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Objective:To define retrospectively the number of hematologic-oncologic patients who undergo platelet substitution therapy and who develop only one or few HLA antibodies over a longer period of time, as well as to determine the corresponding antigens and to evaluate the substitution principle applied: preparations from whole blood lacking the corresponding antigens. Patients and Methods:Sera of 718 hematologic-oncologic patients were tested after platelet transfusions by means of platelet immunofluorescence and lymphocytotoxicity testing. Results:Long-term single HLA antibodies were detected in approximately 5% of the patients. Transfusion responses to preparations lacking the corresponding antigens were comparable to random substitution in patients without detectable HLA antibodies as well as to compatible substitution in patients with antibodies. Conclusions:Provided that regular antibody screening is carried out and a large pool of HLA-typed donors is available, patients with few HLA antibodies can effectively be substituted with platelets obtained by whole-blood donation from donors lacking the corresponding antigens.

Background:Transmission of viral, bacterial or parasitic infections is a well-known adverse side-effect of blood transfusion. The role of medically important fungi in blood transfusion remains to be elucidated. Since enhanced numbers of colony-forming units of Candida species are being isolated from gastrointestinal and urogenital tract of healthy adults with increasing frequency, symptomless fungaemia in blood donors cannot be excluded. We therefore examined sera of healthy blood donors for serological evidence of current or previous fungal infections or presence of fungaemia. Material and Methods:By using commercially available ELISA, sera of 497 blood donors of different age were examined for presence of Candida albicans antigen and antibody. 330 sera were simultaneously screened for the presence of Aspergillus antibodies. Results:Candida albicans antigen could not be detected in any of the examined sera. 41 of 497 sera showed Candida albicans IgM antibodies, and a broad spectrum of sera had Candida albicans IgG antibodies exceeding cut-off value. Conspicuous values of Aspergillus antibodies could not be detected. Conclusions:Though most of the blood donors investigated showed serological evidence for recent or past immunological confrontation with Candida albicans, the results suggest that transmission of medically important fungi by blood transfusion will continue to be unlikely.

All D+ samples were detected by all monoclonal antibodies without problems. Very few of the 3000 Du samples showed a ± to ++ reaction with some of the IgG type mabs using the Coombs technique. It is still open whether these are specific reactions with weak positive samples, which were negative with polyclonal sera. IgM type mabs detected up to 100% of the Du samples dependent on the Rh-phenotype and the technique used (CCDuee > CcDuee > ccDuEe). Most of the CCDuee samples reacted very strong and should no longer be regarded asDu.

A system for testing the biocompatibility of synthetic oxygen carriers is described including tests of hemolysis, complement activation and proliferation of cell lines or lymphocytes. Of 17 surfactants tested in this system, 6 were compatible in all tests while the other compounds showed individually differing patterns of incompatibility. We conclude that, in order to conduct a meaningful screening, a series of different assays has to be applied. In addition to our system, further assays, covering cytokine induction and phagocytosis should be attempted.

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 ± 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were than purified by adding a gradient (bottom loading). Using this closed system 1098 ± 489 IEQ per gram pancreas were purified with a total cell viability of 67 ± 10% and a β-cell viability of 41 ± 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of β-cells was still 56 ± 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-α to 40 ± 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

