect the expression of hTLR2 in AM. In, contrast to PB macrophages however, levels of hTLR2 mRNA remained constant in AM incubated with LPS. Furthermore, we were unable to detect the expression of either b-defensin in AM. This is in contrast to bovine AM which constitutively express several b-defensins. Together these results suggest that the human airway epithelium participates in pattern-recognition based innate immunity by the inducible production of antimicrobial peptides. Human AM, on the other hand, do not express b-defensins, and their innate immune response may be distinct from macrophages found in other anatomical locations.Funded by grants from the NIH and the Cystic Fibrosis Foundation.

Characterization of chitotriosidase, the chitinase by human phagocytes

Hans Aerts, Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam The Netherlands. j.m.@ertseamc.uva.nl

During a search for markers of lipid laden macrophages in Gaucher patients we discovered that the cells secrete an enzyme that is able to degrade an artificial chitotrioside substrate, and that therefore was named chitotriosidase. Plasma enzyme levels are several hundred fold elevated in patients and are now used in early detection of onset of disease and monitoring of efficacy of therapeutic intervention. Chitotriosidase has been characterized at protein and gene level. The enzyme is massively synthesized by specifically activated macrophages as a 50 kDa protein that is predominantly secreted. About one third is proteolytically processed to a 39 kDa form that accumulates in lysosomes. The 39 kDa enzyme is C-terminally truncated and lacks a chitin-binding domain. The 50 kDa enzyme is also formed in progenitors of neutrophils and stored in their specific granula. Chitotriosidase proves to be structurally homologous to chitinases from various species. The enzyme is able to degrade natural chitin and should therefore be considered as the human analogue of class 18 chitinases. Three other chitinase-like proteins in man have been identified in recent years. All these proteins show a substitution in the catalytic region that results in loss of enzymatic activity but renders a lectin-like property. Crystallographic studies indicate that the 39 kDa catalytic portion (that lacks glycans) shows a TIM-barrel structure. The composition of the chitotriosidase gene, located on chromosome 1q31-32 has been analyzed. One specific 24 bp duplication in exon 10 occurs in various ethic groups : the incidence of carriers being about 30%. Further studies are required to establish whether the relatively common chitotriosidase deficiency imposes some risk factor. We have hypothesized that chitotriosidase might fulfil an antifungal action. Incubation of Candida albicans with 50 kDa chitotriosidase indeed results in bursting of chitin-rich hyphal tips. Moreover, administration of chitotriosidase to mice systemically infected with Candida albicans and Aspergillus fumigatus has a major protective effect. This suggest that chitotriosidase may be part of a primitive defence mechanism against chitin-containing pathogens, analogous to the situation in plants. The physiological role and therapeutic value of the human chitinase is subject of further investigations.

Host defense against Gram-negative bacteria: Lessons from study of BPI.

Weiss J, Department of Medicine, Infectious Diseases, University of Iowa College of Medicine, Iowa City, U. S. A. mailto:jerrold-weiss@uiowa.edu

The ability of multicellular organisms to recognize and respond to lipopoly(oligo)saccharides (endotoxin), unique surface glycolipids of Gram-negative bacteria (GNB), plays an important role in host defense against invading Gram-negative bacteria. This is well illustrated by two closely related LPS-binding proteins, the plasma LPS-binding protein (LBP) and the neutrophil-derived bactericidal/permeability-increasing protein. These two proteins are organized as closely similar, highly elongated 2-domain proteins in which the N-terminal domain confers LPS recognition and the C-terminal domain is needed for transfer of protein-coated LPS-containing particles to distinct host acceptors. However, the structural and functional consequences of the interaction of BPI and LBP with GNB and cell-free LPS is strikingly different. LBP greatly increases the potency of the pro-inflammatory activity of endotoxin whereas BPI is potently cytotoxic toward GNB and promotes bacterial and endotoxin clearance and destruction and hence down-regulation of host responses to GNB and endotoxin. Studies are ongoing to address the question as to how similar recognition of endotoxin can be coupled to such different functional responses.

Activation of the leukocyte NADPH oxidase.

Babior B.M., Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037. babior@scripps.edu

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (0₂^- ) at the expense of NADPH. It is composed of 5 unique subunits (p22^PHOX , p40^PHOX , p47^PHOX , p67^PHOX and gp91^PHOX), and comprises in addition 2 low molecular weight GTPases, namely Rac2 and RaplA, that participate in its regulation. In the resting cell three of the subunits occur as a cytosolic complex while the other two are located in the membrane where they comprise cytochrome b_558, a flavocytochrome. During oxidase activation, p47 ^PHOX, a component of the cytosolic complex, is phosphorylated on 8-9 serine residues and migrates to the membrane bringing the other 2 cytosolic components to the membrane-bound flavocytochrome to assemble the active oxidase. Regulation by the phosphorylation of p47 is complex, with various of the phosphorylated serines performing different functions with respect to the activation of the enzyme. Protein kinase C and Akt phosphorylate p47 ^PHOX and activate the oxidase; other kinases, including protein kinase A, MAPKAP2, Erk, JNK and p21^rac-activated kinase (PAK), cannot convert p47 ^PHOX into a form that activates the oxidase. As to the GTPases, RaplA, which co-purifies with cytochrome b₅₅₈, becomes phosphorylated when neutrophils are activated, but the significance of this phosphorylation with regard to the activation of the oxidase is not clear at this point.

Poster No. 5

Cathelicidin-derived peptides: In vitro and in vivo activity

R. Gennaro^1, M. Benincasa¹, L. Dolzani,^l B. Skerlavaj,² M. Scocchi, ^l A. Risso,² and M. Zanetti², ¹Dept. of Biochemistry & Biophysics, Univ. of Trieste; ²Dept. of Biomed. Sci. & Technol., Univ. of Udine, Via Giorgieri, 1, I-34127 Trieste, Italy, e-mail: gennaro@bbcm.univ.trieste.it

Gene-encoded antimicrobial peptides have been identified from extremely diverse organisms, including vertebrates, invertebrates, plants and bacteria. In mammals, such peptides are part of the innate host defence mechanisms and are present in phagocytic cells and epithelial surfaces. In general, they show a potent in vitro activity and susceptible microorganisms include strains resistant to conventional antibiotics, whose diffusion poses serious problems in the treatment of several infections. In this respect, antimicrobial peptides may provide design templates for the development of novel antimicrobial drugs.

In mammals, professional phagocytes are the store of many antimicrobial peptides that belong to the defensin family (a- and b-defensins) or derive from the cathelicidin family of precursors. The cathelicidinderived peptides vary significantly by sequence, structure, and length and include a-helical, Pro- and Argrich, Cys-rich and Trp-rich peptides. To select among these peptides the most promising for further studies, the in vitro antibacterial activity of members representative of a-helical (SMAP-29, BMAP-27 and BMAP-28), Pro- and Arg-rich (the fragment 1-35 of Bac7), Trp-rich (indolicidin) and Cys-rich (protegrin PG-1) peptides, was tested against many antibiotic-resistant clinical isolates. These included methicillin-resistant S. aureus (MRSA), vancomycin-resistant E. faecalis (VRE), and multi-resistant strains of E. faecium, S. equinus, S. agalactiae, A. baumannii, B. cepacia, S. marcescens and P. aeruginosa. The peptides displayed different potencies against the strains tested, with SMAP-29 and PG-1 showing the broadest spectrum and the highest activity (MIC values in general in the 0.5 - 2 mM range of concentration). SMAP-29 was further tested for its tendency to select resistant mutants and to protect mice i.p. infected with lethal amounts of P. aeruginosa or MRSA. The MIC values of SMAP-29 against E. coli and P. aeruginosa were unchanged even after 20 passages in the presence of a sub-inhibitory dose of peptide. Conversely, conventional antibiotics (nalidixic acid, norfloxacin and gentamycin) easily selected resistant mutants, when tested under the same conditions. These results prompted us to test the toxicity and the antimicrobial activity of SMAP29 in an animal model. The peptide displayed a LD50 of 40 mg\Kg, when injected i.p. in Balb\c mice, and at the much lower doses of 0.2 and 0.8 mg/kg it was able to protect mice from an i.p. lethal injection of respectively P. aeruginosa and MRSA. The potent in vitro and in vivo antimicrobial activity of SMAP-29 against clinically relevant, antibiotic resistant bacteria, suggests that this peptide is a promising lead compound for the development of novel anti-infectious agents.

Poster No. 6

Single-cell Measurement of Granulocyte Respiratory Burst With Digital Imaging Fluorescence Microscopy
S. Szücs^a , G. Vámosi ^b,R. Póka^c, A. Sárváry^a , H. Bárdos^a, M. Balázs^a , L. Tóth ^a, J. Kappelmayer ^d, J.Szöllösi^b, R.Adány
Departments of Hygiene and Epidemiology^a, Obstetrics and Gynaecology^’c, Clinical Chemistry^d, Institute of Biophysics and Cell Biology^b, University Medical School, Debrecen, Hungary, e-mail: mailto:szucs@-jaguar.dote.hu

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion (0²) and hydrogen peroxide (H²0²) production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol-dibutyrate (PDBu) and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in 0^2-/H²0² generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass, fibronectin- and type IV collagen-coated surfaces in response to stimulation with PDBu or N-formyl-methionyl-leucyl-phenylalanine (FMLP). We elaborated a calibration technique for the quantitative measurement of the ethidium-bromide generation mediated by 0^2- within individual adherent granulocytes. The EB production varied between 0.48 - 1.17 attomoles/cell/minute.

Poster No. 7

Antimicrobial (poly)peptides of human nasal secretions.

Alexander M. Cole, Thomas Kang and Tomas Ganz. Department of Medicine, Division of Pulmonary and Critical Care. University of California, Los Angeles - School of Medicine, USA. e-mail:acole@ucla.edu

The antimicrobial properties of nasal fluid were discovered by Alexander Fleming in 1922 and attributed to lysozyme. Since then, there have been few studies characterizing the innate immune function of nasal fluid. Several groups have attributed the microbicidal activity of nasal fluid mostly to two major components, lysozyme and lactoferrin. We revisited this issue by first examining the microbicidal activity of whole nasal secretions using a colony-forming unit (CFU) microassay, wherein bacteria are directly incubated with minimally manipulated nasal secretions. Three patterns of antimicrobial effect were seen: some bacteria were killed (strains of Ps. aeruginosa, S. aureus, B. subtilis), other bacteria (strains of Ps. aeruginosa) lost viability at 3 hrs but recovered and grew by 24 hrs; and the third group of bacteria (E. coli, S. typhimurium) were more resistant and grew slowly throughout the 24 hrs of incubation. Boiling nasal secretions for 10 minutes ablated its antimicrobial activity against a standard CF P. aeruginosa strain, and this activity could not be recovered by reconstitution with physiologic concentrations of purified lysozyme and lactoferrin. Thus other antimicrobial (poly)peptides contribute to the activity of nasal fluid. We studied known components of nasal secretions (lysozyme, lactoferrin, sPLA₂, and SLPI) as well as newly identified components (statherin, b-microseminoprotein, calprotectin, HNP 1-3, HBD-1 and HBD-2) using a combination of protein purification techniques (acid extraction, gel fractionation, and HPLC), gel electrophoresis (acid-urea and Tricine-SDS PAGE), semi-quantitative Western analysis, mass spectroscopy and protein microsequencing. Microbicidal activity was tested against standard test strains of E. coli and L. monocytogenes using a CFU, gel overlay, or radial diffusion assay (Steinberg, D.A., and R.I. Lehrer. 1997. Methods Mol Biol. 78: 169-186), and different sets of antimicrobial components were found to be active against the two targets. Nasal secretions are a complex mixture of antimicrobial components whose contribution to total antimicrobial activity differs depending on the test microbial target.

Poster No.8

THE HUMAN ANTIBACTERIAL CATHELICIDIN, hCAP-18, IS BOUND TO LIPOPROTEINS IN PLASMA.

Sørensen 0., Bratt T., Johnsen A.H., Thorup Madsen M., and Borregaard N.
The Granulocyte Research Laboratory, Dept. of Hematology, The Finsen Centre, and Dept. of Biochemistry, The Centre of Laboratory Medicine and Pathology, Rigshospitalet, Copenhagen, Denmark. e-mail:olesoeren@rh.dk

Cathelicidins are a family of antibacterial and lipopolysaccharide (LPS)-binding proteins present in mammalian neutrophils. hCAP-18, the only known human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is more than 20 fold higher than for other specific granule proteins relatively to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography and hCAP-18 was found in two distinct high molecular weight fractions, which co-eluted with apolipoprotein A-I and apolipoprotein B, respectively. The association of hCAP-18 with lipoproteins was validated by the co-fractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Additionally, the level of hCAP-18 in delipidated plasma was less than 1 % of that in normal plasma. Immunoprecipitation of VLDL, LDL and HDL particles with anti-apolipoprotein antibodies resulted in co-precipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was found to mediated by the LPS-binding C-terminal part of the protein through hydrophobic interactions. The binding of hCAP-18 to lipoproteins ensures a high level of the antibiotic protein, hCAP-18, in plasma, and indicates that lipoproteins may play an important role either as a reservoir of microbicidal proteins/peptides or as a protection against the cytotoxic effects of these peptides.

Poster No. 9

Human skin as source of antimicrobial peptide/proteins

Schröder J.M., Harder J. and Meyer-Hoffert U., Department of Dermatology
Schittenhelmstr. 7 , Kiel, Germany. jschroeder@dermatology.uni-kiel.de

Human skin is continously exposed to microorganisms but rarely infected. It has been speculated that the physical barrier together with acidic pH and microbicidal fatty acids mainly contributes to the innate defense of human skin. Due to the fact that epithelia of plants, insects, vertebrates - including frogs and cattle - produce antimicrobial peptides, we attempted to systematically analyse human skin for antimicrobial proteins and peptides. In order to get as much information as possible we used three different sources for our biochemical analyses:
a) Stratum corneum obtained from healthy people
b) Lesional psoriatic scales
c) Supernatants and cell-extracts of cultured human primary keratinocytes.
Extracts and supernatants were separated by a heparin-HPLC column followed by preparative reversed phase HPLC of bound material and analyses of fractions for antimicrobial activity by the use of the radial diffusion assay and microdilution assay using laboratory strains of E. coli, P. aeruginosa, St. aureus and C. albicans.
As a result we found in stratum corneum, psoriatic scales as well as supernatants of cultured keratinocytes a number of fractions containing antimicrobial activities, which were purified to homogeneiety - when ever possible. HBD-2 was identified as the major E. coli killing peptideantibiotic in psoriatic scales and stimulated keratinocyte cultures but not in stratum corneum extracts. One 11 kDa antimicrobial peptide having strong candidacidal activity was found to represent a major polypeptide in psoriatic scales. Another 13 kDa antimicrobial protein represented on of the dominating E. coli killing activities. Surprisingly there were only few fractions killing St. aureus. We conclude from our investigations that human skin is a rich source of antimicrobial peptides and proteins. Structural analyses will give insight into the molecular nature of these innate defense molecules.

Poster No.10

Time and Concentration Dependent Killing of Bacteria by Lactoferricin B

Sykehusveien (PO Box 56) N-9038 Tromsø, Norway. e-mail: mlabhu@rito.no
U1vatne H., Andersen J.H., Vorland L.H. Dept of Medical Microbiology, University and University Hospital of Tromsø, Norway. E-mail: mlabhu@rito.no

Lactoferricin B (bovine) is a product of gastric pepsin cleavage of bovine lactoferrin, and has been shown to have antimicrobial activity against several microorganisms. This study reveals the bactericidal correlation between Lactoferricin B concentration and time of exposure to Escherichia coli cells. Three E. coli strains were exposed to concentrations of Lactoferricin B, ranging from 0 mg/ml to 100 mg/ml. Aliquots of the bacteria-Lactoferricin B mixtures were drawn at times ranging from 1 minute to 24 hours of exposure. The aliquots were plated on media void of the peptide. After over night growth, the colony forming units pr ml (CFU/ml) were calculated, and the killed fraction of cells determined. This study shows that the bactericidal effect of Lactoferricin B is both concentration and time dependent. When the time of exposure is below two hours, there is a marked difference in killed fraction of cells within the concentration range. When exposing the bacterial cells to Lactoferricin B for more than two hours, the bactericidal effect becomes independent of the concentration. Even below the conventional determined MIC-values, there is a significant decrease in bacterial cell number.

Poster No.11

Synthetic cationic peptides exhibit neutralising activity towards HIV-1 and Candida spp.

Jasper Groenink, Academic Centre for dentistry Amsterdam (ACTA),Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands, e-mail: LGroenink.obc.acta@med.vu.ffi

Mucosal candidiasis, one of the most common opportunistic fungal infections in HIV-1 infected patients, is an early sign of clinical overt AIDS, and an important cause of morbidity, particularly in HIV-infected children. The cationic peptide histatin in human saliva is a natural inhibitor of Candida albicans. We have created modified synthetic peptides based on the functional domain of histatin-5 (KRKFHEKHHSHRGY) with strongly increased fungicidal activity towards Candida spp and Asparagillus fumigatus. These membrane disturbing peptides were analysed for their potential effect on the virus envelope of HIV-1. A series of cationic peptides of 14 amino acids was synthesized with modifications in the histatin motif resulting in increased amphipathicity in either linear or lateral direction. Antifungal activities of these peptides were compared with antiviral activities. A two-fold serial dilution of the series of peptides was incubated with a yeast suspension of 10⁷ CFU/ml or with 500 TCID₅₀ /ml HIV-1. The IC₅₀ towards Candida spp was calculated from the viable counts after plating the incubation mixture, and neutralizing activity towards HIV-1 was determined by analysing the cytopathic effect on MT-2 cells (both syncitia formation and p24 production) after 7, 14 and 21 days. Cytotoxic and hemolytic effects were determined using up to 800 mg/ml of the peptides. In comparison with the natural histatin-5, all peptides showed a striking increase in fungicidal effect (IC₅₀ of 2.5 mM for histatin-5, versus 0.4 -1.6 mM for the modified peptides). Neither histatin-5 nor three out of the six synthetic peptides showed neutralisation of HIV-1, as tested up to 800 mg/ml; two of the peptides appeared able to neutralise HIV-1 with 200 mg/ml, whereas one out of the series exhibits neutralisation at a concentration of as little as 25 mg/ml. However, there is no strict correlation between the antifungal and antiviral activities, since both of the peptides showing HIV-1 neutralisation at 200 mg/ml were in the antifungal assay the extremes (ICd₅₀ of 0.4 vs 1.6 mM/ml). Thus we have found HIV- 1 neutralising peptides that combines antiviral capacity with antifungal activity.

Poster No.12

THE BACTERICIDAL/PERMEABIILITY-INCREASING PROTEIN (BPI) IS MEBRANE-ASSOCIATED IN AZUROPhil GRANULES OF HUMAN NEUTROPHILS, AND RELOCATION OCCURS UPON CELLULAR ACTIVATION.

Jero Calafat^l, Hans Janssen¹, Edward F. Knol². Arne Egesten³, ¹Division of Cell Biology, The Netherlands Cancer Institute, ² Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands, ³Department of Medical Microbiology, Lund University, University Hospital, Malmø, Sweden.arne.egesten@medforsk.mas.lu.se

Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI). Through binding to lipopolysaccharides (LPS), BPI exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy. BPI was found to co-locate with myeloperoxidase (MPO), a marker for azurophil granules and it also showed the same pattern of distribution as CD63, a transmembrane-anchored protein. This suggests that BPI is membrane-associated in the azurophil granules in neutrophils. The presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow. Induction of selective release of azurophilic granules by the Na-ionophore monensin resulted in fusion of endosomes with azurophil granules, resulting in the formation of large vacuoles containing MPO, CD63, and BPI. After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments. The results show that BPI is present in azurophil granules, is probably primarily membrane-associated and is relocated after activation, following the same route as MPO and CD63.

