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Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8(+) and CD 4(+) T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3(+) lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19(+) lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R(2) = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R(2) = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE assay.

Treutiger CJ, Mullins GE, Johansson A-SM,Rouhiainen A, Rauvala HME, Erlandsson-Harris H,Andersson U, Yang H, Tracey KJ, Andersson J,Palmblad JEW (Center for Infectious Medicine;Center for Inflammation and HaematologyResearch; Karolinska Institute at HuddingeUniversity Hospital, Stockholm, Sweden, FinnishRed Cross Blood Transfusion Service; Institute ofBiotechnology, University of Helsinki; Helsinki,Finland, Center for Molecular Medicine, KarolinskaInstitutet, Stockholm, Sweden and North Shore-LongIsland Jewish Research Institute, NY, USA). Highmobility group 1 B-box mediates activation of humanendothelium. J Intern Med 2003; 254: 375–385.Objectives. Severe sepsis and septic shock is aconsequence of a generalized inflammatory systemicresponse because of an invasive infection that mayresult in acute organ dysfunction. Mortality is highdespite access to modern intensive care units. Thenuclear DNA binding protein high mobility group 1(HMGB1) protein has recently been suggested to actas a late mediator of septic shock via its function asa macrophage-derived pro-inflammatory cytokine(J Exp Med 2000; 192: 565, Science 1999; 285:248). We investigated the pro-inflammatoryactivities of the A-box and the B-box of HMGB1 onhuman umbilical venular endothelial cells (HUVEC).Design. The HUVEC obtained from healthy donorswere used for experiments. Recombinant humanfull-length HMGB1, A-box and B-box were cloned bypolymerase chain reaction (PCR) amplification froma human brain quick-clone cDNA. The activation ofHUVEC was studied regarding (i) upregulation ofadhesion molecules, (ii) the release of cytokines andchemokines, (iii) the adhesion of neutrophils toHUVEC, (iv) the activation of signalling transductionpathways and (v) the involvement of the receptor foradvanced glycation end-products (RAGE).Results. The full-length protein and the B-box ofHMGB1 dose-dependently activate HUVEC toupregulate adhesion molecules such as ICAM-1,VCAM-1 and E-selectin and to release IL-8 andG-CSF. The activation of HUVEC could be inhibitedto 50% by antibodies directed towards the RAGE.HMGB1-mediated HUVEC stimulation resulted inphosphorylation of the ELK-1 signal transductionprotein and a nuclear translocation of p65 plusc-Rel, suggesting that HMGB1 signalling is regulatedin endothelial cells through NF-jB.Conclusions. The HMGB1 acts as a potent pro-inflammatory cytokine on HUVEC and the activity ismainly mediated through the B-box of the protein.HMGB1 may be a key factor mediating part of thepro-inflammatory response occurring in septic shockand severe inflammation.Keywords: cytokine, endotoxin, high mobilitygroup 1, human umbilical venular endothelialcells, inflammation.

The mode of action of urea in inhibiting infection of stumps of Norway spruce and Scots pine by Heterobasidion annosum was investigated. Stem discs from both species were treated with either urea, ammonium salt, ammonia or potassium hydroxide. Neither urea per se nor ammonium ions affected the growth of H. annosum, but the fungus was inactivated in wood at pH≥7. Urea treatment of pine stem discs or stumps caused a significant rise in both pH (to pH>8) and ammonia content within 24 h in sapwood but not in heartwood. In spruce, rises in pH started in the outer sapwood and were followed by sequential increases progressively towards the heartwood. After urea application to freshly-cut pine and spruce stumps, ammonia formation and pH rise started immediately, and initially there was a correlation between pH rise and ammonia content in the wood. High pH and high ammonia content were maintained for at least 3–4 months in warm humid weather. Ammonia was released to a greater extent in spruce and pine bark than in the adjacent sapwood but without a corresponding rise in pH. Studies on the urease activity suggested that the urea was initially decomposed by urease of host rather than microbial origin. The rate of ammonia decomposition and pH rise was found to be strongly dependent on temperature.