AusblickDiese berechtigte Hoffnung sowie die wissenschaftliche Eigenständigkeit, nicht zuletzt aber auch die wissenschaftliche Kooperationsbereitschaft, ohne die eine gedeihliche Weiterentwicklung der universitären Transfusionsmedizin und klinischen Hämotherapie nicht denkbar sind, rechtfertigen die universitäre Transfusionsmedizin in Forschung und Lehre. Die Transfusionsmedizin ist mit ihren verschiedenen, zukunftsweisenden Aufgabengebieten [s. Seifried E, Müller MM: Transfusionsmedizin – von der Blutspende zur Biotechnologie; in diesem Heft] so umfassend geworden, dass zur Wahrnehmung von Forschungs- und Entwicklungsaufgaben im Gebiet der klinischen Hämotherapie ihre Eigenständigkeit in jeder Beziehung gerechtfertigt erscheint. Mögen auch Interessenskonflikte zwischen verschiedenen Disziplinen auf lokaler Ebene einer selbständigen wissenschaftlichen Profilbildung der Transfusionsmedizin vor Ort entgegenwirken, zumal wenn wirtschaftliche Nutzung und seltener wissenschaftliche Leistungen im Vordergrund stehen, so kann doch zur Weiterentwicklung moderner Therapiestrategien in Gänze nur der wissenschaftliche Charakter der universitären Transfusionsmedizin beitragen. Nur beide Aspekte – Wissenschaftlichkeit und Wirtschaftlichkeit mit hoher transfusionsmedizinischer Effizienz – können die aktuellen und zukünftigen Herausforderungen der klinischen Hämotherapie bewältigen und das Fortbestehen dieses wichtigen Bausteins der modernen Hochleistungsmedizin in Diagnostik und Therapie, den Ausbau interdisziplinärer Forschung und Entwicklung sowie die wissenschaftliche Reputation eines Universitätsklinikums sichern.

SummaryAusblick Diese berechtigte Hoffnung sowie die wissenschaftliche Eigenständigkeit, nicht zuletzt aber auch die wissenschaftliche Kooperationsbereitschaft, ohne die eine gedeihliche Weiterentwicklung der universitären Transfusionsmedizin und klinischen Hämotherapie nicht denkbar sind, rechtfertigen die universitäre Transfusionsmedizin in Forschung und Lehre. Die Transfusionsmedizin ist mit ihren verschiedenen, zukunftsweisenden Aufgabengebieten [s. Seifried E, Müller MM: Transfusionsmedizin – von der Blutspende zur Biotechnologie; in diesem Heft] so umfassend geworden, dass zur Wahrnehmung von Forschungs- und Entwicklungsaufgaben im Gebiet der klinischen Hämotherapie ihre Eigenständigkeit in jeder Beziehung gerechtfertigt erscheint. Mögen auch Interessenskonflikte zwischen verschiedenen Disziplinen auf lokaler Ebene einer selbständigen wissenschaftlichen Profilbildung der Transfusionsmedizin vor Ort entgegenwirken, zumal wenn wirtschaftliche Nutzung und seltener wissenschaftliche Leistungen im Vordergrund stehen, so kann doch zur Weiterentwicklung moderner Therapiestrategien in Gänze nur der wissenschaftliche Charakter der universitären Transfusionsmedizin beitragen. Nur beide Aspekte – Wissenschaftlichkeit und Wirtschaftlichkeit mit hoher transfusionsmedizinischer Effizienz – können die aktuellen und zukünftigen Herausforderungen der klinischen Hämotherapie bewältigen und das Fortbestehen dieses wichtigen Bausteins der modernen Hochleistungsmedizin in Diagnostik und Therapie, den Ausbau interdisziplinärer Forschung und Entwicklung sowie die wissenschaftliche Reputation eines Universitätsklinikums sichern. Copyright © 2004 S. Karger GmbH, Freiburg

During the last years there were new developments in the field of artificial oxygen carriers. Two products which had been in clinical studies were taken off the market. Clinical trials of one further product were halted in January 2001. On the other side two products have filed for approval, and Hemopure™??(Biopure, Cambridge, MA, USA), a bovine, polymerized hemoglobin, gained approval in April 2001 in South Africa. Besides the products in clinical studies, there are also new hemoglobinbased oxygen carriers in preclinical testing. Due to binding of NO donators as well as increase of viscosity and molecular size they should possess reduced vasoactivity and reduced gastrointestinal effects. This article gives an overview of clinical studies, new developments, and side effects such as antigenicity, hemoglobin-endotoxin interactions and vasoactivity.

In this report, we investigate the intracellular fate of selected members of the genus Legionellawithin the monocytic cell line Mono Mac 6 cells. By means of electron microscopy and immunocytochemistry, we could show that Legionella pneumophilaas well as Legionella longbeachaeare able to induce ribosome‐studded phagosomes which associate with the rough endoplasmic reticulum (RER), whereas Legionella micdadeiremains to be located within smooth phagosomes but also shows signs of RER association. In addition, we could demonstrate a remarkable correlation between the phagosome type and the morphological phenotype of intracellular bacteria: within ribosome‐studded phagosomes, bacteria generally lacked the outer coat of low electron density whereas bacteria within the smooth phagosomes still possessed this outer coat. The virulence factors responsible for inhibition of phagosome maturation and their distribution within the genus Legionellaas well as the biological significance of the morphological difference of bacteria within smooth and ER‐associated phagosomes remain to be investigated.