Poster No.13

Effect of neutrophil granule proteins on the release of secretory leukocyte proteinase  inhibitor (SLPI) from human bronchial epithelial cells in vitro.  
P.S. Hiemstra, S. van Wetering, A.C. van der Linden, M.A.J.A. van Sterkenburg ¹J. SchalkwiJk, Dept. of Pulmonology, Leiden University Medical Center,  ¹Dept. of Dermatology, University Hospital Nijmegen, The Netherlands, e-mail: phiemstra@pulmonology.azl.nl

Airway epithelial cells are considered to be a major site of synthesis of proteinase inhibitors and antimicrobial peptides. SLPI is an 11.7 kD cationic protein that is produced locally in the lung by subsets of airway epithelial cells and neutrophils. SLPI was originally identified on basis of its capacity to inhibit neutrophil elastase (NE). Recent studies demonstrate that SLPI also displays broad-spectrum antimicrobial activity, and may have LPS-neutralizing activity. Relatively little is known about the mechanisms that regulate SLPI expression in epithelial cells. Previous studies demonstrated that NE decreases SLPI release from airway epithelial cell lines. Therefore the aim of the present study was to investigate the effect of neutrophil degranulation products on SLPI production by subcultures of primary bronchial epithelial cells (PBEC). PBEC were incubated for several time periods with various concentrations of NE and neutrophil defensins, and SLPI production was analyzed by Elisa and Northern blot analysis. The results revealed that neutrophil defensins cause a two-fold increase in SLPI release, that was not accompanied by a significant increase in SLPI mRNA. In contrast to defensins, NE caused a marked significant decrease in SLPI release (> 90 % decrease at 10 mg/ml), whereas there was a trend towards an increase in SLPI mRNA; this effect of NE was prevented by its inhibitor, a1-proteinase inhibitor. In contrast, NE caused a slight, but significant increase in the release of the related cationic elastase inhibitor elafin/SKALP. Therefore, the inhibitory effect of NE on SLPI protein release appears to be specific for this antimicrobial proteinase inhibitor. Analysis of the cell lysate of NE-treated cells revealed that the NE-induced decrease in SLPI release was accompanied by an increase in the amount of cell-associated SLPI. At present it is unknown whether this is due to retention of SLPI inside the cell or at the epithelial surface. Therefore, whether this effect of NE is detrimental to the cell (by reducing SLPI secretion) or beneficial (by forming a protective coat at the epithelial surface), remains to be studied.

Poster No. 15

Phospholipase A2 in Systemic Fungal Infections

Nevalainen TJ, Salonen JH, Rintala E, and Nikoskelainen J Departments of Pathology and Internal Medicine, University of Turku, Turku, and Pori Central Hospital, Pori, Finland. timo.nevalainen@utu.fi

Group II phospholipase A2 (PLA2) is an acute phase protein with anti-bacterial properties. Earlier, we found high concentrations of PLA2 in sera of haematological patients suffering from septic bacterial infections. In the current study, we measured the serum levels of PLA2 by an immuoassay weekly during hospitalisation of 10 patients with haematological malignancy and systemic fungal infection. Three patients had lethal invasive aspergillosis confirmed by histopathology and fungal culture at autopsy. Cavitating pulmonary infiltrates and persistent aspergillus antigenaemia in serum, urine and broncho-alveolar lavation fluid suggested the diagnosis of aspergillosis in one patient who survived. Five patients had candidiasis. Lung and brain biopsies verified mucormycosis in one patient. Twenty-three patients with haematological malignancy but without infection served as controls. The concentration of PLA2 in serum correlated to the severity of the disease and to the concentration of Creactive protein measured in the same serum samples. The mean peak values for serum PLA2 were: 1006 mg/1 (range 690-1550 mg/1) in aspergillosis, 1707 mg/1 (range 110-5430 mg/1) in candidiasis, 306 mg/1 in mucormycosis and 32 mg/1 (range <10-125 mg/1) in uninfected haematological patients. The upper limit for the normal reference range for serum PLA2 is 11 mg/1. As a rule, serum PLA2 values decreased close to the normal level following successful antifungal treatment. The values remained at highly elevated levels in 6 of the 7 patients who died. In conclusion, the concentration of PLA2 is highly elevated in systemic fungal diseases and persistent high serum PLA2 values are associated with severe life-threatening forms of the disease.  

Session II:  Plants.

Pathogenesis-related proteins, the b-1, 3-glucanases

Iglesias, A. V. and Meins F. Jr., Friedrich Miescher-Institute, Maulbeerstrasse 66CH-4058 Basel, Switzerland. meins@fmi.ch

Interactions between plants and pathogens reflect an elaborate coevolution of recognition, defense, and counter-defense mechanisms. Several plant genes are expressed as part of the hypersensitive reaction (HR) of resistant plants to pathogen infection. Some of these genes appear to serve a general defense function independent of the inciting pathogen. Genes encoding antifungal class I  b-1,3-glucanases (bGLU I) and chitinases, on the other hand, appear to be tailored for defense against a particular class of pathogens, fungi.  Three structural classes of plant b-1,3-glucanases are induced as part of the HR to viral, bacterial, and fungal pathogens. We have focussed on the bGLU I of tobacco. These bGLU I are synthesized as preproenzymes in the endoplasmic reticulum where the N-terminal signal peptide is removed and the C-terminal, vacuolar targeting signal is N-glycosylated. The proenzyme is then transported via the Golgi compartment to the vacuole where the C-terminal extension is removed and mature bGLU I accumulates. bGLU I exhibit complex transciptional regulation. High level expression associated with pathogenesis appears to depend on the interaction of EREBP transcription factors with the an ethylene-responsive element in the promoter.Studies of bGLU I-deficient mutants of tobacco and N. sylvestris generated by antisense transformation suggest a role for bGLU I in viral pathogenesis. Unexpectedly, these mutants showed markedly reduced susceptibility to infection by tobacco mosaic virus (TMV) and tobacco necrosis virus. Intercellular trafficking via plasmodesmata of TMV and a cucumber mosaic virus 3a movement protein (MP)-green fluorescent protein (GFP) fusion as well as systemic spread of a recombinant GFP-potato virus X were delayed in the tobacco mutant suggesting that increased virus resistance is due, at least in part, to restricted cell-to-cell virus movement. Callose, which is a substrate for b-1,3-glucanase, is a physical barrier to virus spread. Induced callose deposition, which is a dynamic process involving synthesis and degradation, was greatly increased in the mutant. We propose that GLU I helps viruses spread by degrading callose, which decreases callose deposition and permits MP-induced increases in the size exclusion limit of plasmodesmata. Our results suggest novel ways for protecting plants against viral infection and raise the intriguing possibility that viruses can use a defense response of the host against fungal infection—­production of b-1,3-glucanases—to promote their own replication and spread.  

Roles of antimicrobial peptides and other factors in plant-pathogen interactions

García-Olmedo F., Rodriguez-Palenzuela P., Molina A., Alamillo J.M., López-Solanilla E., Miguel E, PozaCarrion C, Aguilar I, and Llama-Palacios A., Department of Biotechnology - UPM, E.T.S. Ingenieros Agrónomos, 28040 Madrid, Spain. olmedo@bit.etsia.upm.es

Little is known about the effectiveness of defence peptides and other potential antimicrobial factors in the inhibition of pathogen growth in planta. A number of peptide families isolated from plants have been shown to have in vitro antibiotic properties. Genes encoding these peptides show expression patterns - both during development and upon pathogen infection - that are consistent with a defence role. Further evidence of this role can be obtained through the observation of altered tolerance to pathogens in plants overexpressing or under- expressing these genes. Transgenic over-expression of defence peptide genes might be potentially useful to engineer resistance of plants to relevant pathogens. To dissect pathogen response to plant peptides an other defence factors, pathogen mutants that are sensitive to them in vitro have been obtained and their virulence in planta has been investigated. A decrease in virulence of these mutants would be consistent with the hypothetical defence role of a given peptide or factor. We have begun a molecular dissection of the plant defence factors by isolating bacterial mutants altered in their sensitivity to them. The hypothesis that a given defence factor is important would be supported by a decrease of virulence in this type of mutant. Two genetic determinants of bacterial resistance against peptides have been identified: the rfaF gene of Ralstonia solanaceaurm, which is involved in LPS biosynthesis (1), and the sapA-F (sensitive to antimicrobial peptides) operon from Erwinia chrysanthemi, which determines selective resistance to certain peptides (2). In both types of mutants, virulence of the pathogen was drastically reduced. Both types of mechanisms are also known to be important for virulence in animal pathogenic bacteria. A similar approach has been followed to elucidate the potential direct antimicrobial role of active oxygen species. Additionally, scavengers of reactive species have been used to discriminate among the hypersensitive responses elicited by avirulent strains of different bacterial species.
1. Titarenko, E, Lopez-Solanilla, E, Garcia-01medo, F. and Rodriguez-Palenzuela, P. (1997)
J. Bacteriol.179:6699-6704. 2. L6pez-Solanilla, E., Garcia-01medo, F., and Rodriguez -Palenzuela, P. (1998) Plant Cell 10: 873-876.

Jasmonates as mediators of plant immunity

Broekaert W.F., Thomma B.P.J.H., Penninckx I.A.M.A., Tierens K.F.M., Francois I., Cammue B.P.A.. F.A.Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, Heverlee-Leuven, Belgium. willem.broekaert@agr.kuleuven.ac.be

To defend themselves against potentially pathogenic microbial invaders, plants produce a variety of antimicrobial compounds, some of which are inducible while others are preformed. We are using various mutants of the plant Arabidopsis thaliana to study the contribution of these different compounds to resistance against microbial pathogens.
A defensin gene, a chitinase gene and a hevein-like gene, all encoding antimicrobial proteins, are switched on systemically throughout Arabidopsis plants upon local infection. Induction of these genes requires concomitant activation of the ethylene and jasmonate signalling pathways. Mutants affected in perception of either the ethylene or jasmonate signals are unable to activate these genes. Such mutants are also markedly more susceptible than wild-type plants to the fungal pathogen Botrytis cinerea. Conversely, enhanced resistance to B. cinerea can be achieved by pretreating plants with methyl jasmonate prior to inoculation.
Another set of systemically pathogen-inducible genes includes genes encoding the antimicrobial proteins PR-1, b-1,3-glucanase and thaumatin-like protein. This set of genes requires salicylic acid for induction and mutants in the salicylate, pathway are more susceptible to the fungal pathogen Peronospora parasitica, but not to B. cinerea. Protection against P. peronospora can be provided by pretreating plants with salicylate analogs but not with methyl jasmonate.
A third defense response of Arabidopsis that we study is the pathogen-inducible production of camalexin. Camalexin is an antimicrobial sulphur-containing indole derivative that is produced only in a few cell layers surrounding pathogen infection sites. Induction of its biosynthesis was found not to rely directly on hormone pathways involving either ethylene, jasmonic acid or salicylic acid, but rather to involve perception of reactive oxygen species generated during infection. A camalexin biosynthesis mutant, pad3-1 (Glazebrook and Ausubel, 1994, Proc. Natl. Acad. USA, 91, 8955), was shown to be much more susceptible than wild-type plants to infection by the fungus Alternaria brassicicola, but not to B. cinerea. nor to P. peronospora. Hence, camalexin plays a crucial role in resistance to A. brassicicola.
A general conclusion that can be drawn from our work is that the defense system of plants must be seen as a set of superimposed shields, each of which provides efficient protection to particular but not all types of pathogens.

Poster No.16

APOCYNIN: A POTENT, PLANT-DERIVED NADPH-OXIDASE INHIBITOR

E. van den Worm¹, C.J. Beukelman¹, A.J.J. van den Berg^l, B.H. Kroes’, R.P. Labadie^l and H. van Dijk ², ¹Medicinal Chemistry, Faculty of Pharmacy, Universiteit Utrecht, The Netherlands,  ²Eijkman-Winkler Institute for Microbiology, Infectious Diseases, and Inflammation, Faculty of Medicine, Universiteit Utrecht, The Netherlands. vanderworm@pharm.uu.nl

The production of reactive oxygen species (ROS) by activated human neutrophils plays an important role in many inflammatory diseases, in some cases resulting in excessive tissue damage.
Apocynin (4^’-hydroxy-3’-methoxy-acetophenone) was isolated in our laboratory in the late 1980’s by means of activity-guided isolation from the medicinal plant Picrorhiza kurroa. It proved to be a potent inhibitor of the ROS-generating NADPH-oxidase (IC50 mM). Additional interesting aspects of apocynin are its very low toxicity and the fact that it does not interfere with the killing capacities of neutrophils. These properties of apocynin make the compound a potential anti-inflammatory compound. Although its exact mode of action is not yet fully understood, some interesting experiments have been performed confirming its anti-inflammatory potential. Apocynin prevented colonic inflammation in acute and relapsing experimental colitis in rats and was found to inhibit inflammation-induced cartilage damage in human articular cartilage tissue. In these cases, ROS produced by the activated NADPH-oxidase, are thought to play an important role. Apocynin has also been reported to inhibit TNF-a release by both endotoxin- and peptidoglycan-stimulated human monocytes. Structure-activity relationship studies were used to get insight in apocynin’s mode of action. We are aiming at three goals: 1) elucidation of the mechanism of action, 2) improvement of activity by modifying the molecular structure and 3) modification of the activity-spectrum. Promising results obtained by modification of the molecule will be presented.

Poster No. 17

NADPH oxidase activation and assembly during phagocytosis

William M. Nauseef, Frank R. DeLeo, Lee-Ann H. Allen, Michael Apicella
Inflammation Program and Departments of Medicine and Microbiology, University of Iowa and Veterans Administration Medical Center, Iowa City, IA 200 Hawkins Dr. USA mailto:william-nauseef@uiowa.edu

Generation of superoxide (0₂) by the NADPH-dependent oxidase of polymorphonuclear leukocytes (PMNs) is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB) and compared it with that elicited by serum-opsonized zymosan (OPZ). NMB- and OPZ-dependent generation of reactive oxygen species (ROS) by PMNs peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of ROS by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal co-localization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). By contrast, termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane and/or phagosome-associated p47phox. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and POZ phagosome despite the continued presence of flavocytochrome b. These data suggest that: 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome, and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes without requisite dephosphorylation of membrane-associated p47phox.

Poster No.18

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.

Wim van ‘t Hof, Academic Centre for Dentistry Amsterdam (ACTA), Van der Boechorststraat 7, 1081 BT Amsterdam The Netherlands. W.van_t_hof.obc.acta@med.nl

The mechanism of antimicrobial action of cationic amphipathic peptides is generally believed to proceed through the formation of pores in the microbial cell membrane. This mechanism of action also explains the selective membrane activity of these peptides for prokaryotic over eukaryotic cell membranes. In contrast, the antimicrobial peptide histatin 5 from human saliva is highly active towards the eukaryotic yeast Candida albicans and much less active against simple prokaryotic bacteria. CD and 2D-NMR studies have shown that this peptide has little tendency to form an amphipathic helix. We found that this peptide, acts through a different mechanism.
C. albicans spheroplasts, obtained by enzymatic degradation of the cell wall, were as sensitive towards histatin 5 as intact blastoconidia. Histatin 5 permeabilized the C. albicans cell membrane, it promoted the uptake of the fluorescent dye propidium iodide. Studies with membrane potential-dependent markers showed that permeabilization of the membrane was only transient. Contrary to more amphipathic peptides, histatin 5 barely affected the membrane potential indicating that the membrane integrity is not lost and that leakage from the cells may be a secondary effect. Confocal laser scanning microscopy revealed that propidium iodide distribution showed a granular pattern, suggesting binding to mitochondrial rather than to nuclear DNA. The mitochondrial membrane potential was rapidly dissipated by addition of histatin 5 as monitored by the release of the mitochondrial membrane potential-dependent marker rhodamine 123. This specific targeting was corroborated by colocalization of FITC-labelled histatin 5 and a highly specific mitochondrial marker in double staining studies.
C. albicans can survive blocking of the mitochondrial respiration by switching to fermentation. In this case, the cytoplasmic membrane potential remains intact, whereas the mitochondrial membrane potential is significantly decreased. Inhibitors of mitochondrial respiration, culturing in anaerobic conditions, or elevated salt concentrations inhibited both the uptake of FITC-labelled histatin and the killing activity in a similar fashion. Our data indicate that histatin 5 is able to cross the cytoplasmic membrane of C. albicans without seriously disturbing the membrane integrity. Once translocated over the membrane, histatin 5 targets to the metabolically active mitochondria. Irreversible damage to the mitochondria subsequently leads to cell death.

Poster No. 19

Protection by Group II Phospholipase A2 Against Bacterial Infections

Veli J. 0. Laine*, David S. Grasst^†, and Timo J. Nevalainen*, *Department of Pathology, University of Turku and Turku University Hospital, 20520 Turku, vellai@utu.fi

Finland; ⁺Chrysalis DNX Transgenics, Princeton, New Jersey 08540, USA Group II phospholipase A2 (PLA2) is an enzyme that has marked antibacterial properties in vitro. In order to define the role of group II PLA2 in the defense against bacterial infection, we studied host responses in transgenic mice expressing human group II PLA2 and group II PLA2-deficient C57BL/6J mice in experimental E. coli and S. aureus infections. After the administration of bacteria, the transgenic mice showed increased expression of group II PLA2 mRNA in the liver and increased concentration of group II PLA2 in serum, whereas the PLA2-deficient mice completely lacked the PLA2-response. Expression of human group II PLA2 resulted in reduced mortality and improved the resistance of the mice by killing the bacteria as indicated by low numbers of live bacteria in their tissues. Human group II PLA2 was responsible for the bactericidal activity of transgenic mouse serum against S. aureus. These results suggest an important role for group II PLA2 in the innate immunity against bacterial infection.

Integration of Induced Resistance Responses in Plants

L.C. van Loon, Institute of Biology, Utrecht University, Sorbonnelaan 16, Utrecht, The Netherlands. L.C.vanloon@bio.uu.nl

Plants possess general (“non-host”) resistance against non-pathogens and pathogens from other species, cultivar- specific resistance against specific races of pathogens, and can develop induced (“acquired”) resistance as a result of prior limited infection with a pathogen. Expression of resistance may be associated with accumulation of antimicrobial phytoalexins and cell wall reinforcement around the site of pathogen ingress, as well as both local and systemic induction of pathogenesis-related proteins (PRs). The extent of elicitation and the rapidity and magnitude of the ensuing defense responses are considered to largely determine the expression of resistance.
Upon encounter with an avirulent pathogen, various signals are being generated which give rise to a coordinate induction of an integrated set of defenses, exemplified by the hypersensitive reaction (HR). The localized tissue necrosis characteristic of the HR is accompanied by an increase in the endogenous level of salicylic acid (SA), which potentiates local defense reactions and is required for the induction of PRs and systemic acquired resistance (SAR). Also increased are the production of jasmonic acid (JA) and ethylene, which can likewise induce or stimulate defense reactions. Tobacco plants transformed with a mutant etrI gene from Arabidopsis are insensitive to ethylene and retain resistance gene-mediated defense against tobacco mosaic virus (TMV), but are impaired in the systemic accumulation of PRs and show reduced SAR when challenged with TMV. Moreover, they have lost non-host resistance to naturally non-pathogenic soilborne fungi, such as Pythium sylvaticum. A systemic resistance induced in radish and Arabidopsis by nonpathogenic rhizobacteria (ISR) is not associated with the accumulation of PRs and independent of SA, but requires sensitivity to both JA and ethylene. Both SAR and ISR require functioning of the nprI gene, in contrast to a SA-independent and JA- and ethylene-dependent resistance of Arabidopsis to Alternaria brassicicola. Complemenarity of responses is indicated by combining SAR and ISR which leads to greater induced resistance than by each alone. These results suggest that different signals function in complementary and partly overlapping pathways to activate multiple resistance mechanisms in varying combinations.