Summary The xylem surface of seedlings, stem material and roots of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were inoculated with strains of Heterobasidion annosum s. str. and H. parviporum s. str. The depth of necrosis in wounded spruce increased at a linear rate for at least seven weeks of incubation, but the rate of necrotic spread was significantly faster in infected wounds. In wounded pine the necrosis was maintained at a more superficial level for several weeks. Both spruce and pine sapwood were initially infected by hyphae of both species. In spruce, the hyphae advanced at a constant rate behind the necrotic front. On the contrary, after 1–2 weeks living H. parviporum hyphae were rare in pine rays. Heterobasidion annosum hyphae survived in pine rays, phloem and tracheids, despite a heavy accumulation of phenolics and resins and were able to penetrate into the sapwood at a linear rate although slower than infections in spruce. Histochemistry and quantitative estimates demonstrated that peroxidase activity was initially higher in spruce sapwood than in pine. Within three days of incubation, the activity in spruce sapwood disappeared concurrently with deepening necrosis. However, in pine, in both control and infected samples, there was a significant increase in peroxidase activity in the area surrounding the superficial necrosis, up to the wound surface and in the cambium and phloem around the wound. After wounding and infection, the content of soluble protein increased significantly in wood of older trees but not in seedlings. Infection resulted in an increased formation of lipophilic extractives in both spruce and pine but to a significantly greater degree in the latter, whereas the amount of hydrophilic compounds decreased in both. High-performance liquid chromatography (HPLC) analyses of lipophilic extracts showed that inoculation of pine with the two species of Heterobasidion increased the amounts of pinosylvin, its monomethylether and several other phenolics as also resinous compounds. The results obtained may be relevant in explaining the known higher resistance of Scots pine to H. parviporum. Resume Des semis, tiges et racines d'epicea commun (Picea abies) et de pin sylvestre (Pinus sylvestris) ont ete inocules au niveau du cambium avec des souches d’Heterobasidion annosum (Fr.) Bref. et Heterobasidion parviporum Niemela & Korhonen. La profondeur de necrose a partir des blessures a presente une augmentation lineaire pendant au moins sept semaines d'incubation chez les epiceas, mais l'extension de la necrose a ete significativement plus rapide pour les blessures infectees. Dans le cas des blessures sur pins, la necrose s'est limitee a un niveau superficiel pendant plusieurs semaines. Aussi bien chez les pins que les epiceas, l'aubier a commenceaetre infecte par les hyphes des deux especes d’Heterobasidion. Chez les epiceas, les hyphes ont progressea un taux constant derriere le front de necrose. Au contraire, les hyphes vivants de H. parviporumetaient rares dans les rayons apres une a deux semaines chez les pins. Les hyphes de H. annosum ont survecu dans les rayons, phloeme et tracheides de pins malgre une intense accumulation de resines et phenols et ont pu penetrer dans l'aubier, a un taux lineaire plus faible que dans le cas des infections sur epicea. Une etude histochimique et des estimations quantitatives ont montre que l'activite peroxydase etait initialement plus forte dans l'aubier d'epicea que sur pin. Apres 3 jours d'incubation, l'activite dans l'aubier d'epicea a disparu, en meme temps que la necrose devenait plus profonde. Au contraire chez les pins, aussi bien temoins qu'infectes, l'activite peroxydase a significativement augmente dans la zone entourant la necrose superficielle jusqu’a la blessure de surface, et dans le cambium et le phloeme autour de la blessure. Le contenu en proteins solubles a augmente significativement apres blessure et infection dans le bois des arbres mais pas chez les semis. L'infection s'est traduite par une baisse des composes hydrophiles chez les deux especes et par la formation accrue d'extraits lipophiles, a un niveau toutefois plus important chez les pins. Les analyses par HPLC des extraits lipophiles ont montre que l'inoculation des pins avec les deux especes d’Heterobasidion s'est traduite par une augmentation des teneurs en pinosylvine, de son monomethylether et de plusieurs autres composes phenoliques et resiniques. Les resultats obtenus pourraient expliquer la plus grande resistance, prealablement connue, du pin sylvestre aHeterobasidion parviporum. Zusammenfassung Die Xylemoberflache von Samlingen, Stammabschnitten und Wurzeln der Fichte (Picea abies) und Kiefer (Pinus sylvestris) wurden mit Heterobasidion annosum s.str. und H. parviporum inokuliert. Bei Fichten, die nur verletzt waren, nahm die Tiefe der Nekrose wahrend mindestens 7 Wochen linear zu; in infizierten Wunden breitete sich die Nekrose dagegen signifikant schneller aus. In verletzten Kiefern blieb die Nekrose fur mehrere Wochen auf einen oberflachennahen Bereich beschrankt. Beide Baumarten wurden kurz nach der Inokulation von Hyphen beider Heterobasidion-Arten infiziert. Bei der Fichte breiteten sich die Hyphen mit einer konstanten Wachstumsrate hinter der nekrotischen Front aus. Bei der Kiefer wurden lebende Hyphen von H. parviporum dagegen nach 1–2 Wochen nur selten in den Holzstrahlen gefunden. Die H. annosum-Hyphen uberlebten in den Holzstrahlen, im Phloem und in den Tracheiden der Kiefer trotz einer starken Akkumulation von Phenolen und Harzen, und sie konnten sich im Kiefern-Splintholz zwar langsamer als in der Fichte, aber doch mit einer linearen Wachstumstrate ausbreiten. Histochemische Reaktionen und quantitative Analysen zeigten, dass die Peroxidaseaktivitat im Splintholz der Fichte anfangs hoher warals in dem der Kiefer. Bei der Fichte verschwand diese Aktivitat innerhalb von 3 Tagen und gleichzeitig entwickelte sich eine Nekrose. Bei der Kiefer wurde dagegen sowohl in der Kontrolle als auch in den infizierten Proben ein deutlicher Anstieg der Peroxidaseaktivitat in der Umgebung der oberflachlichen Nekrose bis zur Wundoberflache sowie im Kambium und im Phloem in Wundnahe beobachtet. Nach Verletzung und Inokulation stieg der Proteingehalt im Holz alterer Baume signifikant an, bei Samlingen war dies jedoch nicht der Fall. Die Infektion fuhrte auch zu einer verstarkten Bildung von lipophilen Extraktstoffen (bei Kiefer signifikant mehr als bei Fichte), wahrend die hydrophilen Verbindungen bei beiden Baumarten abnahmen. HPLC-Analysen der lipophilen Extrakte zeigten, dass bei der Fichte durch beide Heterobasidion-Arten die Bildung von Pinosylvin, seinem Monomethylather, verschiedenen Phenolverbindungen und von harzartigen Verbindungen deutlich erhoht wurde. Diese Befunde konnen zur Erklarung der hoheren Resistenz der Kiefer gegen H. parviporum beitragen.