ABSTRACTThis study evaluated the effects of rrr--tocopherol (500 IU/day, 8 days) on in vivocytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by rrr--tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. rrr--Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to rrr--tocopherol supplementation.

We found that human peripheral B and T cells differed in the surface expression of α2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galß1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the α2-6 sialylated model gangliosides IV⁶NeuAcnLc₄Cer (2-6 SPG), VI⁶NeuAcnLc₆Cer (2-6 SnHC), VIII⁶NeuAcnLc₈Cer (2-6 SnOC), and X⁶NeuAcnLc₁₀Cer (2-6 SnDC). We found that, in addition to their common requirement of an α2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar α2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

Recent investigations have provided substantial evidence that increased concentrations of proinflammatory parameters, such as total leukocyte count, interleukin-6 (IL-6), fibrinogen, and C-reactive protein, are related to the incidence and progression of coronary heart disease (CHD). However, interpretations for this finding such as chronic bacterial or viral infection have been discussed controversially. Therefore, the purpose of this study was to investigate the role of fatty acids and fatty acid subclasses on systemic inflammatory parameters in subjects not being on special medication or receiving specific fatty acid supplements. Plasma fatty acid profiles were determined by gas chromatography in 64 healthy male subjects aged 22–34 years; the P:S ratio as the quotient of polyunsaturated fatty acids and saturated fatty acids was then calculated. Proinflammatory target variables were total leukocyte count, Il-6, fibrinogen, and C-reactive protein. Inflammatory parameters were negatively correlated with polyunsaturated fatty acids and positively with saturated fatty acids; no significant correlations were found with monounsaturated fatty acids. Analysis of distribution within P:S quartiles revealed that leukocyte count (p<0.01), Il-6 (p<0.05), cortisol (p<0.05), C-reactive protein (p<0.05), and fibrinogen (p<0.05) were significantly higher in the lowest P:S quartile than in the highest P:S quartile. From these data it can be concluded that even in young, healthy individuals, low levels of polyunsaturated fatty acids and high levels of saturated fatty acids in plasma are associated with increased concentrations of pro-inflammatory parameters. The amount of epidemiological evidence indicating a cardioprotective effect of a high P/S ratio is constantly increasing. Therefore, future investigations should evaluate the role of a high P/S ratio not only with regard to metabolic risk factors such as lipoprotein levels but also with respect to inflammatory parameters. Die Bedeutung von Entzündungsprozessen in der Pathogenese der Atherogenese wird in zunehmendem Maße erkannt. Vor allem die Parameter Leukozytenzahl, Interleukin-6 (IL-6), Fibrinogen und C-Reaktives-Protein (CRP) waren in klinischen und prospektiven Studien signifikant mit der Inzidenz und Progredienz der Arteriosklerose bzw. mit der Wahrscheinlichkeit eines koronaren Ereignisses (Myokardinfarkt, kardiovaskuläre Mortalität) assoziiert. Die Ursachen für die individuell unterschiedliche Ausprägung der Entzündungslage sind jedoch noch nicht abschließend geklärt. Ziel der vorliegenden Untersuchung war es daher, den Einfluß der Fettsäurezusammensetzung des peripheren Blutes auf die Entzündungskonstellation zu untersuchen. Die Zielvariablen waren Leukozytenzahl, IL-6, Fibrinogen und CRP. Um Störgrößen wie Rauchen oder akute sowie chronische Erkrankungen auszuschließen, wurde die Untersuchung bei 64 gesunden jungen Männern (Alter 28,3±6,2 Jahre; BMI 22,1±1,7) durchgeführt. Alle Zielvariablen waren negativ mit den mehrfach ungesättigten Fettsäuren (FS) und positiv mit den gesättigten FS korreliert; die einfach ungesättigten Fettsäuren zeigten keine signifikante Korrelationen mit den Entzündungsparametern. Eine Aufteilung der Fettsäurespiegel entsprechend der Quartilen für den Quotienten zwischen mehrfach ungesättigten FS und gesättigten FS (P/S-Quotient) ergab jeweils einen signifikanten Unterschied für die Zielvariablen zwischen der jeweils niedrigsten und höchsten P/S-Quartile (Leukozyten p<0,01, IL-6 p<0,05, Fibrinogen p<0,05, CRP p<0,05). Aus diesen Ergebnissen kann gefolgert werden, daß auch bei gesunden jungen Männern ein niedriger P/S-Quotient im Serum mit einer signifikant gesteigerten Entzündungslage assoziiert ist. Da zum einen viele epidemiologische Untersuchungen eine deutliche Beziehung zwischen P/S-Quotient und koronarer Morbidität bzw. Mortalität aufgezeigt haben, und zum anderen vermehrt über die entzündliche Genese von kardiovaskulären Erkrankungen diskutiert wird, sollte der Beeinflussung der Entzündungsreaktion durch Fettsäuren vermehrt Beachtung geschenkt und durch weitere klinische und prospektive Studien untersucht werden.