Pathogen recognition, signal transduction, and gene expression changes mediated by the Pto disease resistance pathway in tomato.

Martin G, Bogdanove A, D’Ascenzo M, Frederick R, Gu Y, Halterman D, He X, Kantety R, Kim YJ, Loh YT, Lyman J, McCormack R, Riely B, Sessa G, Subrahmanyam. T, Thilmony R, Yang C. The Boyce Thompson Institute and the Department of Plant Pathology, Cornell University, Ithaca, NY, USA. gbm7@cornell.edu

We are studying the molecular basis of pathogen recognition, signal transduction, and gene expression changes involved in plant disease resistance. Towards this goal, we use various approaches including genetics, molecular biology, biochemistry, and more recently genomics, to examine the interaction between tomato and the causative agent of bacterial speck disease, Pseudomonas syringae pv. tomato. Resistance to bacterial speck disease in tomato is governed by a gene-for-gene interaction in which the tomato Pto gene determines recognition of bacterial strains expressing the avrPto avirulence gene.
We have previously shown that the molecular basis of recognition specificity in bacterial speck resistance is the physical interaction of the AvrPto and Pto proteins. By using chimeric Pto proteins and site-directed mutagenesis, we found that specific residues located in kinase subdomain VIII are required for Pto interaction with AvrPto and also for recognition specificity in a tobacco leaf transient assay.
We are also characterizing several classes of genes that encode Pto-interacting (Pti 1) proteins. Pti4/5/6 encode putative transcription factors that bind a cis element that is present in the promoter region of many genes encoding “pathogenesis-related” (PR) proteins. We are examining the transcriptional regulation of the Pti4/5/6 genes, the localization of their proteins in the plant cell, and the phosphorylation of the Pti4/5/6 proteins by Pto and the possible effects of this modification on their activity.
To examine genome-wide gene expression mediated by the Pto pathway and other pathogen-responsive pathways we have initiated a collaborative project to use cDNA microarrays. The cDNAs for these arrays are derived from libraries developed from tomato leaves after infection with diverse pathogens or treatment with various elicitors. 

Poster No. 20

INTERACTION OF PHYTOSTEROLS WITH THE COMPLEMENT SYSTEM

C.J. Beukelman¹, Henriette C. Quarles van Ufford¹, B.H. Kroes¹, A.J.J. van den Berg^l, P.C. Aerts², F.J.M. Beurskens² , R.P. Labadie^l & H. van Dijk^{1,2 1}Department of Medicinal Chemistry, Pharmacognosy section, Faculty of Pharmacy, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands and ²Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Faculty of Medicine, Utrecht University, the Netherlands. c.j.beukelman@pharm.nl

Since ages natural products are used for the production of medicinal preparations. Besides of local importance, these medicines may serve as valuable sources of most interesting (new) molecules. Phytosterols are ubiquitously present in the plant kingdom, in common food and, more in particular, in nutritional supplements. During activity-guided isolation, using complement-mediated hemolysis as readout system, we repeatedly identified a series of three hardly separable phytosterols b-Sitosterol, campesterol and stigmasterol) as potent anticomplementary principles. However, we did not aim at further purification of the individual components, since they become insoluble on isolation.
b-Sitosterol, one of the most prominent phytosterols, showed complement consumption resulting in IC50 values of about 65 mM. A tenfold increase of in vitro activity (IC50 from 65 mM to 5 mM) could be reached by the addition of escin (100 mg/ml), a solubilizing agent without intrinsic anticomplementary activity. In component-specific assay systems, identical activities were observed at the level of Cl and C4 inhibition (IC50 = ± 160 mM); activity at the level of C2 could be excluded. Preliminary in vivo experiments in Fl (BALB/c x Swiss) mice suggest an interaction with one of the classical pathway inhibitors. Extension of the in-vivo experiments and characterisation of the mode of action of phytosterols are subject of our current research.

Poster No. 21

Protective Effect of Plantago major L. Pectin Polysaccharide against Systemic Streptococcus pneumoniae Infection in Mice

Hetland G¹, Samuelsen A.B.², Løvik M.^1,3, Paulsen B.S.², Aaberge I.S.³, Grodeng E-C ^l,  and Michaelsen T.E.^2,3 Departments of ¹Environmental Medicine and ³ Vaccinology, National Institute of Public Health, Oslo, Norway, and ²Institute of Pharmacy, University of Oslo, Norway geir.hetland@folkehelsa.no

The putative anti-infection effect of a soluble pectin polysaccharide, PMII, extracted from the leaves of Plantago major, was examined in inbred NIH/OlaHsd mice experimentally infected with Streptococcus pneumoniae serotype 6B. Serotype 6B is known to result in a more protracted infection when injected intraperitoneally in susceptible mice than more virulent serotypes like type 4. PMII or LPS, which possibly contaminated the PMII preparation, was administered i.p. either once 3 days before challenge or once to thrice from 3 to 48 hours after challenge. The number of bacteria in blood and the mouse survival rate were recorded. Pre-challenge administration of PM11 or LPS showed a dose-dependent protection against S. pneumoniae type 6B infection. However, injection of the substances after establishment of the infection had no effect. The data demonstrate that the polysaccharide fraction PMII from P. major protects against pneumococcal infection in mice when administered systemically pre-challenge and that the protection is independent of that of a possible LPS contaminant.

Poster No. 22

Alterations in Functional State of Circulating Granulocytes in Association with Tumor Progression in Animal Model

S. Szûcs(a), M. Kávai(b), Cs. Varga(a), P. Kertai(a), Zs. Pocsai(a), Zs. Karányi(c) and R. Ádány(a),
 (a)Dept. of Hygiene and Epidemiology, (b)Third Dept. of Internal Medicine, (c)First Dept. of Internal Medicine, University Medical School, Debrecen, Hungary sszucs@jaguar.dote.hu

Although it is well established, that neutrophils can participate in the anti-tumor immunity, little is known about their functional state in tumor bearing hosts. Recently, insufficiencies in granulocyte oxidative burst have been described in patients with chronic neutrophilic leukemia, and a reduced superoxide anion (0₂ ^-) production by neutrophils in response to phorbol-myristate-acetate (PMA) have been reported in patients with oral and lung cancer. However, the changes in functional state of granulocytes attributed to malignant proliferation from the early phase to the advanced stages can be detected only in animal models. Therefore, the aim of our work was to monitore the PMNL, functions including 0₂ ^- production and phagocytosis in a rat model of mesoblastic nephroma (Ne/De). Mesoblastic nephroma cells were implanted under the renal capsule of Fischer 344 rats. Peripheral blood was taken from the animals and granulocytes were separate every second day after tumor cell implantation. Superoxide anion production in response to PMA and phagocytosis of yeast particles were measured. Both phagocytosis and PMA induced 0₂ ^- generation were found to be enhanced in the first period but they became significantly reduced in the advanced stage of cancer. The suppression of PMNL functions was accompanied with the tumor development and an increased number of granulocytes in the peripheral blood. Studies were also carried out on neutrophils isolated from normal rats and the cells were treated with plasma samples obtained from tumor-bearing animals at different stages of Ne/De. Incubation of the normal cells with plasmas separated on the 2^nd and 8^th days of tumor growth influenced neither the 0₂ ^- generation nor the phagocytosis. Plasma preparations obtained on the 14^th day significantly inhibited both 0₂ ^- production and phagocytosis by normal neutrophils. The alterations in 0₂ ^- production and phagocytosis by circulating neutrophils can be observed in close association with tumor growth, thus they may be considered as indicators of tumor progression.

Poster No. 23

Galectin-3, a new inflammatory mediator

¹Jenny Almkvist, ¹Elisabeth Feuk-Lagerstedt, ¹Claes Daffigren ²Hakon Leffler, and ¹Anna KarIsson. ¹Dept of Medical Microbiology and Immunology, Göteborg University and ²Department of Medical Microbiology, Lund University, Sweden. anna.karlsson@microbio.gu.se

Galectin-3 is a mammalian lectin with affinity for b-galactoside-containing glycoconjugates, preferentially poly-N-Acetyl-lactosaminoglycans. Several facts suggest that galectin-3 participates in inflammatory responses. It is produced by macrophages, mast cells and epithelial cells, and its level and rate of secretion is augmented by inflammatory mediators such as LPS and IFN-g. The extracellular galectin-3 may act by cross-linking glycoconjugates, mediating processes such as cell-cell adhesion, cellbacteria aggregation and cell-matrix interactions, thereby inducing intracellular signals and modulating cellular function. We have investigated the effect of galectin-3 on neutrophils regarding the ability to induce a respiratory burst, i.e., production of superoxide anion by the neutrophil NADPH-oxidase. This activity is of outmost importance for proper killing of infectious agents, but may also contribute to tissue damage during inflammatory processes. Our data show that primed neutrophils respond to galectin-3 by NADPH-oxidase activation. Such priming may be achieved by in vivo extravasation into an inflammatory site, interaction with bacterial LPS, or by pretreating the neutrophils with stimulating agents such as fMLP (in vitro priming). The molecular basis for the induction of galectin-3 responsiveness appears to be an increased exposure of galectin-3 receptors on the cell surface, caused by granule fusion with the plasma membrane. We have identified the galectin-3-receptor- storing granule to be the gelatinase granules, and CD66a and CD66b have been identified as potential galectin-3 receptor candidates. In conclusion, we suggest that galectin-3 perform activities which enhance or regulate the inflammatory process by interaction with primed neutrophils.

Poster No. 24

Human Mannan-Binding Protein Induces Aggregation and Superoxide Production of Human PolymorphonuClear Leukocytes

Kazuhide Uemura, Harumi Mori, and Toshisuke Kawasaki. Department of Biological Chemistry and CREST, JST, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan, mailto:uemura@pharm.kyoto-u.ac.jp

Mannan-binding protein (MBP, also called mannose-binding lectin, MBL) is a member of collectin family that participates in innate immunity. We have previously found that recombinant human MBP has anti-tumor activity to SW1116 cells inoculated on the back of athymic nude mice (Ma, et al., PNAS 1999, 96, 371-375). This anti-tumor activity of MBP appears to be complement-independent. Furthermore, MBP did not show any cytotoxicity in vitro by itself. These facts suggest that MBP kills tumor cells with the help of some kind of immune cell (MBP-dependent cell mediated toxicity, MDCC).
In order to clarify the mechanisms of MDCC, we have investigated the role of polymorphonuclear leukocytes (PMNs) in MDCC using polyvinyl-mannose coated plastic wells. Recombinant human MBP effectively bound to the wells in mannose- and EDTA-inhibitable manner. This ligand-bound form of MBP induced aggregation and superoxide production of PMNs isolated from human blood. The MBP-induced PMN aggregation and superoxide production were completely inhibited by pertussis toxin, suggesting the involvement of chemoattractant receptors. These results suggest that the presence of putative receptor for MBP, which induces chemoattractant and/or superoxide production of PMNs.

Poster No. 25

Genetic and Immunity in Resistance against Salmonella in Chicken

Carl-Henrik Brogren, Jeannette Dan Møller and Susana Alvarez Herrero Department of Microbiological Safety,. Institute of Food Safety and Toxicology, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark. chb@vfd.dk

Studies with 6 inbred lines of chicken have shown, that several genes are linked to resistance against Salmonella. Among these genes, the chicken analog of the NRAMP-1 macrophage expressed gene play an important role. NRAMP-1 code for a functional phagocytic factor especially important for Salmonella resistance in the early stage of life, before more MHC controlled genes take over and dominate by immune prevention. This early innate immunity have been studied in experimental experiment, where one week old chicks were challenged orally with live Salmonella typhimurium SL-1344.
The six lines consist of 3 lines (line 1, line 21 and line 131, from Foulum. Research Center, Denmark) with congenic sublines carrying the MHC haplotype B19/19 or B21/21. This experimental construction allows separation of the non-MHC gene effect from classical immune-associated MHC effect. Apart from differences observed in fecal bacterial secretion, also titers of Salmonella-LPS antibodies varies among the genetic line. A functional in vitro assay to measure the uptake of bioluminescent Salmonella-LUX or fluorescent Salmonella-GFP was established from cultures of intraperitonal macrophages. Bioimaging by intensified CCD-camera is used as a tool to visualize and measure macrophage uptake of bacteria.
In order to map the macrophage function to the NRAMP-1 genotype, several attempt to type the NRAMP-1 gene in the inbred lines of chickens have be validated. Based on PCR-amplified fragments of the gene both the single stranded conformational polymorphism (SSCP) technique and denaturing gradient gel electrophoresis (DGGE) were used.
In conclusion innate immunity through an early phase non-specific macrophages based phagocytic function play an important role in Salmonella resistance in chicken. This indicates, that selection of genetic background can be an important preventive factor. MHC-linked immunity also play an important role in the Salmonella resistance obtained by vaccination, naturally induced by oral route, but it might be enhanced by acquired immunity, e.g. based on DNA immunization, which is presently under investigation

  Poster No. 26

INNATE KILLERS OF MENINGOCOCCI: NEUTROPHIL AND COMPLEMENT SYSTEM

Platonov A.E. and Vershinina I.V. Central Institute of Epidemiology, Moscow, Russia id@glasnet.ru

The main host defense mechanisms against systemic bacterial infections, particularly, meningococcal disease (SMD) are well-known, namely complement system, specific antibodies, phagocytosis, but their relative contribution and the cooperation is less clear. Our own investigations and the analysis of publications suggest that the relative risk to contract SMD is about 5000 for late complement component deficient (LCCD) individuals, about 80 for individuals without specific meningococcal antibodies, and at least 2 for individuals with inefficient phagocytosis in comparison to the individuals with normal complement, high level of specific antibodies, and efficient phagocytosis, correspondingly. To mimic in vitro the conditions of bacteremia in meningococcal disease, we elaborated a model where different concentration of meningococci were incubated at 37⁰C in 90% human serum with or without human polymorphonuclear leukocytes (PMNLs) in physiological concentration. The number of viable bacteria and the hemolytic complement activity was measured at time intervals until 24 h. Several strains of serogroup A, B, W135 and uncapsulated meningococci were tested. If meningococci were incubated without PMNLs in serum samples obtained from patients with LCCD, this was the best growth medium for meningococci. After the addition of human PMNLs to meningococci in LCCD serum, the growth was partly inhibited or even reversed to bacterial elimination. The rate of bactericidal effect of PMNLs correlated with concentration of serogroupspecific IgM and, to some extent, IgG. No significant bactericidal effect was demonstrated by PMNLs in heat-inactivated serum samples in spite of the concentration of specific antibodies, suggesting that the action of antibodies was complement-mediated, possibly via interaction bacteria - Ig - C3 component - C3 receptor at PMNL, surface. Meningococci were killed hundred times more efficient when incubated, even without PMNLs, in human serum with normal complement activity, although this process depended on a number of bacteria, strain type and donor of serum. Thus the phagocytosis may be considered as the last defense reserve against complement-resistant meningococci, modulating the probability to contract SMD and its severity.

Poster No. 27

Abnormal functional and phagocytic activity of macrophages from bronchoalveolar lavage in rats when exposed to low g-irradiation

Tarasova E.E. ¹, Kilchevskaya E.V. ¹ and Kuzovkova N.A^2
1Clinical-Diagnostical Department, Mother and Child Care Institute, and ²Department of Ecological Immunology, Institute of Epidemiology and Microbiology, Minsk, Belarus Mlada7@nsys.by

We have studied functional activity of macrophages from bronchoalveolar lavage (BAL) in reaction of spontaneous and zymozan activated NBT-test and their phagocytic activity in phagocytosis reaction in experimental animals exposed to low ionizing radiation. Sex-immature rats (females) of herd breeding were exposed to single-dose g-radiation from device “IGUR 1” with the source ¹³⁷CS . Dose-rate radiation intensity was 5.4 cGr/min. Total dose of irradiation was 1.0 Gr. BAL samples were taken in 3, 10, 20 and 30 days after termination of g-irradiation.
In 3 days after termination of g-irradiation it was noticed significant decrease in total amount of cells in BAL in the rats and in 30 days their amount was 3 time less than that in intact rats.
In 3 days after finish of g-irradiation index of spontaneous NBT-test was in 3.5 time higher versus that in intact rats. At the same time index of zymozan activated NBT-test was elevated only by 1.7. As result, stimulation coefficient (the ratio between indices of spontaneous and zymozan activated NBT-test) was equal 2 in exposed rats, and it was equal 4 in intact rats. The indices under study remained constant till the end of experiment.
Already in 3 days after single-dose g-radiation of rats we have found both reliable decrease amount of active phagocytes and disorders of absorbant abilities of BAL macrophages. Parallelly abnormality of digestive ability was revealed. This abnormality also remained till the termination of experiment.
Thus, single dose g-radiation in 1 Gr causes fast rise of metabolic processes intensity in macrophages in experimental animals. At the same time, low-level stimulation coefficient indicates a decrease of macrophage defence abilities in response to intervention of bacterial or viral infection into a body, and disorders in their phagocytic activity may cause a persistent existence and growth of microbions in a body.

Session III: Collectins and Mannose Receptors.

Introduction to the collectins and C1q - opsonins involved in acquired and innate immunity.

Reid, K.B.M., Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. kbmreid@bioch.ox.ac.uk

The serum complement system protein Clq shows an overall similarity in structure to a subgroup of the C-type lectins, the ‘collectins’ - which includes the serum proteins, mannose binding lectin (MBL), conglutinin and CL-43 as well as the lung surfactant proteins SP-A and SP-D. Most of these proteins, as well as the serum ficolins, are composed of 4 to 6 subunits, each subunit having an Nterminal triple-helical collagen-like region and a C-terminal, trimeric, cluster of binding domains. Of these proteins, only Clq and MBL initiate activation of the complement system on binding to appropriate targets. Clq-mediated activation can proceed by innate routes (via, for example, its binding to C-reactive protein) or by acquired immunity (via its globular ‘heads’ binding to Fc regions of antibody IgG or IgM). The globular regions of Clq recognise peptide motifs in their targets and thus bring about the activation of the C1r and Cls proteases, whereas the C-type lectin domains of MBL bind to carbohydrate structures on pathogens and activate complement via the newly described MBL-associated proteases, MASP-1 and MASP-2. The C-type lectin domains of SP-A and SP-D also bind to carbohydrate structures on pathogenic microorganisms which allows these collectins to agglutinate their targets and mediate killing and clearance via phagocytic cells. The ficolins’ globular binding regions are composed of fibrinogen-like domains, which can also bind to carbohydrate structures on microorganisms and enhance their phagocytosis by polymorphonuelear leukocytes.
Two of the collectins (SP-A and MBL) and Clq have been shown to bind via their collagenlike regions to a putative receptor, ClqRp, found on neutroPhils and monocytes. A number of other putative receptors, specific for SP-A and SP-D, have also been described on macrophages. It is probable that Clq- and MBL-mediated opsonic effects are mainly the result of the coating of targets with activated complement components whereas SP-A, SP-D and the ficolins may rely primarily on the direct triggering of phagocytic cells.

Genetics of mannose-binding lectin.

Hans O. Madsen, Tissue Typing Laboratory, Department of Clinical Immunology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark. hom@rh.dk

Mannose-binding lectin (MBL) is a liver-produced, C-type serum lectin that plays an important role in innate immunity. Upon binding to certain carbohydrate moieties on various pathogens, MBL may mediate phagocytosis by receptors on phagocytes and use MBL serine proteases (MASP) - 1 and -2 to activate the MBL pathway of complement.
MBL is a multi-chain molecule of up to six subunits and each subunit consists of three identical polypeptide chains that contain a cysteine-rich region, a collageneous region, a “neck” region, and a carbohydrate binding domain.
MBL is encoded by two genes, but in man only one of the genes is functional. In man, MBL deficiency and low levels of serum MBL are strongly associated with the presence of variant MBL alleles that encode three different variants of the MBL polypeptide. Each variant contains a disruption of the collagen-like structure, which leads to a profound reduction of functional MBL in individuals that are heterozygous for variant alleles and to MBL deficiency in individuals homozygous for variant alleles. The variant alleles are highly frequent in normal, healthy populations, but the frequency varies in different ethnic groups, where the MBL variants are present in 20% up to 70% of the individuals.
In addition to the structurally variant alleles there exist other variants situated upstream of the gene, and they also influence the serum MBL level, thereby giving rise to a two-sided regulation of the MBL expression. These observations on the genetics of human MBL combined with studies on MBL genes in other primates provide the basis for proposing a model for the evolution of the MBL genes. 

Mannose-binding lectin in health and disease.

Malcolm W. Turner, Institute of Child Health, University College, Guilford Street, London U.K., m.turner@ich.ucl.ac.uk

Mannose-binding lectin (syn: mannan-binding lectin, MBL) is a collectin found in the serum of mammals and birds. This versatile macromolecule mimics many of the functional characteristics of IgM, IgG and Clq and is able to bind to the repeating sugar arrays on many microbial surfaces through multiple lectin domains. There is increasing evidence that the protein plays an important role in immune defence, particularly during the phase of primary contact with a microorganism. Not all organisms are equally susceptible and the presence of intact LOS structures appears to impair binding. Following binding the most significant effector function expressed by MBL appears to be complement activation through two serine proteases (MASP-1 and MASP-2). In contrast, the significance of direct interactions with collectin receptors on phagocytic cells remains to be established.
Serum levels of MBL are determined by a set of polymorphisms clustered in exon 1 of the gene and are further modulated by various promoter region polymorphisms. The exon 1 mutations lead to secondary structural abnormalities of the collagenous triple helix and a failure to form biologically functional higher order oligomers. There is an increased incidence of infections in individuals with such mutations, an increased susceptibility to recurrent miscarriage and an association with the autoimmune disorders SLE and rheumatoid arthritis. Furthermore, the rate of progression of some diseases appears to be accelerated in those with MBL mutations. Nevertheless, MBL genotyping of various populations has led to the suggestion that there may be some biological advantage associated with absence of the protein. These and other findings suggest that the concept of MBL as a protein involved solely in first line defence is an oversimplification and the protein should rather be viewed as having a range of activities including disease modulation.

  Ficolins

Misao Matsushita, Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan, mmatsu@cc.finu.ac.jp

F icolins were originally identified as tranforming growth factor-b1-binding proteins on porcine uterus membranes. They consist of subunits, each containing both collagen-like and fibrinogen-like domains, and form a hompolymer. So far, three forms of ficolins characterized by the presence of both domains have been identified in human. 1) Several research groups independently discovered a human serum protein homologous to porcine ficolins on the basis of the assays of their interest. Liver is the primary site of its synthesis. This ficolin (called P35, EBP-37, L-ficolin or hucolin) is an N-acetylglucosamine (G1cNAc)-, elastin- and corticosteroid-binding protein. Upon binding to the carbohydrates of Salmonella typhimurium, P35/EBP-37/L-ficolin/hucolin enhanced phagocytosis of the bacteria by neutrophils, suggesting an opsonin effect. 2) A ficolin called the Hakata antigen is also present in sera. The Hakata antigen showed lectin activity for G1cNAc. 3) There exists a ficolin (P35 -related protein, M-ficolin or ficolin-1) synthesized in lung and peripheral blood cells. P35-related protein/M-ficolin/ficolin-1 has been demonstrated to be expressed on certain monocytic cells. Although little is known regarding the physiological roles of three forms of human ficolins, it is possible that ficolins are collagenous lectins which form a family like collectins and are involved in innate immunity.

Poster No. 28

Lectin-dependent complement system in the solitary ascidian, Halocynthia roretzi

Fujita T, Sekine H, Matsushita M, Endo Y, Azumi K*, and Mizuochi T.
Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan. *Faculty of Pharmaceutical Science, Hokkaido University, Sapporo, Japan. Department of Applied Chemistry, Tokai University, Hiratsuka, Japan, tfujita@cc.fmu.ac.jp

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, we have isolated cDNA clones for ascidian MBL-like lectin and purified this lectin from the body fluid from a urochordate, the solitary ascidian, Halocynthia roretzi. Sequence analysis revealed that the C-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) which is homologous to a C-type lectin, but it lacks collagen-like domain as seen in the case of mammalian MBLs. SDS-PAGE of the purified lectin revealed a band of 36 kDa, which binds specifically to the glucose residue but not to mannose or N-acetylglucosamine. This lectin, designated glucose-binding lectin (GBL), associated with ascidian MASPa and MASPb. GBL-MASPs complex cleaves the ascidian C3 which has been recently shown to function as an opsonin. Namely, antibody against ascidian C3 inhibits the opsonic activity which enhances phagocytosis of yeast by ascidian blood cells. The removal of the ascidian GBL-MASPs complex using antibody against GBL inhibits both the binding of C3 to yeast and this opsonic activity. These results indicate that the ascidian complement system, consisting of the lectin-serine proteases complex and C3, functions in an opsonic manner. Thus, the complement system has played a pivotal role in innate immunity by recognizing pathogen and enhancing phagocytosis since before the establishment of acquired immunity.

Poster No. 29

Molecular cloning of a novel human collectin from liver (CL-L1)

Ohtani K., Suzuki Y., Eda S., Kawai T., Kase T., Keshi H., Sakai Y., Fukuoh A., Sakamoto T., Wakamiya N. Research Institute for Microbial Diseases, Osaka University, Osaka Prefectural Institute of Public Health, 3-1 Yamada-oka, Suita, Osaka, Japan, wakamiya@biken.osaka-u.ac.jp

Collectins are a C-lectin family having collagen-like sequences and carbohydrate recognition domains (CRD). These proteins can bind to carbohydrate antigens of microorganisms, and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway and opsonization by collectin receptors. Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1) which has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 bp coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern, Western blot and RT-PCR analyses show that CL-L1 is present mainly in liver as a cytosolic protein and slightly in placenta. More sensitive analysis by RT-PCRs shows most tissues except for skeletal muscle have mRNA. Zoo-blot analysis indicates that CL-L1 is limited to mammals and avian. The chromosome localization study indicates that CL-L1 gene localizes to Chromosome 8q23-q24.1 different from Chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in E.coli affirm that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan-columns. Analysis of the phylogenetic tree of CL-L1 and other collectins indicates that CL-L1 belongs to a fourth subfamily of collectins following the MBP, the surfactant protein A (SP-A), and the surfactant protein D (SP-D) subfamilies including bovine conglutinin and collectin-43 (CL-43). These findings indicate that CL-L1 may be involved in different biological functions.

Poster No. 30

Cloning of gp340, a putative opsonin receptor for lung surfactant protein D

Uffe Holmskov, Jan Mollenhauer#, Jens Madsen, Lars Vitved, Jørn Grønlund, Ida Tornøe, Anette Kliem, Kenneth B. M. Reid§, Annemarie Poustka# and Karsten Skjødt. Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark; #Division of Molecular Genome Analysis, Deutsche Krebsforschungszentrum, Heidelberg, Germany; §Department of Biochemistry, University of Oxford, UK. holmskov@imbmed.sdu.dk

Surfactant protein D (SP-D) is an oligomeric C-type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp340) has been shown to bind SP-D in the presence of calcium in a manner that is independent of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp340 has been established by molecular cloning, which yielded a 7686-bp cDNA sequence encoding a polypeptide chain of 2413 amino acids. The calculated M_r of gp340 is 258,069 Da and the sequence contains fourteen potential N-linked glycosylation sites. The domain organization features 13 SRCR (scavenger receptor cysteine-rich) domains, each separated by a SID (SRCR-interspersed domain), except for SRCRs 4 and 5 which are contiguous. The 13 SRCR domains are followed by a short Thr-rich region, two CUB domains separated by a 14^th SRCR domain and a zona pellucida (ZP) domain. The isolated cDNA clones for gp340 contain no sequence that codes for a transmembrane region. gp340 appears to be an alternatively spliced form of DMBT1, a molecule which lacks three SID and four SRCR domains found in gp 340. RT-PCR analysis showed that the main sites of synthesis of gp340 are lung, trachea, salivary gland, small intestine and stomach. Immunohistochemistry revealed high gp340 expression in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, while other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages SP-D and gp340 were located in the same cellular compartment. Immunoreactive gp340 was also found in epithelial cells of the small and the large intestine and in the ducts of salivary glands. The distribution of gp340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

Poster No. 31

Characterization of the interactions between mannan-binding lectin (MBL) and its associated proteases and comparison with the C1 complex

Steffen Thiel, Steen V. Petersen, Thomas Vorup-Jensen, Misao Matsushita, Teizo Fujita, Cordula M. Stover, Wilhelm J. Schwable and Jens C. Jensenius. Department of Medical Microbiology and Immunology, University of Aarhus, Denmark , Department of Biochemistry, Fukushima, Japan and Department of Microbiology and Immunology, University of Leicester, UK. st@microbiology.au.dk

Clq and mannan-binding lectin (MBL) are initiator molecules of the complement system. Clq binds to antibodies bound to antigens on the surface of microorganisms, whereas MBL directly binds to carbohydrate structures presented by microorganisms. When bound to ligand both Clq and MBL elicit activation of the complement cascade. The four plasma serine proteases C1r, C1s, MASP-1 and MASP-2 (MBL associated serine protease 1 and 2) exhibit the same modular structure and high similarity. There is controversy as to whether MBL can utilize C1r and Cls and vice versa if Clq can utilize MASP-1 and 2. Activation of complement on mannan was dependent on the presence of MBL whereas the activation on IgG required the presence of Clq in the serum tested. When serum deficient of C1r was employed no activation was seen on IgG-coated surfaces whereas activation was seen on mannan. Analysis of proteases associated with MBL and Clq in whole serum was performed by catching MBL, C1q, C1r, Cls or MASP-1 (together with associated molecules) with solid phase antibodies and then analyzing for bound serine proteases. C1r and C1 s were found associated only with C1q, whereas MASP-1 and MASP2 were found associated only with MBL. A third protein, MAp19 (MBL associated protein of 20 kDa) was also found associated with MBL. The interactions of MASP-1, MASP-2 and MAp19 with MBL was distinct from the interaction of C1r and Cls with Clq as high salt concentrations or the presence of calcium chelators could not dissociate the MASPs from MBL. A combination of high salt concentrations and calcium chelators did dissociate MASPs from MBL.

Lung-surfactant protein D

Erika C. Crouch, M.D., Ph.D., Washington Univ. School of Medicine at BJC-North 216 S. Kingshighway, St. Louis, MO 63110, USA crouch@path.wustl.edu

The lungs are subjected to a constant onslaught of potentially infectious agents and toxic particles. For this reason, the upper airways, bronchioles, and alveolar regions of the lung have evolved a complex and multilayered system of defense that includes locally-synthesized, and constitutively secreted defense molecules that accumulate near the air-tissue interface. The surfactant-associated proteins, SP-A and SP-D, are members of a family of collagenous C-type lectins, designated collectins. There is increasing evidence that these epithelial-derived proteins are important components of the innate immune response to microbial challenge and participate in immune and inflammatory regulation within the airspaces of the lung. SP-D is synthesized as tetramers of trimeric subunits. Each subunit consists of an amino-terminal crosslinking domain, an uninterrupted triple helical domain, and a high affinity, trimeric, carbohydrate recognition domain. This molecular organization facilitates bridging interactions between spatially segregated ligands on particles or cell surfaces. SP-D binds to conserved saccharide components integral to many bacterial, fungal, and viral cell walls, and to certain surfactant-associated lipids in vitro. Although binding may facilitate microbial clearance through aggregation or other direct effects on the organism, SP-D can also modulate the host defense functions of leukocytes, alter leukocyte interactions with biologically active bacterial products such as endotoxin, and regulate the proliferation of T-lymphocytes. A characterization of the binding determinants for lung collectins in vitro suggests that they may interact with organisms during distinct phases of the infectious process. Likewise, their interactions with leukocytes probably involve distinct or only partially overlapping binding mechanisms. Alterations in the levels of active proteins within the airspaces and distal airways may alter the metabolism of the airspace lining material, increase susceptibility to infection by specific microorganisms, modify the inflammatory response in the setting of lung infection or injury, and influence the development of clonal immunity.

USE OF GENE TARGETING FOR ANALYSIS OF SP-A STRUCTURE AND FUNCTION IN VIVO.

Jeffrey A. Whitsett, Ann Marie LeVine, James Fisher, Thomas R. Korfhagen. whitjo@chmcc.org

The SP-A gene was subjected to gene targeting, producing heterozygote SP-A (+/-), and homozygote SP-A (-/-) mice. SP-A gene targeted mice live and breed normally under barrier conditions. While SP-A (-/-) mice lack tubular myelin in alveolar spaces, lipid content, composition, clearance and surfactant function are not perturbed in the absence of SP-A.
SP-A deficient mice are susceptible lung infections by various pathogens. SP-A (-/-) mice are unable to opsonize or kill pathogenic bacteria, including Group B streptococcus, Pseudomonas, and Hemophilus. SP-A (-/-) mice are deficient in clearance of respiratory syncitial virus (RSV), and adenovirus, exhibiting increased inflammatory responses charactarized by increased expression of TNF-a, mip-2, and IL-6. Uptake of fluorescence adenovirus by alveolar macrophages is decreased in SP-A (-/-) mice. Treatment of SP-A (-/-) mice with exogenuos SP-A, completely corrects the abnormalities of clearance of both bacteria and virus, ameliorating the increased influx of PMNs, and decreasing cytokine mRNA and proteins during infection. Reduction and deglycosylation of SP-A failed to disrupt the anti-inflammatory and innate defense properties of human SP-A, supporting the likelihood that the collagenous domain and triple-helical structure of SP-A are not required for innate defense properties. Effects of SP-A on host defense and inflammation seen in response to bacterial pathogens were distinct from those seen in SP-D (-/-) mice, in which clearance of bacterial pathogens was unperturbed. SP-D gene targeted mice demonstrated marked abnormalities of lipid homeostasis, associated with increased alveolar pools and production of surfactant phospholipids. Ablation of SP-D in trasgenic mice caused severe pulmonary emphysema, demonstrating unexpected roles of SP-D in alveolar remodeling and lipid homeostasis. Gene targeting in transgenic animals clearly distinguished the functions of SP-A and SP-D in vivo. SP-A functions in innate defense, with activity against a variety of organisms, activating macrophage uptake and clearance, and enhancing killing mediated through oxygen radical production. SP-D regulates surfactant lipid concentrations in the alveolus, and does not appear to play a primary role in bacterial clearance in vivo.

Pattern recognition molecules in host defense.

R. Alan, B. Ezekowitz, Laboratory of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114. ezekowitz.alan@mgh.harvard.edu

The role of innate immunity is to restrict and limit infection. Many molecules and cellular processes conspire and act in concert to defend the host in the first minutes or hours after exposure to an infectious challenge. We have been interested in two mammalian molecules that may be considered as pattern recognition molecules in that they appear to distinguish the patterns of carbohydrates that adorn certain microorganisms selectively. The serum mannose-binding protein may be considered as an ante-antibody and acts like a broad spectrum polyvalent antibody. The macrophage mannose receptor by contrast is a membrane protein that mediates endocytosis and phagocytosis and appears to play a role in first line host defense. Furthermore, we have used Drosophila as a model system to identify putative primitive pattern recognition molecules. I will discuss our progress in these areas of investigation.

Poster No. 32

The association of variant Mannose-Binding Lectin genotypes with inflammation and outcome in rheumatoid arthritis.

Graudal N, Madsen HO, Tarp U, Svejgaard A, Jurik AG, Graudal HK, Garred P. The Tissue Typing Laboratory of the Department of Clinical Immunology, Copenhagen, University Hospital, Copenhagen, Denmark, and the Departments of Rheumatology and Radiology, Aarhus University Hospital, Aarhus, Denmark.  

Growing evidence indicates that innate immune defence molecules are involved in the pathogenesis of autoimmunity. Deficiency of mannose-binding lectin (MBL) has ben shown to be a weak predisposing factor for systemic lupus erythematosus. The present study was performed to investigate the possible association of MBL genotypes with the outcome of rheumatoid arthritis (RA).
Methods: MBL-genotypes and plasma concentrations were determined in 140 RA patients who were followed prospectively for up to 32 years after onset of RA.
Results: MBL deficient patients (two defective MBL alleles, or one defective allele combined with a low-expressive variant of the normal allele) had a significantly worse disease activity and outcome than patients with a significant MBL production. The relative risk of a severe radiographic event defined as 30% of maximal radiographic destruction was 2.8 (95% CI: 1.7-4.8) in the MBL insufficient group compared with the MBL competent group (p < 0.0001). This radiographic event ocurred in 50% of the MBL competent patients within 17 years, while it ocurred 9 years earlier in the MBL insufficient patients, i.e. within 8 years (within 17 years, p < 0.00004).
Interpretation: MBL insufficiency was a highly significant risk factor for poor outcome in RA. The perspective is that MBL may be a potential drug for the treatment of RA.

Poster No. 33

Molecular cloning of a human transmembrane molecule (M160) belonging to the scavenger receptor superfamily

Jørn Grønlund, Lars Vitved, Karsten Skjødt and Uffe Holmskov. Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark. j.groenlund@imbmed.ou.dk

A new member of the scavenger receptor cysteine rich (SRCR) superfamily was identified in the process of cloning another member of this protein family called gp340 from lung cDNA libraries. gp340 is a macrophage-associated molecule and a putative receptor for lung surfactant protein D. Other members of SRCR superfamily are located on T-cells, B-cells and macrophages and it has been suggested that they play a role in the immune defense system. The primary structure of M140 has been established by molecular cloning, which yielded a 4575-bp cDNA sequence encoding a polypeptide chain of 1451 amino acids. The calculated M_r of M160 is 158 kDa and the sequence contain 16 potential N-glycosylation sites. The domain organization features 12 SRCR domains of 110 amino acids. All the SRCR domains except SRCR 11 contain 8 cystine residues and belong to group B of the SRCR. SRCR 11 contains 6 cysteine residues and belongs to group A of the SRCR. The SRCR domains are followed by a transmembrane region of approximately 26 amino acids and a cytoplasmatic domain. The cytoplasmatic domain is found in two forms. A dominant form of 70 amino acids and an alternative spliced form of 40 amino acids. The dominant form contains four possible phosphorylation sites. These phosphorylation sites are all lost in the alternative spliced form. RT-PCR analysis on 19 different tissues showed that the main sites of synthesis of M160 are the spleen. Signals of varying intensity were found of the dominant form in all tissues, while the alternative spliced form was only found in spleen, thymus and the small intestine. M160 shows high homology to human M130 and to bovine WC1. M130 is a macrophage differentiation marker containing nine SRCR domains while WC1 mainly is expressed on T-cells and contains 11 SRCR domains. RT-PCR analyses have shown M160 to be expressed by alveolar macrophages and in the monocytic cell line U937 but not in COS cells and Raji cells.

Poster No. 34

Mammalian collectins: Structure and post-translational modifications.