We found that human peripheral B and T cells differed in the surface expression of alpha2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galss1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the alpha2-6 sialylated model gangliosides IV6NeuAcnLc4Cer (2-6 SPG), VI6NeuAcnLc6Cer (2-6 SnHC), VIII6NeuAcnLc8Cer (2-6 SnOC), and X6NeuAcnLc10Cer (2-6 SnDC). We found that, in addition to their common requirement of an alpha2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar alpha2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

The ability of Legionellaspecies to multiply within human mononuclear phagocytes is usually regarded as being associated with their pathogenicity. Activation of host cells results in inhibition of intracellular Legionellamultiplication. The most effective substance to induce macrophage activation, both in vivoand in vitro, is interferon‐γ. In addition, some evidence exists that macrophage‐derived cytokines may contribute to the host defense against L. pneumophila, but the production of pro‐ and antiinflammatory cytokines by monocytes after infection with different Legionellaspecies has not been reported with regard to their ability to multiply within the host cells. We therefore examined the production of TNF‐α, IL‐1, IL‐6, IL‐8, IL‐10 and TGF‐β by Mono Mac 6 cells after infection with Legionellaspecies of different human prevalence that differ in their ability to replicate within this macrophage‐like cell line. After infection, Mono Mac 6 cells showed a cytokine response with time kinetics characteristic for the cytokine. Maximum cytokine levels produced differed with Legionellaspecies, but were not related to intracellular multiplication rates. Moreover, LPS‐tolerant Mono Mac 6 cells, which failed to produce cytokines, showed intracellular increase or decrease of bacterial numbers identical to that of untreated Mono Mac 6 cells. By FACS analysis, an up‐regulation of CD14 (LPS receptor) and CD54 (ICAM‐1) could be demonstrated. We conclude that, in the Mono Mac 6 cell line, induction of macrophage‐derived cytokines after infection with members of the genus Legionellamimics an inflammatory reaction without association with intracellular multiplication rate.

Monoclonal CDw60 antibodies recognize glycolipid antigens with restricted surface expression on human leukocytes. They allow us to define new functional subpopulations of T lymphocytes and are able to induce costimulatory signals. In this report, we describe the molecular composition of CDw60 glycolipid antigens derived from different human leukocyte subpopulations. The glycolipids were isolated and their structures were identified by immunochemical methods. All molecules containing the CDw60 determinant were found in the disialoganglioside fraction. They were O-acetylated derivatives of the gangliosides II3 (Neu5Ac)2-LacCer (GD3), IV3 (Neu5Ac)2-nLc4Cer (DSPG), and VI3 (Neu5Ac)2- nLc6Cer (DSnHC), respectively. The most common CDw60 glycolipid antigen in human leukocytes was 9-O-acetyl GD3. In a comparison of various cell types, the highest concentration of 9-O-acetyl GD3 on a per cell basis was determined in granulocytes and in blood T lymphocytes, whereas B lymphocytes, thymus cells, and monocytes contained considerably smaller amounts of this molecule. Polar CDw60 antigens such as 9-O-acetyl DSPG and 9-O-acetyl DSnHC were only detected in granulocytes.