Højrup P*, Leth-Larsen R*^†, Berg T, Mortensen H D* and Holmskov U^†. Molecular Biology* and Immunology and Microbiology, Institute of Medical Biology^† University of Southern Denmark, Odense University, Denmark. mailto:php@pr-group.ou.dk

The mammalian collectins are all based on a common framework of trimers of heavily posttranslationally modified polypeptide chains of similar structural organization. These trimers are then usually combined into higher oligomers.
In an effort to understand the ultrastructural organization, we have for some years been working on the complete structural elucidation of all five known collectins. Although the framework is similar, the post-translational modifications vary among the collectins:
Only two collectins, SP-A and SP-D, contains an N-linked carbohydrate. In SP-D this is situated in the collagen-like domain and is dominated by a bi-antennary sugar that is not capped by sialic acids. In SP-A a sialic acid capped N-linked bi-antennary structure is also found, but located in the carbohydrate recognition domain and much larger structural variants are also present. In all the collectins, the collagen-like domain contains both constant and heterogeneously modified proline (hydroxyproline) and lysine (hydroxylysine) residues. The hydroxylysine residues are further derivatized with a disaccharide structure (Lys-O-Gal-Glc). The lysine residues in SP-A are not hydroxylated.
The cysteine rich N-terminal region of the collectins is responsible for the organization of the ultrastructure (single arm, cruciform or bouquet). CL-43 has an unusual 1-1, 2-2, 1-2 disulphide bridging that results in a single arm structure. In SP-D the base linking unit seems to be six polypeptide chains, conglutinin seems to be held together by an unusual cysteine linked glycolipid while the exact linking of MBL and SP-A has not yet been determined.

Poster No. 35

Structural characterization of human and bovine surfactant protein D.

Leth-Larsen R*^†, Holmskov U^† and Højrup P* Molecular Biology* and Immunology and Microbiology, Institute of Medical Biology^† University of Southern Denmark, Odense University, mailto:Denmark.leth-bjerg@get2net.dk

Characterization of the primary structures of human and bovine SP-D show extensive post-translational modification. Mass spectrometric analysis of intact SP-D confirms this as the mass obtained is considerably higher than predicted from the amino acid sequence. Furthermore, the mass spectra show very wide peaks, both of SP-D subunits and reduced polypeptide chains, indicating that the nature of post-translational modifications is very heterogeneous. The great heterogeneity of the SP-D species is mainly caused by modifications in the collagen-like region, and the modified residues have been characterized by mass spectrometry and N-terminal sequencing to be either always modified or partially modified. The characteristics of modified residues are hydroxylation of proline and lysine residues placed in the Yaa-position of the Gly-Xaa-Yaa repeat. Lysines are usually further 0-glycosylated with galactose-glucose (Glucosyl-galactosyl-hydroxylysine). Human SP-D contains nine modified lysine residues and sixteen hydroxypro lines. Bovine SP-D contains seven modified lysine residues and eighteen hydroxyprolines. The partially modified residues seem to be approximately 50 % hydroxylated/glycosylated. We have further characterized an N-linked glycosylation to Asn 70 in human SP-D to be a complex type bi-antennery structure, but containing minor fractions of mono- and tri-antennery structures. The structure is partially modified with fucose and N-acetylglucosamine linked to the core. Quite surprisingly no sialic acids are present on the glycan. Like most of the modifications, the N-glycosylation is also partial.  

Poster No. 36

Localization of lung surfactant protein D (SP-D) on mucosal surfaces in human tissues and demonstration of SP-D in saliva

Jens Madsen, Anders Schlosser, Anette Kliem, Ida Tornøe, Claus Koch*, Karsten Skjødt and Uffe Holmskov, Department of Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense University, Odense, Denmark, and *Statens Seruminstitut, DK-2300, Copenhagen, Denmark. jensmadsen@hotmail.dk

Lung surfactant protein D (SP-D) is a collectin mainly produced by alveolar type II cells that initiates effector mechanisms of innate immunity by binding of microbial carbohydrates. SP-D mRNA was searched for in a panel of human tissues by reverse transcription-polymerase chain reaction (RT-PCR). Lung tissue was the main site of synthesis but a clear message was amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney and pancreas mRNA. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland and stomach. The sequence of SP-D derived from glandula parotic mRNA was identical to that of lung SP-D. Immunobistochemical analysis showed the presence of SP-D in alveolar type 11 Cells on and within alveolar macrophages, in epithelial cells of the large and small ducts of glandular parotis, sweat glands, glandular lacrimalis, in the epithelial cell of the gall bladder and in the small hepatic canaliculi and in the ducts of the exocrine part of pancreas. The presence of SP-D in saliva was demonstrated by quantitative ELISA, carbohydrate affinity chromatography and by Western blotting. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung.

Poster No. 37

Interactions of rat surfactant protein A and D with Candida albicans

Bianca A.W.M. van Rozendaal^l, Annemiek B. van Spriel², Jan G.J. van de Winkel² and Henk P. Haagsman ^1,3. ¹Department of Biochemistry and Cell Biology, ²Department of Immunology, and ¹Department of the Science of Food of Animal Origin, Utrecht University, Utrecht, The Netherlands. Rvanrozendaal@vet.uu.nl

Pulmonary surfactant proteins A and D (SP-A and SP-D), members of the collectin family, are implicated in innate host defense of the lung against pathogens. Interactions of SP-A and SP-D with bacteria and viruses have been described. Much less is known about their interaction with fungi. We have studied the interactions of SP-A and SP-D with Candida albicans. To study the interactions of SP-A and SP-D with Candida albicans, antibodies against rat SP-A and SP-D were used to detect the binding to Candida albicans, and the binding was quantified by flow cytometry. We observed that SP-D binds to Candida albicans in the presence of calcium ions, agglutinating them into large complexes. Binding was inhibited in the presence of EDTA or competing sugars (maltose and mannose). In contrast, we found no SP-A binding to Candida albicans. Furthermore, incubation of Candida albicans with SP-D resulted in decreased phagocytosis of Candida albicans by alveolar macrophages, probably due to the size of the agglutinated Candida albicans. We conclude that the lung collectin SP-D might play an important role in the first-line defense against fungi in the lung, by agglutinating these fungi.

Poster No. 38

Functional Studies of the Peptidoglycan Recognition Protein

Kang D., Liu G., Lundström A., Gelius E. and Steiner H. Department of Microbiology, Stockholm University, Stockholm, Sweden. eva@mibi.su.se

The Peptidoglycan Recognition Protein (PGRP) gene is strongly expressed after a bacterial infection in Trichoplusia ni. The cDNA has been cloned from T ni as well as from mouse and humans. Deduced protein sequence comparison of both murine and human PGRP show that they share 43% identity with T ni PGRP. Recombinant T. ni PGRP, as well as recombinant murine PGRP, binds insoluble peptidoglycan from Micrococcus luteus. Thus the function is conserved from insects to humans. We have also demonstrated that the protein binds strongly to live Micrococcus luteus, but not to Escherichia coli. Recombinant PGRP has not shown any bactericidal activity neither to gram positive nor to gram negative bacteria. However, growth of low numbers of some Staphylococcal strains was inhibited by PGRP, whereas growth of Escherichia coli was not affected.

 Poster No. 39

Serum collectins in domestic food animals. A review of the proteins, their genes, biological functions, regulation and variation

Ingvartsen, KL, Hansen, TK, Holmskov, U^§, Lovendahl, P and Madsen, HJ, Danish Instute of Agricultural Sciences, Research Centre Foulum, Denmark. ^§Institute of Medical Biology, Odense University, Denmark.  KlausL.Ingvartsen@agrsci.dk  

The objective of the present review is to give a status concerning important infectious diseases in dairy cattle, pigs and poultry and to review the group of serum collectins, their genes, biological functions, regulation, variation and their potential involvement in infectious disease resistance in livestock animals.
Infectious diseases are of major concern in the cattle, swine and poultry industries. Infectious diseases are very cost-intensive, and in addition they compromise the welfare of the animals and cause excessive use of antibiotics. The extensive use of antibiotics has been criticised because of the risk of development of antibiotic-resistant zoonotic organisms and the residual antibiotics in food animal products. Intensive breeding programs focusing solely on increased production and efficiency have been suggested to have adverse effects on infectious disease defence mechanisms. This may, at least in part, be due to depression of the innate immune system that serves as the first line of defence and plays a key role in the host’s surveillance against infectious challenges. Collectins are now believed to be important constituents of the innate immune system. The collectins bind to a wide range of bacteria, fungi, virus and parasite antigens in humans and laboratory animals where they participate in the killing and clearance of the targets. Collectins may play a similar important role in livestock. Mannan-binding lectin (MBL) is found both in cattle, pigs and chicken while conglutinin and CL-43 have been found only in cattle. In periparturient dairy cattle, conglutinin appears to be reduced and recently, a strong negative genetic correlation between plasma conglutinin levels and the incidence of respiratory diseases has been found in calves. In pigs and chickens, MBL have been characterised and more isoforms has been detected. However, little is known about the effects of collectins on disease resistance in farm animals. A conceptual framework is proposed for the involvement of the innate immune system in the susceptibility to infections in domestic food animals and how it may interact with genotype, nutrition, metabolism and the endocrine system.

Poster No. 40

Mannan-binding lectin in serum affects the replication of influenza A virus in animal lungs

Kase T, Suzuki Y, Eda S, Kawai T, Maeda, A, Okuno, Y, Ohtani K, Sakamoto T, Wakamiya N, Osaka Prefectural Institute of Public Health, Research Institute for Microbial Diseases, Osaka University, 1-3-69 Higashinari, Osaka, Osaka, Japan, kase@iph.pref.osaka.jp

Mannan binding lectin (MBL) is thought to play important roles in the innate immunity of animals. We have reported that human MBL was able to directly inhibit the infection with influenza A virus without complement in vitro (Immunology in press). Human MBL was able to neutralize the infectivity of influenza A viruses by binding to hemagglutinin (HA) and neuraminidase (NA) molecules and to prevent viral spreading to contiguous cells by interfering budding process or viral release. However, the antiinfluenza viral activity of MBL in vivo is still unknown. To determine the function of MBL in vivo, we tested the susceptibility of mice and hamster to influenza A viruses. A/Ibaraki/1/90 (H3N2) virus of fresh isolated virus which is lectin sensitive, can proliferate in the hamsters lung, but cannot do in Balb/c and SCID mice lung. A/Adachi/2/57 (H2N2) virus which is lectin insensitive, can replicate in lungs of both hamsters and Balb/c mice. We hypothesized that the difference of susceptibility to influenza A viruses between hamsters and mice was based on MBL in the serum. The mice sera was able to neutralize A/Ibaraki/1/90 virus but not A/Adachi2/57 virus, while hamsters sera has no ability to neutralize against both viruses. We measured the concentrations of mannan binding lectins isolated in animals sera. The mice sera had 15 mg/ml of MBL, while that of hamsters was 1.2 mg/ml. We found that purified mice MBL added the neutralizing activity to the raw hamster sera. These results showed that lectin insensitive virus could grow in all animal lungs, but lectin sensitive virus could proliferate only in the low concentration of serum MBL. Our findings suggest that the MBL in the serum would affect the replication of influenza A virus in vivo.

Poster No. 41

An opsonin with mannan-binding lectin (MBL)-like activity in the Atlantic salmon, Salmo salar.

Gunnlaugsdóttir B, Gudmundsdóttir S, Arason GJ* Fish Disease Laboratory, Keldur Institute for Experimental Pathology, University of Iceland, and *Department of Immunology, National University Hospital, Reykjavik, Iceland garason@rsp.is  

Mannan-binding lectin (MBL) is a calcium-dependent serum lectin with opsonic activity towards Saccharomyces cerevisiae and several types of bacteria. A molecule with functional resemblance to MBL has been found in mammals down to marsupials. In an attempt to extend these studies to more primitive species, we searched for sugar-specific proteins with opsonic and/or agglutinating properties in the Atlantic Salmon, Salmo salar. We report that (1) salmon serum restores opsonic activity of a MBL-deficient human serum to 50-80% of normal levels; this ability is lost/greatly reduced upon heat-inactivation of the human/salmon component. (2) Full opsonization is achieved with salmon serum when human PMNs are replaced by salmon phagocytes; this activity is heat-sensitive. (3) Agglutination of S. cerevisiae or zymosan is achieved in about 50% of sera taken from fingerling group, with titers ranging up to 1:640. Titers as well as the proportion of positive sera appear lower in broodfish populations. (4) Agglutination of S. cerevisiae is inhibited by mannose or glucose but not galactose. When purified (and relatively mannan-depleted) zymosan is used, glucose is more effective for inhibition. (5) EDTA does not inhibit agglutination. (6) The agglutinin may be precipitated with ammonium sulphate and removed by absorbtion with zymosan followed by glucose elution. (7) Partial purification has been achieved by absorbing serum on a column with mannan-Sepharose and eluting with 10 mM EDTA. The most prominent molecule of the resulting fraction migrates in unreduced form as a large (>200 kDa) molecule. This band disappears after reduction, leaving major bands corresponding to ca. 30-32 kDa and < 14.3 kDa. Minor bands are also seen, before and after, reduction.
The above properties, i.e. multivalent, sugar-specific absorbtion onto microbial cell walls, indicate a mannose/glucose specific lectin. Fractionation results suggest the presence of a large protein composed of 32 kDa polypeptide chains linked together by disulphide bonds. The results of opsonic and agglutinating studies suggest resemblance to mammalian MBL; bivalent cations are required for binding to mannan-coupled sepharose but apparently not to zymosan.

Poster No. 42

The Y and X variants of the MBL promoter are distinguished by the ability to bind a nuclear protein.

Lars Nørgaard, Hans 0. Madsen, Arne Svejgaard and Peter Garred, Tissue Typing Laboratory, Department of Clinical Immunology, Rigshospitalet, Denmark.  larsnorgaard@hotmail.com

Human mannose-binding lectin (MBL) is a liver-derived C-type serum lectin involved in innate immunity. On binding to specific carbohydrate structures on various microorganisms, MBL may mediate phagocytosis and utilise MBL associated serine proteases (MASPs) to activate the complement system by the MBL pathway.
Previous studies have identified a number of polymorphic positions in both the structural and regulatory part of the MBL gene. In particular, two G/C polymorphisms situated in positions -550 (the H/L variants) and -221 (the Y/X variants) of the MBL promoter have been demonstrated to have a profound effect on the MBL serum concentration. Based on these observations, the HY, LY and LX haplotypes have been identified as high, intermediate and low producing MBL promoters, respectively. Using a gene reporter assay the effect of the G/C polymorphism in position -221 was confirmed, i.e. the Y variant was recognised as a high producing promoter variant compared to the X variant. However, the differences found were not as pronounced as those observed in vivo, indicating that other levels of regulation are involved. Furthermore, we have applied the electrophoretic mobility shift assay (EMSA) in order to identify regions of the MBL promoter capable of interacting with nuclear proteins. The results showed that a small DNA fragment covering the positions around the G/C polymorphism of the Y variant was able to bind two nuclear proteins purified from HepG2 cells, a hepatoma cell line. By substituting the G in position -221 with a C, i.e. creating the X variant, the binding to one of these proteins was substantially diminished.
These findings indicate that low MBL serum concentrations associated with the X variant of the MBL promoter are caused by a diminished ability to bind a nuclear factor.

Poster No. 43

Recombinant MASP-2 is self-activating and cleaves complement factor 4 (C4)

Thomas Vorup-Jensen, S. V. Petersen, S. Thiel & J.C. Jensenius. Dept. of Med. Microbiology & Immunology, University of Aarhus, Denmark vorup@biobase.dk

Recombinant synthesis of the two mannan-binding lectin (MBL) associated serine proteases (MASP-1 and MASP-2) requires eukaryotic processing of disulfide bridges and potentially chaperone-mediated folding in order to obtain functionally active proteins. We have expressed MASP-2 and MBL in mammalian cell lines under serum-free conditions. Recombinant MASP-2, incubated with either recombinant MBL or native MBL (depleted of MASP by acid elution), causes C4b deposition in mannan-coated microtitre wells. Also, conversion of proenzymatic rMASP-2 into the enzymatic state occurs in an MBL-dependent manner on carbohydrate-coated surfaces while there is no evidence of other proteins being part of this process. Thus, MASP-2 may be a self-activating as well as a C4 cleaving component of the MBL/MASP complex. The structural resemblance between MBL and C1q, the non-enzymatic part of the Cl complex, together with the shared domain organisation of the MASPs and the two Cl serine proteases C1r and Cls, suggested the activation sequel of the MBL/MASP complex to be similar to that of the Cl complex, i.e., with MASP-1 activating MASP-2 in analogy with C1r activating the C4 cleaving Cls. Our findings, however, point to the MBL pathway requiring only MASP-2 and not MASP-1 for activation of C4 and C2.  

Poster No. 44

GENERATION OF ANTIBODIES TOWARDS MASP-1 AND MASP-2

Steen Vang Petersen, Poulsen, K., Stover, C.M., Koch, C., Vorup-Jensen, T., Thiel S., and Jensenius J.C. Department of Medical Microbiology and Immunology, University of Aarhus, Denmark [svp@microbiology.au.dk] .Department of Microbiology and Immunology, University of Leicester, UK. Statens Serum Institut, Copenhagen, Denmark.

Mannan-binding lectin in association with mannan-binding lectin associated serine protease-1 and -2 (MASP-1 and MASP-2) can activate the complement system upon binding to carbohydrate structures on the surface of bacteria. We want to raise antibodies against the MASPs to assist resolving the unanswered questions about structural, and functional aspects of the MBLectin pathway of complement activation. Lacking homogeneous purified native protein we initiated the expression of MASP-2 and MASP-1 in E. coli.
MASP-2 was produced as a fusion protein with thioredoxin, and purified by affinity chromatography on a matrix coupled with 4-aminophenylarsine oxide utilising the affinity of thioredoxin for arsenic. Animals were immunised with the fusion protein and antisera tested by Western blotting and time resolved immunofluorometric assay (TRIFMA) for reactivity against MBL/MASP purified from human plasma. Polyclonal antisera from rabbits, chickens, rats and mice were obtained. A mouse x mouse fusion resulted in three monoclonal antibodies reacting with denatured MASP-2 in a TRIFMA assay and/or blot.
Full length MASP-1 was produced with a C-terminal fusion of His₆. Animals immunised with purified recombinat protein gave no response towards native protein as assessed by Western blotting and TRIFMA.
As immunisation with proteins expressed in E. coli did not result in antibodies with reactivity against native proteins we decided to use partially purified MASP as antigen. All animals immunised responded to the immunisation. A mouse x mouse fusion was made and from this we obtained several antibodies with reactivity towards native MASP.

Poster No. 45

Mechanisms of Modulation of Mannose Receptor Expression on Macrophages and Dendritic Cells by Immune Mediators.

Brian S. Egan and Virginia L. Shepherd, Departments of Biochemistry and Medicine, Vanderbilt University, Nashville, TN. virginia.l.shepherd@vanderbilt.edu

Studies from a number of laboratories have demonstrated that tissue macrophages express a receptor on their surface - the mannose receptor (MR) - which mediates the phagocytosis of a variety of pathogens and endocytosis of extracellular peroxidases and hydrolases. Recent work has demonstrated that immature dendritic cells also express this receptor, and that MR function in this cell type appears to involve efficient capture of antigens. Expression of the MR is absent on circulating monocytes. Treatment of these cells with granulocyte/macrophage colony-stimulating factor (GM-CSF) promotes MR-positive macrophage differentiation, while culture in the presence of GM-CSF plus interleukin-(IL)-4 produces MR-positive immature dendritic cells. MR expression on both of these differentiated cell types is tightly regulated by exposure to immune mediators. Treatment of macrophages with anti-inflammatory agents such as dexamethasone (Dex) or IL-4 up-regulates MR expression, while treatment with pro-inflammatory agents such as interferon-gamma (IFN-gamma) decreases MR levels. Exposure of dendritic cells to proinflammatory agents including IFN-gamma, lipopolysaccharide (LPS), or tumor necrosis factor-alpha (TNF) results in decreased MR expression. In the present study, we have examined potential mechanisms involved in MR regulation on these two cell types using a rat alveolar macrophage cell line (NR8383) and a murine immature dendritic cell line (FSDC). Treatment of NR8383 cells with Dex resulted in a 2.5-fold increase in MR ligand binding activity, MR protein, and MR mRNA. mRNA stability was not altered by Dex treatment, while the rate of MR transcription was increased by 2.5-fold, suggesting that Dex alters MR expression on macrophages at the level of transcription. Treatment of FSDC cells with IL-4 increased MR activity and protein by 2.5-fold, while TNF, LPS, and IFN-gamma all decreased MR ligand binding and protein expression. Further studies are underway to examine the control of MR promoter activity in these cell lines. (Supported by NIH grant HL55977)

Poster No. 46

cDNA cloning, mRNA Expression and Carbohydrate Analysis of Porcine Surfactant Protein D

¹Van Eijk M, ²Haagsman H.P., ¹Van de Lest C.H.A., ³Reid K.B.M. and ³Lawson P.R. ¹Department of Biochemistry, Cell Biology & Histology and ²Department of the Science of Food of Animal Origin Utrecht University, Utrecht, The Netherlands, ³MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, England. m.vaneijk@vet.uu.nl

Lung surfactant protein D (SP-D) belongs to a subgroup of mammalian lectins known as the collectins. These proteins appear to play an important role in innate non-clonal defence due to their ability to bind a variety of pathogens, to influence macrophage activity and, to regulate inflammation. This host defence system is of importance in pulmonary defense since inhaled pathogens are a constant threat for the cause of respiratory diseases. In modern swine production pulmonary diseases are regarded as the most serious disease. As a first step towards understanding the role of SP-D in porcine pulmonary defense, the primary structure of porcine SP-D was analyzed by cDNA cloning. The derived amino acid sequence shows that the mature protein is 358 residues long being 76% identical to the human and 73% identical to the mouse protein. One of the distinctive features found in the sequence of porcine SP-D as compared to other cloned species, was the presence of a potential N-glycosylation site in the carbohydrate recognition domain (CRD). Enzymatic treatment and sugar analysis of mannan affinity purified native protein from lung lavage confirmed that the lectin domain is N-glycosylated, contributing to the larger mass of the porcine SP-D monomer being 52 kDa in contrast to other SP-D species (43 kDa). Mono-Q anion exchange chromatography on native SP-D showed the presence of a variety of porcine SP-D isoforms, characterized by differences in the sialic acid content of N-linked moieties of the differently charged mono-isomers. These properties of porcine SP-D could have implications by enhancing the binding potency of SP-D to a large variety of pathogens. In order to study the expression of SP-D in non-Pulmonary tissues, Northern blot analysis was performed on RNA extracts obtained from a wide range of porcine tissues using, a radiolabelled DNA probe that codes for the CRD of porcine SP-D. The presence of a 1.3 kb signal in lung but also in, duodenum, jejunum, ileum and ileal mucosa further supports the function of SP-D as a host defence molecule in mucosal surfaces other than the lung.

Poster No. 47

Microfibril-associated protein 4 (MFAP4) is present in lung washings and binds to the collagen region of lung surfactant protein D

Mette Lausen, Nicholas Lynch¹, Anders Schlosser, Ida Tornøe, Børge Teisner, Antony C. Willis², Erika Crouch*, Wilhelm Schwaeble¹ and Uffe Holmskov.
Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense University, Denmark; the *Department of Pathology, Washington University, St. Louis, MO, USA. the ¹Department of Microbiology and Immunology, University of Leicester, Leicester, UK and the ²Department of Biochemistry, University of Oxford, Oxford, UK. holmskov@imbmed.ou.dk

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-PAGE the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa, indicating that the protein exists as a 250 kDa oligomer of 66 kDa dimeric subunits. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Calcium-dependent binding was demonstrated between bMFAP4 and lung surfactant protein SP-D. No binding was seen if the SP-D were digested with collagenase or to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D indicating that the interaction between bMFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 binds equally well to recombinant wildtype SP-D and to a recombinant form of SP-D where the N-glycosylation. site at position 70 in the collagen region of SP-D is missing. bMFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. hMFAP4 was cloned and the coding region spanned 255 amino acids. An N-terminal region of 16 amino acids containing an Arg-Gly-Asp sequence and one cysteine residue is followed by a single fibrinogen-like domain of 239 amino acids showing high homology to fibrinogen domains found in ficolins. One potential calcium binding is found in the fibrinogen domain. Recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. Our findings indicate that MFAP4 may have two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.

Poster No. 48

Genomic characterization of collectin 43 (CL-43)

Søren Hansen, Dorte Svendsen, Lars Vitved, Karsten Skjødt and Uffe Holmskov. Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark. shansen@imbmed.ou.dk

CL-43 is a member of the collectin family together with conglutinin, mannan-binding lectin (MBL), lung surfactant protein A and D (SP-A and SP-D). CL-43 and conglutinin have only been found in bovidae, while the other members are characterized in many other species. CL-43 is a serum protein and like the rest of the collectins CL-43 binds glycoconjugates on the surface of microorganisms thereby inducing effector mechanisms like aggregation and probably also opsonization for phagocytosis. CL-43 is composed of three identical polypeptide chains linked together through two N-terminal cysteine residues. The N-terminal region is followed by a collagen region, an a-helical coiled-coil neck region and a C-terminal calcium dependent carbohydrate recognition domain.

We have previously characterized the cDNA coding for CL-43. We now describe the partial characterization of gene for CL-43 and present evidence for a CL-43 gene with a coding sequence spanning approximately 8 kilobases. The genomic sequencing demonstrated that CL-43 is composed of six translated exons. The signal peptide/N-terminal segment of 138 bp, the neck-region of 84 bp and the carbohydrate recognition domain of 384 bp are each encoded by a single exon. This organization is also found in the other collectins. The collagen region is encoded by three exons of 108bp, 72bp and 117bp, respectively and CL-43 thus. lacks an exon corresponding to the second translated exon of SP-D and conglutinin. The structure of the CL-43 gene suggests that CL-43 together with conglutinin arose by partial duplications of an ancestral SP-D gene after the divergence of the bovidae from other mammals.

Characterization of the promotor region of CL-43 is in progress and these data will show how CL-43 is regulated. Combined with measurements of the concentration of CL-43 in plasma, the genomic structure will allow for genetic analysis of possible polymorphism of the CL-43 gene.

Session IV: General Topics.

Transmembrane pore formation: an overview of the concept and its unexpected involvement in the pathogenesis of atherosclerosis.

Sucharit Bhakdi, Institute of Medical Microbiology and Hygiene, Augustusplatz, D-55101 Mainz, Germany. mailto:makowiec@mail.uni-mainz.de

The concept that mammalian cells can be damaged by pore-forming proteins originated from work in the complement field, when the terminal C5b-9 complex was isolated and characterized as a channel-structured protein oligomer (1). The discovery followed that certain microbial toxins analogously inflict damage to the host organism by inserting pore-forming toxin molecules into target membranes (2). Pore-formers exert their detrimental effects not only via their cytocidal action, but also by deregulating target cell function. Thus, membrane damaging effectors of the immune system and of invading microorganisms can both provoke local and systemic pathological sequelae (3). Investigations into the mechanism of pore formation have culminated in the elucidation of the molecular structure of the staphylococcal alpha-toxin pore (4), and in the delineation of individual steps underlying assembly of the functional oligomer (5).
Our work on complement has unexpectedly led to the formulation of a novel hypothesis on the pathogenesis of atherosclerosis. When LDL is stranded in the subendothelium, it is enzymatically degraded (but not oxidized) to yield a particle that binds C-reactive protein, activates complement and induces macrophage foam cell formation and cytokine release. These processes are initially meaningful because they enable the stranded lipoprotein to be removed from the vessel wall, but they become harmful when the cholesterol removal system is overloaded. Thus, atherosclerosis is a novel type of immunological disease that is sustained through overactivation of complement and macrophages by an altered autologous molecule, whereby the terminal complement sequence assumes a hitherto unrecognized role in promoting pathology (6).
(1) Bhakdi, S. and Tranum-Jensen, J. 1978. PNAS 75: 5655. (2) Bhakdi, S., and Tranum-Jensen, J. 1987. Rev. Physiol. Biochem. Pharmacol. 107: 147. (3) Bhakdi et al. 1996. CTMI 216: 101. (4) Song et al. 1996. Science 274: 1859. (5) Bhakdi, S. et al. 1996. Arch. Microbiol. 165: 73. (6) Bhakdi, S. 1998. Ann. Med. 30: 503.

The role of nitric oxide in innate immunity

Bogdan C, Schindler A, Röllinghoff M, and Diefenbach A, Institute of Clinical Microbiology, Immunology and Hygiene, University of Erlangen,, Wasserturmstrasse 3, D-91054 Erlangen, Germany. mailto:christian.bogdan@mikrobio.med.uni-erlangen.de

Macrophages and neutrophils, but also fibroblasts, endothelial cells and epithelial cells generate nitric oxide from the aminoacid L-arginine, when stimulated with cytokines or certain microbial products such as lipopolysaccharide. The enzyme responsible for the production of NO in this context is the type 2 or inducible nitric oxide synthase (NOS2 or iNOS) that is (up)regulated by both transcriptional and posttranscriptional mechanisms. The established main functions of NO/NOS2 in the immune system are the control of intracellular pathogens and the regulation of proliferation, survival and cytokine production of lymphocytes. Because interferon (IFN)-g, a cytokine produced by type 1T-helper cells, natural killer cells or macrophages in response to interleukin (IL)-12 and IL-18, has been established as the key inducer of NOS2, and most microbes suppress rather than promote the expression of NOS2, the production of NO by NOS2 has been thought to be a component of the adaptive immune response, but not an event of the first line defense. In this lecture, evidence will be presented that NOS2-derived NO forms an integral part of the innate immune system. Using the mouse model of cutaneous leishmaniosis, we could demonstrate that during the first 24 hours of infection (a) NOS2 is expressed by macrophages, (b) type 1 interferon (IFNa/b) rather than IFN-g is the inducer of NOS2, and that (c) NOS2-derived NO is a prerequisite for the activation of NK cytotoxicity and the rapid upregulation of IFN-g production by NK cells and thereby prevents the early dissemination of the parasite from the skin to visceral organs. A further molecular analysis revealed that NOS2-derived NO is required as a cofactor for the IL-12-induced activation of Tyk2 kinase and the subsequent phosphorylation of the Stat4 transcription factor in NK cells. Thus, NO is a critical regulator of IL-12 responsiveness and cytokine production in innate immunity.

Bacterial defence strategies against host antimicrobial products

Eduardo A. Groisman, Howard Hughes Medical Institute/Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, United States. groisman@borcim.wustl.edu

The production of antimicrobial peptides is a prevalent host defense strategy used by a wide variety of animal and plant species. Microorganisms, having coexisted with their hosts for millions of years, have evolved strategies that enable them to avoid or withstand the various microbicidal activities of their hosts. The insect pathogens Serratia marscecens and Bacillus thuringiensis are resistant to cecropins, a family of antimicrobial peptides present in the hemolymph of the Cecropia moth and other insects. Our studies of the Gram-negative pathogen Salmonella enterica have uncovered several peptide resistance determinants which allow Salmonella to colonize host tissues that are rich in antimicrobial peptides. Some of these determinants alter the surface properties of the microbe, making its lipopolysaccharide less anionic by substituting the ester-linked phosphate groups with 4-aminoarabinose and ethanolamine. Other determinants mediate the acylation of the lipid A portion of the LPS decreasing permeability to cationic antimicrobial peptides. A group of genes responsible for resistance to protamine encode seven proteins that include an ATP-binding cassette transporter and the components of the major K⁺ uptake system in enteric bacteria, suggesting that hypersensitivity to protamine may be due to the inability to take up K⁺ at high rates. Two regulatory systems, designated PhoP/PhoQ and PmrA/PmrB, govern resistance to antimicrobial peptides in Salmonella. Phenotypic modulation of these regulatory systems by environmental Mg²⁺ and pH alters antimicrobial peptide susceptibility >1,000 in wild-type Salmonella. Drugs that interfere with these regulatory systems may increase susceptibility to antimicrobial peptides.

Interactions of Herpesviruses with the host immune system.

Hidde L. Ploegh, Department of Pathology, Harvard Medical School, Boston, Mass. ploegh@hms.harvard.edu

The interactions between hosts and their pathogens are the result of millions of years of coevolution. The continued existence of both host and pathogen relies on the precarious balance established in the course of evolution. Consequently, a deeper understanding of this interplay is likely to provide new insights in both host defenses and the pathogen’s survival strategies. Large DNA viruses such as the members of the poxvirus and herpesvirus family are masters at the art of immunological deception. Their genomes encode homologs of cytokines their receptors and molecules that interfere with the induction of programmed cell death. Above and beyond, they have evolved more proprietary countermeasures that have shed light on some very basic aspects of cell biology, notably proteolysis and the turnover of membrane proteins.
In the foreseeable future, the complete genome sequences of all major pathogens of medical and agricultural relevance will be available. In many cases, this information will set the stage for a more refined genetic analysis of host-pathogen interactions. Genomics will also provide the substrate for more direct structural analysis of newly identified gene products crucial to the survival strategy of the pathogen in question. It is widely believed that this information will reveal new principles of cell biology, with the attendant possibility of manipulating the newly identified molecular interactions with the help of small molecules.

Poster No. 49

Identification, characterization and molecular cloning of initial host-parasite signals.

Annelie Tjernlund, Department of Infectious Diseases, P1 651, 144 51 Rönninge, Sweden. Annelie.Tjernlund@impi.ki.se

We have recently identified a new immune system induced factor, ISRAF ( immune system released activating factor) which induces immune cells to produce cytokines. ISRAF is produced one minute after subcutaneous injection of the parasite Trypanosoma Brucei (T.b.). We believe that there is a cross-talk between the nervous system and the immune system and that this cross-talk leads to the production of ISRAF. These believes are based on the fact that if we cut the splenic nerve there is no detection of ISRAF after injection of T.b. It is known that primary and secondary immune responses are not isolated from other organ systems in the body, and they can be anticipated to communicate with the immune system in different ways. Among these communications are regulatory interactions between the central nervous system (CNS) and the immune system. A channel of contact between these two system is noradrenergic sympathetic innervation of the primary and secondary lymphoid organs. b and a-adrenergic receptors on lymphocytes and on other immunocompetent cells also play a role of contact between the CNS and the immune system.
The aims of this study is to characterise ISRAF and examine the effects of the sympathetic nervous system in early host-parasite interactions and the role of the splenic nerve in primary immune signals.  

Poster No. 50

Human bladder epithelial cells express Toll-like receptors and recognise lipid A acylation

Fredrik Bäckhed, Staffan Normark, Agneta Richter-Dahlfors, Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden. fredrik.backhed@mtc.ki.se

When pathogenic bacteria infects a host their initial site of interaction is the epithelial lining of the airways, the gastrointestinal tract or the urinary tract. Due to the very rapid multiplication of bacteria, a rapid induction of the innate immune system is of utmost importance to the host, otherwise the infection will quickly become deleterious. The interaction can be achieved by the interactions between conserved molecules present on the microbe, and specific receptors in the cell membrane of the target cell. Our data show that epithelial cells from the urinary bladder express at least three such receptors; the Toll-like receptors TLR2, TLR3 and TLR4. These receptors are homologues to the Toll receptor in Drosophila, known to stimulate production of anti-microbial peptides upon recognition of conserved microbial features. Using defined bacterial mutants of Escherichia coli (pathogenic and non-pathogenic strains) we will proceed to identify the bacterial ligands for the receptors.
The lipid A moiety of LPS has been shown to be the ligand of TLR2. An E. coli strain defected in lipid A acylation induces lower amounts of pro-inflammatory cytokines compared to the isogenic wild type strain. We hypothesise that this effect is mediated by the inability of TLR2 to recognise its ligand.

Poster No. 51

Host-derived antimocrobial compounds can be substrates for bacterial efflux

Shafer W, Rouquette C, Lee E-H, Veal W, and Balthazar J, Department of Microbiology and Immunology, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, USA. wshafer@emory.edu

Pathogenic microorganisms must breach several lines of host defense in order to cause disease in a susceptible host. At the first line of host defense, particularly at mucosal surfaces and fluids, are a number of antimicrobial agents that have antibiotic-like action. These substances include free fatty acids, bile salts,  steroidal hormones, and antimicrobial peptides/proteins. In an effort to learn how bacterial pathogens might resist or subvert the action of these host-derived “antibiotics”, we have studied the Gram-negative pathogen, Neisseria gonorrhoeae (the gonococcus). The gonococcus is a strict human pathogen that has caused the sexually transmitted disease, gonorrhea, for thousands of years. Its capacity to cause repeated infections in some humans suggests that little if any protective immunity develops as a result of infection and that host-derived antimicrobial agents that operate at the mucosal surfaces against pathogenic bacteria may be ineffectual against gonococci. Research in our laboratory has identified the ability of efflux pump systems, normally associated with providing bacteria with resistance to antibiotics as a mechanism by which gonococci can resist host-derived antimicrobial compounds. The gonococcus possesses two different efflux pumps that seem to recognize and export host antimicrobial agents. The efflux pump encoded by the mtrCDE operon and the closely linked mtrF gene appears to have the capacity to recognize hydrophobic antibiotics, antibacterial peptides (such as the human cathelicidin peptide LL-3 7 and the porcine-derived peptide, Protegrin-1), detergent-like compounds, bile salts and steroidal hormones that can become available at mucosal surfaces. We have found that the genes encoding this efflux pump are subject to both negative and positive transcriptional systems. The MtrR repressor protein is a DNA-binding protein that reduces transcription of mtrCDE, while MtrA is a member of a family of bacterial transcriptional activators that is used by the gonococcus when it senses the presence of antimicrobial compounds. During certain infections, the invading gonococcus is likely to encounter toxic fatty acids. We have found that a second efflux pump, encoded by the farAB (fatty acid resistance) locus, can act independently of the mtrCDEF efflux pump system to provide resistance to long-chained fatty acids, which are often present at high levels in vivo. This efflux system bears striking similarity to an efflux pump system possessed by other Gram-negative bacteria that recognize antibiotics and uncoupling agents. We propose that the efflux pumps possessed by Neisseria gonorrhoeae arose through evolution as a means to resist those antimicrobial agents that are produced by the human host to protect mucosal surfaces from pathogenic bacteria.

Poster No. 52

20:10, a novel peptide bridging immune- and stress response in Drosophila melanogaster

Sophia Ekengren and Dan Hultmark*, Dept. of Dev Biol., Stockholm University, Sweden; *UCMP, Umeå University, Sweden. sophia@molbio.su.se

The 20:10-gene was found and cloned in a screen for genes activated upon an immunechallenge. The gene encodes a novel kind of peptide that is synthesized in the fatbody and secreted into the hemolymph in response to a microbial challenge. In addition, other more stress-related stimuli such as heat shock, dehydration and internal wounding also stimulate gene activity. However, sterile injections do not activate transcription. Preliminary data suggest 20:10 to be regulated via the p38 MAP kinase signalling pathway. This pathway is known to participate both in the inflammatory response and in stress-induced signalling in man. Drosophila p38, which is suggested to have a down-regulatory effect on the immune response, has a clear negative effect on 20:10 transcription. The 20:10 protein does not seem to have any direct antimicrobial effect. Its induction is relatively slow compared to the antimicrobial peptides, and the peak of expression is not reached until 16 hours after immune stimuli. We speculate that 20:10 may have a function, directly or indirectly, as a mediator of homeostasis upon exposure of bacteria and/or other perilous situations.