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.

Loss of certain red blood cell antigens has been described in patients with acute myelocytic leukemia (AML). This paper describes the loss of blood group A antigen in a patient with AML. The acute leukemia in this patient was preceded by a myelodysplastic syndrome for several months. At the time of diagnosis the patient's red cells showed the 0 Rh(D)+ phenotype. After induction of complete remission with two courses of Daunorubicin, Cytosin-arabinosid, and Etoposid his blood group reverted to A2. Serological studies including saliva analysis revealed that the original blood group was very likely A.

New surfactants have been synthesized with perfluorohexyl-groups as hydrophobic tail, a polyethyleneglycol-methylether as hydrophilic head and a prolongator between them. The prolongator units are -C4H8-, -C2H4OC5H10-, -C2H4OC3H6C(O)- and -CH2CH(OEt)CH2-. The surface tension, CMC and the interfacial tension water/perfluorocarbon were measured and the surface excess concentrations of the surfactant molecules were calculated. The influence of the surfactants on in vitro proliferation of human cell lines Hela and Molt 4 was investigated as a parameter for biocompatibility. The influence of the surfactants led to a reduction of cell proliferation depending on the concentration and chemical nature of the surfactant. Thus the prolongators changed the properties of the surfactants. They reduced the interfacial tension water/perfluorocarbon and improved the biocompatibility of surfactants.

An unusual case of a 37°C-active irregular anti-A1 is reported. Apparently consisting mainly of IgG, the antibody appeared in an A2B recipient only two days after massive transfusion of A1-cells in absence of previous transfusion. It was associated with severe hemolysis and renal failure which was reversed after exchange transfusion.

A patient who had been admitted to hospital for surgical treatment of inguinal hernias was found to have a blood group phenotype of A1B in the presence of a non-auto-anti-B. No previous records of the patient's blood group were available. The serological workup including absorption and saliva inhibiton studies yielded a high probability for an acquired B-antigen which is known to be often associated with carcinoma of the colon. Subsequent coloscopy revealed the presence of a carcinoma of the sigmoid, unaccessable to palpation. To our knowledge this is the first report in the literature that the serological diagnosis of an acquired B-antigen led to the detection of a hitherto undetected carcinoma.

Clostridium difficile toxins A and B isolated from strain VPI 10463 were tested for induction of cytokine release by human monocytes. Toxin B at 10(-12) M activated human monocytes as measured by release of interleukin-1 (IL-1), tumor necrosis factor (TNF), or IL-6. These effects of toxin B were heat labile (51 degrees C, 30 min). Toxin B was as effective as bacterial lipopolysaccharides in inducing IL-1 beta but less effective in inducing TNF or IL-6. Toxin B and lipopolysaccharides were synergistic in induction of IL-1 beta, TNF, and IL-6. The toxin A preparation used was 1,000-fold less active than toxin B. Apart from the difference in activity, the two toxins showed identical patterns of reaction and there was no synergism between them. A short pulse with toxin B was sufficient to trigger IL-1 release. Toxin B was also extremely toxic for monocytes. The toxicity and the induced proinflammatory monokines (IL-1 and TNF) may contribute to the pathogenic mechanisms of C. difficile infection and pseudomembranous colitis.

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

ABSTRACTLegionella pneumophila, a gram-negative bacterium causing Legionnaires’ disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limuluslysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophilalipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in theLimulusamoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella entericaserovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

Interaction of endotoxin (lipopolysaccharide [LPS]) with human lipoproteins is known to prevent the LPS-induced activation of human monocytes and release of cytokines (monokines). LPS was exposed to lipoprotein classes separated by ultracentrifugation and to apolipoprotein A-I. Then monocytes were added, and the LPS activation of monocytes was determined by measuring the induced monokines. Failure of LPS to induce monokine release was called LPS inactivation caused by lipoproteins or apolipoproteins. The LPS inactivation is shown to be a function of low-density lipoproteins. High-density lipoproteins inactivate LPS to a much lesser extent. The very-low-density lipoproteins cannot inactivate LPS. Lipid components seemed not absolutely required for LPS inactivation, because purified human apolipoprotein A-I without its physiological lipid complement also inhibits LPS-induced monokine release.