Innate Immunity: Common Themes and Diversity

Robert L. Lehrer, M.D., Dept. of Medicine, UCLA School of Medicine, Los Angeles, California 90095-1690, USA. rlehrer@ucla.edu

Innate immunity is a powerful host defense system, containing some elements that antecede the divergence of animal and plant lineages. Eli Metchnikoff, a Russian biologist, first recognized and championed the importance of phagocytes. Paul Ehrlich, a physician-chemist who advocated the primacy of humoral factors, promulgated a side chain theory, deduced the existence of receptors, and provided theoretical constructs needed to develop practical antitoxic immunotherapy and to design potent chemotherapeutic agents (for syphilis). After this strong start, the field of innate immunity languished for much of this century, while adaptive responses of T and B cells occupied center stage. With improved technology to characterize proteins and their genes, interest in innate defenses recently reawakened.
Phagocytes derive antimicrobial properties from an ability to encapsulate microbes or ingest and sequester them inside phagocytic vacuoles whose content of protons and nutrilites can be modulated. Phagocytes also expose microbes to an onslaught of noxious oxidants generated by NADPH oxidase and/or by inducible nitric oxide synthase. Many phagocytes also contain antimicrobial proteins and peptides that can be delivered to phagocytic vacuoles or secreted - acting as endogenous antibiotics. Epithelial cells and secretory glands also produce antimicrobial polypeptides, thereby providing mucosal surfaces in the respiratory, digestive and genitourinary tracts with strong local defenses.
Invertebrates use oxidants derived from phenoloxidases and many antimicrobial peptides for host defense. Multiple antimicrobial peptides produced by the fat body of insects appear in hemolymph within hours of injury or infection. Other antimicrobial peptides are induced locally. Plants have complex innate immune responses, including generation of oxidants by NADPH oxidase and local and systemic induction of antimicrobial peptides - some with structural resemblance to antimicrobial peptides produced by insects. Studies in Drosophila have provided insights into the regulation of antimicrobial peptide synthesis, and remarkably similar regulatory systems operate in vertebrates and plants.
As the pathways and molecules involved in innate responses to infection become better defined, many useful horticultural, medical and veterinary applications are likely to result.

Poster No. 53

Bactericidal Activity of Normal Cord Serum (NCS) against Gram-negative Rods with Sialylated Lipopolysaccharide (LPS)

Mielnik G, Jankowski S, Doroszkiewicz W, and Gamian A. Institute of Microbiology, University of Wroclaw, Department of Biology, Medical University, Institute of Immunology and Experimental Therapy, PAS, Wroclaw, Poland

The complement system plays an important role in protection of higher organisms against bacterial infections. NCS has relatively low bactericidal activity because of its low innate level of proteins of complement system. The structure of LPS is one of factors determining the susceptibility of bacteria to complement.
The scope of this work was a comparison of the susceptibility to the bactericidal action of NCS of strains in which the presence of sialic acid in LPS was stated. Two E. coli strains (PCM 1217 and PCM 2386) of three tested can propagate in 75% NCS. Percent of cells alive was in these cases 734 and 340 respectively. The strain E. coli PCM 1186 was sensitive to 25% NCS. The strain of H. alvei (PCM 2386) was resistant and the strain (PCM 1186) was susceptible to NCS. The strain Citrobacter freundii 037 PCM 2346 was completely resistant and the strain S. toucra 048 PCM 2359 was susceptible to NCS. Further investigations should explain the role of sialic acid present in LPS in resistance of bacteria against bactericidal action of serum.

Poster No. 54

Dynamic change of chemotactic cytokine LECT2 during regeneration following partial hepatectomy of mice.

H. Nagai ^1,2, S. Yamagoe¹, H. Ohtake³, M. Ohtomi² , T. Uchida⁴ , A. Mabuchi⁵, *K. Suzuki^1
1Biodefense Laboratory, Department of Bioactive Molecule, National Institute of Infectious Diseases (NIID),Tokyo, Japan, ²Faculty of Science, Toho University, ³Dokkyo University, School of Medicine, ⁴Nihon University, School of Medicine, ⁵Nippon Medical School.  ksuzuki@nih.go.jp

We have isolated a novel chemotactic cytokine LECT2, which is 16 kDa protein expressed in human leukemia cells SKW-3. LECT2 is mainly expressed in hepatocytes in liver tissue of human and cell lines of hepatocytes also expressed. In addition, LECT2 is also produced in hepatocytes of mouse. To know the role of LECT2 in vivo, we determined the change of LECT2 in regeneration of partial hepatectomy of mice. According to method of Higgins and Anderson’s mouse liver was eliminated. At 0 to 24 hrs after the hepatectomy, the remained liver was isolated and fixed. Expressions of LECT2 protein and LECT2 mRNA in the remained tissues were observed by techniques of immunocytochemistry and in situ hybridization, respectively. LECT2 protein and LECT2 mRNA localized in most of hepatocytes in liver with diffusely profile under the normal conditions. However, LECT2 protein completely disappeared in the remained liver tissue at 0.5 hr after the hepatectomy. Thereafter, LECT2 protein was detected in a few regions at 2hrs. Then, hepatocytes expressing the protein gradually increased in the tissue. At 12hrs most of hepatocytes expressed LECT2 protein in the liver as well as normal tissues. Distribution of hepatocyte expressing LECT2 mRNA during regeneration of hepatectomzed liver tissue recovered with almost similarity to the profile of LECT2 protein, whereas not complete disappearance at 0.5 hr. These results suggest that LECT2 may contribute as a trigger during the regeneration of liver, especially in the early events. *K. Suzuki will present this paper.

Poster No. 55

Marco Colonna, Basel Institute for Immunology, Basel, Switzerland. colonna@BII.ch

Immunoglobulin-like transcripts (ILTs) encode several novel Ig-SF receptors which are structurally and functionally related to killer cell inhibitory receptors (KIRs) and are preferentially expressed on monocytes and granulocytes. ILTs are characterized by 2 or 4 homologous extracellular Ig-SF domains, some of them are specific for MHC class I molecules, and display different transmembrane and cytoplasmic domains. One subset of ILT receptors displays long cytoplasmic tails containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Another subset of ILT receptors contains short cytoplasmic domains that lack kinase homology or recognizable motifs for signaling mediators. These ILT receptors activate monocytes and granulocytes and associate with Fc receptor gamma chain to transduce stimulatory signals. A third subset of ILTs has no transmembrane and cytoplasmic domains and may be secreted as soluble receptors. We have recently found that inhibitory ILTs specific for MHC class I molecules can inhibit production of inflammatory cytokines and chemokines by monocytes. Thus, a dysfunction of the inhibitory ILT-class I interactions may facilitate an inappropriate secretion of inflammatory cytokines by myeloid cells, contributing to the pathogenesis of chronic inflammatory autoimmune diseases. On the other hand, viruses can produce class I-like molecules and thereby mimic inhibitory ILT-class I interactions, in order to escape from immune responses.

Poster No. 56

Increase of MPO-ANCA titer in ICSBP knock-out mice

Toshiyuki Matsuoka^1,2, Ydd Hashimoto^l, Akiko Okawara¹, Keiko Ozato³, Takao Arai², Kazuo Suzuki^l , ¹Biodefense Laboratory, Department of Bioactive Molecule, National Institute of Infectious Diseases, Japan. ²Department of Applied Biological Science, Science University of Tokyo, Japan. ³Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development NIH, U.S.A. matu@nih.go.jp

Auto-antibody against neutrophils, anti neutrophil cytoplasmic antibody (ANCA) is believed to play a role in vasculitis. Major ANCAs, MPO-ANCA and PR3-ANCA, target myeloperoxidase (MPO) and proteinase-3 (PR3) contained in granules of neutrophils, respectively. Vasculitis and glomerulonephritis (GN) are known as diseases that show high MPO-ANCA titer, and thus MPO-ANCA has been used as a serum. marker. Recently, to elucidate the role of MPO-ANCA in nephritis and vasculitis, several mouse models have been established which show high MPO-ANCA titer. In addition to MPO-ANCA titer, activated neutrophils seems to be involved in the etiology of vasculitis. It suggests that mice with abnormal neutrophil function may be associated with production of high MPO-ANCA titer. It is reported that mice with a targeted mutation of the ICSBP (Interferon consensus sequence binding protein) gene show an increase in neutrophils and leukernia like infiltration into kidneies. Therefore, we postulated that ICSBP-/- mice have high levels of MPO-ANCA. MPO-ANCA titer in ICSBP+/+, +/- and -/- mice was measured by MPO-ELISA. The antibody titer in ICSBP -/- is higher than that in ICSBP+/+ and +/-. We also observed that CD69⁺CD4⁺ T-cells in spleen are increased in ICSBP-/- mice. MPO-ANCA titer in the mice might be relevant to the increased CD69⁺CD4⁺ T-cell populations in ICSBP-/-spleen.

Poster No. 57

Different disoRders of innate immunity in patients with chronic inflammatory diseases

Tarasova E.E.¹ and Kuzovkova N. A² , ¹Clinical-Diagnostical Department, Mother and Child Care Institute, and ²Department of Ecological Immunology, Institute of Epidemiology and Microbiology, Minsk, Belarus. Mlada7@nsys.by

We studied innate immunity status of patients (aged 14-30), who are sufferring from chronic (2-5 years) inflammatory diseases which are hardly treated with traditional antibacterial therapy. Immune status valued by quantitative and functional methods: CD3+, CD4+, CD8+ and CD22+, levels IgG, A and M, total haemolytic activity CH50 and contents of effective molecules Clq-C5-components of classic and factors B and D of alternative pathways of complement system activation, phagocytic neutrophil activity.
All examined patients did not revealed deflections in T-cell chain of immunity. Based on humoral and nonspecific chain of immunity status patients were divided into three groups. The 1^st group patients (6 persons) revealed a significant reduction of CD22+ number (3,0±0,34%, P<0,01), of IgG (2,5±0,21 g/1, P<0,001), A and M (traces), of phagocytic neutrophil activity (phagocytic index (Phl) - 24,0±2,5%, phagocytic number (PhN) - 1,8±0,11, P<0,00 1) and of content of all components of complement system (more than 95% versus norme). In 2^nd group patients (16 persons) it was specific a reduction of IgG content (6,5±1,8 g/1, P<0,01), of absorbant ability (PhN - 3,8±0,41, P<0,01) and digestive ability of neutrophil (index of phagocytic completeness (IPhC) - 0,8±0,13), of C3- and C5-components content (by 1.5-2). 3^d group patients (5 persons.) had a high level of IgG (23,4±2,3 g/1, P<0,01), high content of all components of complement system and, as a result high CH50 level (194,0±12,6 c.u., P<0,01), depression of absorbant (PhN - 2,0±0,21, P<0,01) and, particularly, digestive abilities of neutrophils (IPhC - 0,1±0,04).
Thus, patients suffering from the same diseases and having the same clinical patterns revelead different immunity disorders which are more indicative for phagocytic neutrophil activity and for complement system. This fact should be considered when therapy is administered.

Poster No. 58

THE INNATE ANTIVIRAL DEFENSE SYSTEM IS PERSISTENTLY ACTIVATED IN TYPE 1 DIABETES

V. Bonnevie-Nielsen, P. M. Martensen, J. Justesen, K. Levin, H. Beck-Nielsen, A. Worsaa and T. Dyrberg. University of Southern Denmark, Odense University, Odense University, University of Aarhus, Odense University Hospital, Novo Nordisk, Denmark. bonavita@dadlnet.dk

Type 1 diabetes results from autoimmune destruction of the pancreatic a-cells. The factors initiating this destruction have not yet been identified although increased frequencies of virus antibodies support a viral hypothesis. Recently, attention has been increasingly focused on the significance of the innate antiviral defense system in directing adaptive immune responses. We hypothesized that the pathogenesis of type 1 diabetes may involve an aberrantly regulated immune response to endogenous or exogenous viruses. In the innate antiviral immune defense, alpha-interferon (interferon-a induced proteins are the main responsible effector molecules in degradation of infectious virus particles before major cellular damage occurs. Hence it was the aim to examine the activity of the innate, antiviral defense system expressed as two antiviral enzymes, the IFN-a inducible, ds/ss RNA-dependent 2´,5´ oligoadenylate synthetase (2´,5´AS), the autophosphorylating proteinkinase p68 and the virus inhibiting MxA protein. Activities of 2´,5´AS and proteinkinase p68 were determined in lymphocyte homogenates by ³²P-labelled ATP and quantified by b-counting or phosphorimaging. The results show that lymphocytic 2´,5´AS activity was significantly and selectively increased in type 1 diabetics, both in those with recent-onset (n, p<0.005) and with long-standing disease n$, p<0.001) as compared to healthy controls (ne). Activity of 2´,5´AS was not elevated in patients with type 2 diabetes or multiple sclerosis, thus excluding hyperglycemia or autoimmunity per se as inducing upregulation of enzyme activity lymphocyte levels of proteinkinase p68 and MxA, two other IFN-a inducible antiviral proteins, were similar in type 1 diabetic patients and controls. This suggests that within the innate immune system processes may be activated without being caused by a general induction of e.g. IFN-a dependent pathways. In type 1 diabetes the chronically increased 2´, 5´AS activity is not increased due to IFN-a but rather is an aberrant response to viruses or RNA molecules, originating from exogenous or endogenous sources.

Poster No. 59

Expression of the high affinity phagocyte receptor FcyRI (C1)64) on neutrophil granulocytes during bacterial infections.
An early linkage between Innate and Specific Immunity?

Fjaertoft G, Ewald U, Foucard T, Håkansson L, Venge P. Department of Women’s and Children’s Health and Department of Clinical Chemistry,Uppsala University Hospital, Uppsala, Sweden. Gustavfjaertoft@pediatrik.uu.se

By its capacity for phagocytosis and killing of micro-organisms, the neutrophil granulocyte plays an important role in defence against infections. To enable neutrophil phagocytosis, the micro-organisms have to be opsonised by complement and/or specific antibodies. At least partly due to their immature immune system, newborn infants, and especially the preterm ones, have an increased susceptibility to serious infections. In accordance with this, neutrophil granulocytes from preterm infants have a reduced capacity to upregulate the complement receptor CR3, and also express the two Fcg-receptors, FcgRIII (CD16) and FcgRII (CD32), to a significantly lesser extent, compared with granulocytes from adults. The high affinity Fcg-receptor FcgRI (CD64), which binds monomeric IgG, is normally expressed only to a very low extent by neutrophils. During bacterial infections, however, neutrophils from adult patients significantly increase their expression of FcgRI. Stimulation through FcgRI is a highly effective way to improve various aspects of neutrophil function including phagocytosis. In the present study the expression of FcgRI on neutrophils from preterm (n=9) and term (n=3) newborn infants, children (n), and adults (n=6) during the early phase of an acute bacterial infection was investigated. Our results showed that neutrophils from newborn infants with bacterial infection expressed FcgRI to a significantly higher extent than both non-infected preterm (p<0.001) and term (p<0.001) newborn infants, and that neutrophils from preterm neonates expressed FcgRI to the same extent as neutrophils from term neonates and older infants, children, and adults.
Expression of FcgRI probably represents an important mechanism to improve neutrophil phagocytosis as well as other aspects of neutrophil function during bacterial infections, especially in preterm infants.

Poster No. 60

High Serum Mannose Binding Lectin Concentration in Japanese Leprosy Patients

Suzuki Y, Ohtani K, Kase T, Eda S, Kawai T, Keshi H, Sakai Y, Fukuoh A, Sakamoto T,

Wakamiya N, Osaka Prefectural Institute of Public Health, Research Institute for Microbial Diseases, Osaka University, National Leprosarium Oku-Komyou-en, 1-3-69 Higashinari, Osaka, Osaka 537-0025, Japan, suzuki@iph.pref.osaka.jp

Mannose Binding Lectin (MBL) is a kind of animal collectin, which consists of Ca⁺⁺ dependent carbohydrate recognition domain and collagenous domain. This protein is thought to have immunological activities against microbes through its binding to the sugar moiety. In this study, we have measured the MBL concentration in the sera of leprosy patients in Japan to get the insights on the role of MBL in leprosy. The concentration of MBL in the sera of 388 healthy controls and 128 leprosy patients were measured by using newly constructed ELISA system in our laboratory. Leprosy patients gave average MBL concentration of 1.52 mg/ml. This is higher than that of healthy controls (0.97 mg/ml). And there was no difference between lepromatous, borderline and tuberculoid leprosy patients. The striking difference between leprosy patients and healthy controls was the rate of those who have high MBL concentration (more than 2.0 mg/ml). In leprosy patients, 37.5 % had MBL concentration of more than 2.0 mg/ml. This rate was 4.6 times higher than that in healthy controls (8.2%). These results suggested that high concentration of MBL might be one of the risk factors for leprosy infection. We are trying to find any polymorphism in promotor lesions and exon-1 of MBL gene.

Poster No. 61

KNOCKED-OUT HUMAN BEINGS.
ARE LATE COMPLEMENT COMPONENTS THE SUPERFLUOUS ELEMENTS OF INNATE IMMUNITY?

Platonov A.E. and Vershinina I.V. Central Institute of Epidemiology, Moscow, Russia   id@glasnet.ru

The antibody dependent effects are consider to be vital for host defense against systemic meningococcal disease (SMD), that correlates with and explains a high incidence of SMD at early childhood and a usefulness of specific immunization. However, on the one side, the pathogenic meningococci interact in the first place with complement system in vitro and in vivo and, on the other side, the individuals with late complement component deficient (LCCD) are extremely susceptible to SMD. Since 1988 we have identified a large group of 49 LCCD individuals in Russia and have studied the molecular genetic basis of the deficiency, the epidemiological and clinical features of SMD in LCCD individuals, their immune state and immune response to vaccination with meningococcal vaccine. Using this group as a model, the importance of late complement components for human physiology and, particularly, for innate and acquired immunity may be evaluated. These data will be presented at the Benzon Symposium in detail. In brief, the LCCD individuals are not knocked out or seriously disabled. Although any of them has contracted from one to five episodes of SMD, individuals with LCCD are generally regarded by themselves and their physicians as clinically well between SMD episodes. There is no other specific pathology, either infectious or somatic, that may be clearly linked to LCCD. They do not experience any social or professional limitation; one women worked at an emergency ambulance during last decades contacting with numerous meningococcal patients that, together with our other observations, is arguing for the partial resistance of LCCD individuals to SMD. Taking into account that the effective system of late complement components had been developed already in ancient mammals whereas N. meningitidis is an exclusively human pathogen, one may speculate that the initial task of late components was a defense against other unknown, or even disappeared microorganism(s). Currently the individuals with homozygous LCCD may be under negative selection pressure due to SMD but the effects of heterozygous LCCD would be neutral or even beneficial.

Poster No. 62

Protective Effect of b-Glucan against Systemic Streptococcus pneumoniae Infection in Mice

Hetland G¹, Ohno N², Aaberge IS³, and Løvik M^1,3. Departments of ¹Environmental Medicine and ³Vaccinology, National Institute of Public Health, Oslo, Norway, and ²Tokyo University of Pharmacy & Life Science, Japan. geir.hetland@folkehelsa.no

The anti-microbial effect of a soluble b-1-3-D-glucan from Sclerotinia sclerotiorum fungus (SSG) was examined in inbred NIH/OlaHsd mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. Serotype 4 is more virulent than type 6B, which results in a more protracted infection when injected into mice. SSG was administered i.p. either once 3 days before challenge or once to thrice from 3 to 48 hours after challenge. The number of bacteria after plating of blood samples, and the mouse survival rates were recorded. Pre-challenge SSG administration showed a protective, dose-dependent effect against both S. pneumoniae type 4 and 6B infection. SSG injected as a single dose 24 hours after inoculation of bacteria had a curative effect against type 6B but not type 4 pneumococcal infection. In some experiments SSG was also administered orally before infection with serotype 6B, but showed no effect, possibly due to too low doses. In SSG treated mice surviving challenge with serotype 6B infection, there were higher levels of anti-pneumococcal-type 6B IgM antibodies than in mice given PBS pre-challenge and a positive correlation between anti-6B IgM levels and SSG dose. However, since there was no cross-reactivity between SSG and S. pneumoniae type 6B, the finding may be due to an adjuvant effect of SSG. The data demonstrate that the b-glucan SSG protects against pneumococcal infection in mice when administered systemically.

Poster No. 63

Non opsonic binding of Mycobacteria to Complement receptor type 1 (CR1)

Brai M., Clemenza L., Accardo P., Amodeo G. and Dieli F., Institute of General Pathology, University of Palermo, Palermo, Italy. melbrai@unipa.it

The initial contact between inhaled mycobacteria (M) and host cells takes places in the lung, where bacteria are phagocytosed by alveolar macrophages. Complement receptors CR1 and CR3 and mannose receptors play an important role in the adhesion of mycobacteria to the cell targets, in the presence or in the absence of serum opsonins. We studied the binding of recombinant soluble CR1 (rsCR1) (Avant, Needham, MA) to M bovis BCG (2x10⁶ cells) by an ELISA system based on bacteria coated microwells. The non opsonic binding of rsCR1 to BCG was of high affinity (Kd @ 10^-10, 38,550 sites/cell) and saturable. The maximum uptake, (20% of rsCR1 added at a saturating concentration of 0.5 mg/m1) was reached after 30 min. incubation at 37^oC. Several other M strains were studied and gave similar results. Non opsonic binding of rsCR1 to other intracellular pathogens (L. monocytogenes, C. xerosis, N. meningitidis) was also detectable, although to a lower level than for M, whereas a strain of E. coli (InvaF) showed no binding at all. Inhibition studies of the direct binding of rsCR1 to BCG, performed using C3i, C4i and 3 different anti-CR1 moAbs (3D9, J3D3, E11) demonstrated that C3i and 3D9 induce 60% inhibition of binding, C4i and E11 inhibit only 10-20%. No inhibition was obtained with J3D3. Using PPD (20 mg/ml) as a specific competitor, more than 70% of inhibition was observed. Western blot of sonicated M revealed with rsCR1 followed by peroxidase-conjugated anti-CR1 showed two bands in the range of 60-65 kDa. Recombinant M heat shock protein 65 (Hsp65, MA5C), but not M 38 kDa protein, inhibited, in a dose-dependent manner, rsCR1 binding to M. A panel of 11 peptides derived from Hsp65 sequence were used in inhibition experiments. Only peptide 277-293 (100 mg/ml) induced 66% inhibition. In conclusion M binds to CR1 via a direct interaction. Binding is specific, saturable and of high affinity. It is inhibited by C3(H₂-0) and by the blocking anti-CR1 moAb 3D9, suggesting that the ligand sites for C3 and for the mycobacterial ligands on CR1 might overlap. Hsp65 from M tuberculosis might represent the ligand (or one of the ligands) used by the mycobacteria to enter macrophages via CR1. Supported by ISS progetto finalizzato tubercolosi 1997.

Poster No. 64

Peptides binding antibody effector ligands.         

Vigdis Lauvrak¹, Gøril Berntzen¹, Stine Granum¹, Ole Henrik Brekke^1,2 and Inger Sandlie^{1 1} Division of Molecular Cell Biology, Department of Biology, University of Oslo, Norway.  ² Affitech A/S, Oslo Research Park, Gaustadalleen 210349 Oslo. vigdisl@bio.uio.no

Antibodies recruit effector functions by binding soluble complement proteins and cellular Fc receptors (FcR). The antibody effector functions are diverse including activation of the classical complement pathway, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, superoxide release, cytokine release, and antigen presentation.
Peptides able to recruit or block antibody effector functions would be of great therapeutic interest. As a first step to isolate such peptides we have applied the technology of phage display for the selection of peptides binding the first component of the classical complement activation pathway, C1q, and a soluble form of the human high affinity Fc gamma receptor 1 (FcgRI). The biological activity of selected peptides will be discussed.

Late Poster Session

Late Poster No. 2

Regulation on Enterococcal Infections to non-phagocytic cells

Shirai F, Doi S, Kawaguchi M, Murakami A, Sogi A, and  Dohi Y, Department of Microbiology and Infectious Diseases, Division of Allied Health Sciences, Osaka University Medical School, Suita, Japan. E-mail: dohi@sahs.med.osaka-u.ac.jp

Causes of opportunistic infections on compromized hosts were still unclear and remain to be elucidated. We found that Enterococcus faecalis easily infected not only human epithelial cancer cell lines such as Hep-2 and Hep-G2, but also Sephadex G10 column-passed peripheral lymphoid cells including B cells and CD4-positive T cells. We evaluated the number of intra-cellular phagocytized bacteria on non-phagocytic host cells. The numbers increased for several hours after phagocytosis, and then decreased. Estimation of nitric oxide in these cells suggested that the induced nitric oxide might reduce the viable intra-cellular bacteria. Furthermore, the numbers of intra-cellular bacteria were reduced by the presence of non-specific CD4-positive cells, suggesting that some factors from CD4-positive T cells let host cells activated to induce nitric oxide, and as a consequence, intra-cellular viable bacteris decreased. These results strongly suggest that enterococcal infections may be influenced by "non-specific" CD4-positive T cells.                       

  Late Poster No. 3

  A low-frequency Ca²⁺ oscillatory response is rapidly induced in kidney epithelial cells by E. coli encoded a-hemolysin

  Uhlén P¹, Söderholm T², Laestadius Ź, Jahnukainen T¹, Bäckhed F², Normark S², Aperia A¹, Richter-Dahlfors A², ²Microbiology and Tumor Biology Center, and ¹Dept. of Woman and Child Health¹, Karolinska Institute, Stockholm, Sweden.
 E-mail: agneta.richter.dahlfors@mtc.ki.se

  We study innate immune responses induced by host-microbe interactions at epithelial linings. As a model system, we use a vesicoureteral reflux model of pyelonephritis in rats. We identified the proximal tubule cells as the initial site of interaction between the pathogen and the host. When investigating signalling responses in primary preparations of proximal tubule cells, we discovered that uropathogenic bacteria induce a novel type of signalling in their target cells. A low-frequency Ca²⁺ oscillatory response is induced by bacteria-encoded exotoxin a-hemolysin. The importance of hemolysin for bacterial virulence is well documented. The Ca²⁺ response is rapidly induced (within 2-5 minutes), and continues as long as hemolysin is present. Influx of calcium occurs through voltage-dependent calcium channels, presumably triggered by efflux of a monovalent cation via the hemolysin-created pore. The [Ca²⁺] oscillation occurs at a period of 15 minutes. Recently, it was shown that the frequency of [Ca²⁺] oscillations increases the specificity and efficiency of expression of eukaryotic transcription factors (e.g. NF-kB). Accordingly, we hypothesize that the oscillatory calcium response induced by sub-lytic concentrations of hemolysin is involved in the regulation of gene expression involved in the innate response of the host.

Late Poster No. 4

Antibacterial components in bronchoalveolar lavage fluid from sarcoidosis and asthmatic patients

 Agerberth B¹, Lensmar C³, Grunewald J^2,3, Castaños-Velez E⁴, Olsson B², Jörnvall H¹, Wigzell H², Eklund AG³, Gudmundsson GH², ¹Department of Medical Biochemistry and Biophysics, Karolinska Institutet, ²Microbiology and Tumorbiology Center, Karolinska Institutet, ³Department of Medicine, Division of Respiratory Disease, Karolinska Hospital and ⁴Immunopathology Laboratory, Karolinska Hospital, Stockholm, Sweden. E-mail: birgitta.agerberth@mbb.ki.se

Antibacterial peptides and proteins are an integral part of the epithelial defense barrier that makes up immediate protection against bacterial invasion. In human the a-defensins (HNP 1-4) are bacteriecidal effectors in circulating granulocytes, while the b-defensins (HBD 1-2) are mainly synthesized by epithelial cells. The human cathelicidin peptide LL-37 is produced by granulocytes but is also induced in skin epithelia during inflammation. To investigate the importance of these defense effectors in disease, bronchoalveolar lavage (BAL) fluid was analyzed from healthy individuals and sarcoidosis patients. In addition we have compared the antibacterial pattern in BAL fluid from asthmatic patients before and after allergen provocations. Antibacterial activity was detected with enhanced activity in BAL fluid from the sarcoidosis patients, while the activity is reduced after the provocation in the asthmatic patients. The activity was present at several antibacterial componenets, of which we so far have charaterized LL-37, lysozyme, HNP 1-3 and antileukoprotease. By immuno-histochemistry LL-37 and HNP 1-3 have been located in alveolar macrophages. Furthermore, LL-37 was found in bronchial epithelial cells and bronchial glands, indicating a defense role for LL-37 in airway mucosa. In conclusion, the airway epithelium is protected by a complex antibacterial defense system that is activated in sarcoidosis but seems to be diminished during challenge in asthmatic patients. This may in part explain the clinical observations of a low degree of respiratory infections in sarcoidosis patients. However, asthmatic patients may hypothetically be more sensitive for respiratory infections during exacerbations.

  Late poster No. 5

 Porcine Pulmonary Surfactant Preparations Contain the Antibacterial Peptide Prophenin and a C-terminal 18-Residue Fragment Thereof

Johansson J¹, Wang Y¹, Griffiths WJ¹ and Curstedt T², ¹Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm and ²Department of Clinical Chemistry, Karolinska Institutet at Karolinska Hospital, Stockholm, Sweden. E-mail: jan.johansson@mbb.ki.se

Surfactant preparations obtained from porcine lungs by extraction with chloroform/methanol followed by chromatography over Lipidex-5000 are used for treatment of respiratory distress syndrome in preterm infants. These preparations contain about 98% phospholipids and 1-2% of the hydrophobic pulmonary surfactant-associated proteins B and C (SP-B and SP-C). Separation of the proteins in the surfactant preparation by reversed-phase high performance liquid chromatography revealed the presence of members of the cathelicidin family of antibacterial peptides. In addition to SP-B and SP-C, the 79-residue proline-rich peptide prophenin (identical to that isolated from leukocytes), an 80-residue prophenin with an N-terminal pyroglutamic acid residue, and a C-terminal 18-residue fragment of prophenin were found. It appears possible that the presence of prophenin peptides may contribute to the antibacterial properties of surfactant prepartions.

  Late Poster No. 6

 Molecular Analysis of the Horse Cathelicidin Gene Family

Scocchi M^1,2,  Boscolo S², Tomasinsig L², Giulotto E³, Gennaro R⁴, Skerlavaj B¹, Risso A¹ and Zanetti M^1,2, ¹Dip. Scienze e Tecnologie Biomediche, University of Udine, Udine, ²Natl. Lab. CIB, Area Science Park, Trieste, ³Dip. Genetica e Microbiologia, Universita' di Pavia, Pavia, ⁴Dip. Biochimica, University of Trieste, Trieste, Italy. E-mail: marco@icgeb.trieste.it

Cathelicidins are a protein component of innate immunity identified in mammals. Members of this family (15-18 kDa) are primarily expressed in myeloid cells and stored in the secretory granules of neutrophils. Their sequences include a conserved N-terminal propiece belonging to the superfamily of cystatins, and a C-terminal domain (12-100 residues) characterized by a remarkable structural variety that gains antimicrobial activity after the propiece has been cleaved off upon neutrophil degranulation. We analyze here the horse cathelicidin gene family. Three congeners (eCATH-1, eCATH-2, eCATH-3) have been identified as deduced from horse myeloid cDNA. The putative polypeptides display 80-97% identical preprosequences and unique C-terminal sequences of 26, 27 and 40 residues with a cationic character. Southern blotting of genomic DNA from six horses, using a cathelicidin-conserved  probe reveals a polymorphic DNA region  with several hybridization-positive bands. The use of gene-specific probes indicates that one of the genes (i.e., eCATH-1) is absent, or rearranged to such an extent that it is not detected, in 50% of the horses analyzed. Analysis of several horse families suggests that the eCATH-1-positive and -negative variants are inherited as Mendelian alleles. A phylogenetic analysis of the nucleotide sequences of mammalian cathelicidins indicates that the three horse congeners likely originated by gene duplication and divergence from an ancestral gene after perissodactyl and artiodactyl species diverged. The genetic mechanisms that promote diversity in the 3' region of these genes have not been defined. We have noticed however a 100-150 bp region in the 3' UTR of eCATH-3, which is 80-85% identical to untranslated regions of a variety of otherwise unrelated horse genes, and followed by an unstable AAAT microsatellite. One could speculate on its role in gene rearrangement mechanism(s).

 Late poster No. 7

 Application of histatin analogue peptides in combination with a carrier molecule

 Ruissen ALA, van der Reijden WA, van't Hof W, Veerman ECI and van Nieuw Amerongen A, Department of Oral Biochemistry, Department of Oral Biology, ACTA, Amsterdam, The Netherlands. E-mail: ala.ruissen.obc.acta@med.vu.nl

 Various peptides have been developed analogous to salivary histatin 5. These peptides have increased activity against Candida albicans and are less susceptible to ionic strength. They can probably be used for treatment of oral Candida-infections or infections with other micro-organisms to which the peptides are active, like Staphylococcus aureus. Application in combination with a muco-adhesive polymer carrier can inrease their effectiveness as it is described that such polymers can prolong the retention of a drug at the side of application and are able to create a slow release over a longer period of time.
We investigated the effect of several polymers on the activity of one of the peptides (dhvar 1). Further, we coupled this peptide covalently to one of the polymers (xanthan gum). This was performed because we expect that a peptide-polymer conjugate will result in a longer retention of the peptide than if the peptide and polymer are applied in a mixture. We also determined the influence of xanthan gum on the degradation of dhvar 1 by enzymes present in saliva.
  Polymer molecules were able to inhibit the activity of dhvar 1. Xanthan gum and hydroxyethylcellulose caused the lowest inhibition. In general, it appeared that the lower the negative charge of the polymer the less inhibition occurred. This can probably be explained by reduction of the availability due to electrostatic interactions between polymer and peptide as the peptides are cationic. After coupling antifungal activity was still present. In the presence of xanthan peptide degradation was delayed.
  The results pointed out that polymer molecules can inhibit the activity of cationic antimicrobial peptides. Highly negatively charged polymers affect the activity more than weakly negatively charged or uncharged polymers. In addition polymers can provide protection against degradation.

  Late Poster No. 8

  Three dimensional structure of surfactant protein A in association with its lipid ligand

Palaniyar N, McCormack FX and Harauz G, Pulmonary/Critical Care Medicine, Department of Internal Medicine, University of Cincinnati, Ohio, USA and Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada. E.mail: nadesap@ucmail.uc.edu

 Surfactant protein A (SP-A) is a C-type collectin found primarily in the lung, and plays a role in innate immunity. This protein interacts with surfactant lipid monolayers, bilayers, membranes of microorganisms and carbohydrates. SP-A domains involved in the interaction with various ligands have been investigated exclusively by mutagenesis and biochemical methods but high resolution structural details of SP-A:ligand interactions are not available. To determine the structure of SP-A:lipid complexes, we have used single particle electron crystallography and computional 3D-reconstruction in combination with homology-based molecular modeling. Collagen-like domain deleted recombinant rat SP-A was interacted with lipid monolayers in the presence of calcium, and imaged by electron microscopy. Lipid-protein complexes with different angular orientations were computationally categorized, converging 2D averages processed by sinogram correlation function, and projected at every single degree angle. These projections were used to reconstruct the 3D structure of the protein. A homology-based molecular model of neck/CRD of SP-A was also made to fit into the electron density map of the protein. These results showed that the SP-A neck/CRD trimers existed in a non-symmetric organization when it interacted with lipid monolayers. Compared with the known structure of the other lung lectin, SP-D, trimers of the globular domains of SP-A differ in a magnitude of its asymmetric arrangement. This non-symmetric organization of globular domains of lung lectins may be related to their ligand binding specificity.

  Late Poster No. 9

  The bacterial protein YopJ antagonises immune responses at several levels

  Meijer L, French NS, Wolf-Watz H¹, and Pettersson S, Center for Genomics Research, Karolinska Institute, Stockholm, Sweden and ¹Department of Cell and Molecular Biology, University of Umea, Umeå, Sweden. E-mail: neil.french@cgr.ki.se

The Gram-negative bacterium, Yersinia pseudotuberculosis (YP), evades eukaryotic responses to lipolysaccharide (LPS) by translocating number of bacterial effector proteins into eukaryotic cells. This process allows extracellular Yersinia to neutralise inflammatory cells. We have previously shown that one of the effector proteins, YopJ, is capable of perturbing the expression of the pro-inflammatory cytokines IL1, IL8 and TNFa by blocking NF-kB activation. We now show that YopJ can also inhibit activation of the transcription factor CREB and thus dampen mitogen-induced proliferation of mouse splenocytes. YopJ mediates the inhibition of CREB activity by blocking its phosphorylation. By using specific chemical inhibitory compounds, we show that the YopJ-mediated perturbation of CREB phosphorylation occurs on the p38 MAP kinase pathway. We are currently attempting to identify the eukaryotic target protein(s) for YopJ. It will be interesting to assess whether YopJ subverts NF-kB and CREB activation through a single target or through independent, pathway-specific proteins.

Late Poster No. 10

Elafin has antimicrobial activity against Gram positive and Gram negative respiratory pathogens

Simpson AJ, Maxwell AJ, Govan JRW, Haslett C, Sallenave J-M, Rayne Laboratory, Respiratory Medicine Unit & Cystic Fribrosis Laboratory, University of Edinburgh Medical School, Edinburgh, UK. E-mail: j.sallenave@ed.ac.uk

Elafin (elastase specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung. Using synthetic peptides corresponding to full-length elafin (H₂N-¹AVT…..⁹⁵Q-OH), the NH2-terminal domain (H₂N-¹AVT…..⁵⁰K-OH), and the COOH-terminal domain (H₂N-⁵¹PGS…..⁹⁵Q-OH), we demonstrate that elafin's anti-elastase activity resides exclusively in the COOH-terminus. Several characteristics of elafin suggest potential antimicrobial activity. The antimicrobial activity of elafin, and of its two structural domains, was tested against the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 mM elafin, and 48% killing of S. aureus by 25 mM elafin. For both organisms, full-length elafin was required to optimise bacterial killing. These findings represent the first demonstration of co-existent anti-proteolytic and antimicrobial functions for elafin.

Late Poster No. 11

Overproduction of NO Induced by Curli-expressing Escherichia Coli Causes the Fall in Blood Pressure in Mice

Bian Z¹, Thoren P², Yan Z-Q³, Hansson G³ and Normark S¹, ¹Microbiology and Tumorbiology Center, ²Department of Pharmacology and Physiology, ³Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden. E-mail: zhao.bian@mtc.ki.se

Human septic shock is characterized by cardiovascular collapse following a systemic infection with microbial pathogens. Much evidence indicates that nitric oxide (NO) mediates the hyporesponsiveness of the peripheral circulation in septic shock. E. coli isolates from patients with sepsis are capable of expressing curli at 37⁰C, which causes cytokines induction in vitro. Here, we demonstrate the pathophysiological role of curli during E. coli peritonitis and septic shock in mice, in respect of the fall in blood pressure and the elevated levels of nitrite/nitrate (NO end-products) in the peripheral circulation. Both a significant reduction of blood pressure and an elevation of plasma nitrite/nitrate were documented when the normal mice were challenged with curli-expressing wt E. coli (5x10⁸ CFU, i.p.), as compared to that with a csgBA mutant. However, the blood pressure remained stable throughout the 8 h observation in NOS2 mice following wt E.coli challenge. In vitro study, curli-expressing wt E. coli activated the type 2 NO synthase in vascular smooth muscle cells, resulting in a higher level (P<0.05) of NO induction than that its isogenic mutants did. Thus, a genetically defined bacterial cell wall component (curli) other than LPS may play an important role during septic shock by means of activating NOS2 and NO overproduction.