Toxicity of lipopolysaccharide (LPS) (endotoxin) is, to a large extent, mediated by the activation of monocytes/macrophages and subsequent release of monokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). It is known that LPS binds readily to serum lipoproteins and that LPS-lipoprotein complexes are less toxic than unbound LPS. Here we present data analyzing the impact of the LPS-serum interaction at the cellular level. By measuring IL-1 TNF-alpha, and IL-6, the interaction of different LPSs or lipid A with human serum could be shown to prevent the activation of human monocytes. The amounts of LPS inactivated by normal human serum did not exceed 10 ng/ml. The LPS-inactivating capacity of serum was shown to be a function of the lipoproteins. Other serum components, such as naturally occurring anti-LPS immunoglobulin G, complement, or nutritive lipids, had no significant influence in our system. Our experiments suggest that serum lipoproteins control endotoxin-induced monocyte activation and monokine release.

Zusammenfassung: Fragestellung: Gemäß den Richtlinien zur Hämotherapie kann präoperativ gespendetes Eigenblut auch als Vollblut gelagert und transfundiert werden. Im Vergleich zu den Komponenten kommt es bei Vollblut jedoch zu einer stärkeren qualitativen Minderung während der Lagerung. Dies wird hauptsächlich auf die Kontamination mit Leukozyten und Thrombozyten zurückgeführt. In der vorliegenden Studie wurde deshalb untersucht, welchen Einfluß die Filtration von Vollblut vor Lagerung auf die Qualität der Konserve hat. Methodik: Vollblut von gesunden Dauerspendern (n=14, 500 ml, 70 ml CPDA-1) wurde über einen setintegrierten Vollblutfilter (Leukotrap A1, Fa. Pall, Dreieich) gefiltert und über die Lagergrenze von 35 Tagen hinaus bis 49 Tage gelagert. Die Qualität der Vollblutkonserven wurde anhand hämatologischer und biochemischer Parameter zu verschiedenen Zeitpunkten der Lagerung bestimmt. Ergebnisse: Unsere Ergebnisse zeigen, daß die Qualität von gefiltertem Vollblut der von Buffy-coat-freien Erythrozytenkonzentraten vergleichbar ist. Schlußfolgerung: Krankenhäuser ohne Möglichkeit zur Komponentenherstellung steht mit dem Leukotrap A1-Vollblutfiltersystem in der Eigenbluttherapie eine interessante Option zur Verfügung, deren klinische Eignung im Rahmen von Studien überprüft werden sollte.

We report the results of a study on red cell antibody screening in 2329 sera. We compared the Solidscreen II solid-phase test with the Solidscreen II enzyme tests (papain and bromelin) and the gel centrifugation test (DiaMed/bromelin).The sensitivity of the Solidscreen II papain test is higher than that of all other methods. Seven out of 9 antibodies in the Kidd system could not be detected in any other test, an important fact for optimal patient support.

The lipopolysaccharide(LPS)-induced release of cytokines from human monocytes can be prevented by previous interaction of serum with LPS. This inactivation is a function of lipoproteins. Here we show that LPS can be inactivated by low-density lipoproteins (LDL) as well as by high-density lipoproteins (HDL). The effects of heparin and EDTA on LPS inactivation by serum are also described.

Background: Despite availability of potent anti-bacterial, anti-mycotic drugs and hematopoietic growth factors invasive bacterial or fungal infections remain a severe threat to neutropenic patients after chemotherapy or hematopoietic stem cell transplantation (HSCT). Granulocyte transfusions (GT) from granulocyte colony stimulating factor (G-CSF)-stimulated donors have been shown to increase the leukocyte count prior to expected hematopoietic regeneration, resulting in a shorter period of neutropenia and thus offer a therapeutic option for the control of severe infections. However, published studies rely on clinical observations of individual cases as no statistical comparison with control groups could have been established. The aim of this study was to define the clinical benefit and the leukocyte increment/duration of neutropenia after randomized administration of leukocyte transfusions in immunocompromized neutropenic patients. Patients and methods: Between 1999 and 2005 80 patients with underlying hematological diseases with or without allogeneic or autologous HSCT were randomized to receive either G-CSF, anti-infective treatment according to local standards with or without therapeutic or prophylactic application of GT from G-CSF-stimulated volunteer donors. The mean age was 47 years (range, 14 – 62 y). Indications were either fever in neutropenia and pulmonary infiltrates or soft tissue infiltration (therapeutic) or the history of invasive fungal infection during episodes of neutropenia following earlier chemotherapy courses and anticipated neutropenia of > 10 days (prophylactic). Results: 10 centers participated in the trial, however only five centers recruited patients (n=80) for randomization during the study period. This corresponds to ~50% of the expected sample size of 160 patients, hence results are statistically insignificant. Patient characteristics were comparable within the randomized cohorts (underlying disease, stage of disease, indication for GT, lenght of neutropenia). No significant difference in the clinical outcome was found between patients who received either therapeutic or prophylactic GT from G-CSF stimulated donors or no GT. The probability of survival on day 28 was 85% in both groups. Furthermore, no difference in the incidence and causes of death could be identified within the compared cohorts. Conclusion: The high percentage of infection clearance and survival in patients with severe infections in both groups contrasts with published results and own experiences. We speculate whether this is due a bias in including predominantly patients into the randomized study who presented with relatively favourable prognostic factors, as in the observation period numerous GT were performed in the participating centers without randomization. Most likely, existing clinical evidence for the benefit of this therapeutic measure was sufficient in many cases to exclude patients in serious conditions from randomization. Although well designed, a randomized trial may not always provide the expected results.

Background: Despite availability of potent anti-bacterial, anti-mycotic drugs and hematopoietic growth factors invasive bacterial or fungal infections remain a severe threat to neutropenic patients after chemotherapy or hematopoietic stem cell transplantation (HSCT). Granulocyte transfusions (GT) from granulocyte colony stimulating factor (G-CSF)-stimulated donors have been shown to increase the leukocyte count prior to expected hematopoietic regeneration, resulting in a shorter period of neutropenia and thus offer a therapeutic option for the control of severe infections. However, published studies rely on clinical observations of individual cases as no statistical comparison with control groups could have been established. The aim of this study was to define the clinical benefit and the leukocyte increment/duration of neutropenia after randomized administration of leukocyte transfusions in immunocompromized neutropenic patients.

In this report, we investigate the intracellular fate of selected members of the genus Legionella within the monocytic cell line Mono Mac 6 cells. By means of electron microscopy and immunocytochemistry, we could show that Legionella pneumophila as well as Legionella longbeachae are able to induce ribosome-studded phagosomes which associate with the rough endoplasmic reticulum (RER), whereas Legionella micdadei remains to be located within smooth phagosomes but also shows signs of RER association. In addition, we could demonstrate a remarkable correlation between the phagosome type and the morphological phenotype of intracellular bacteria: within ribosome-studded phagosomes, bacteria generally lacked the outer coat of low electron density whereas bacteria within the smooth phagosomes still possessed this outer coat. The virulence factors responsible for inhibition of phagosome maturation and their distribution within the genus Legionella as well as the biological significance of the morphological difference of bacteria within smooth and ER-associated phagosomes remain to be investigated.

Endotoxin-induced tumor necrosis has been shown to be mediated by a factor termed tumor necrosis factor (TNF). The biochemical nature, source, and mode of induction of TNF have been clarified. TNF is a mediator of activated macrophages that is released into the supernatant by these cells after their stimulation with endotoxin. A number of biologic functions of TNF other than its tumoricidal activity have been demonstrated. In vivo, TNF induces reactions similar to those induced by endotoxin. Hypothermia, elevated hematocrit and plasma lactate levels, and reduced plasma glucose levels have been measured in mice injected with TNF. These reactions typically occur soon after endotoxin injection and are induced with purified recombinant TNF in mice that exhibit a low response to endotoxin. No TNF-induced production of interleukin 1 (which can induce similar effects) was detected in macrophage/monocyte cultures. Therefore, TNF appears to mediate endotoxin effects directly.