Markus Schneemann

Macrophage biology and immunology: man is not a mouse.

A recent article by Shibata et al. [1] and an interview with the senior investigator Kobayashi [2], both in the October 2006 issue of Journal of Leukocyte Biology, discuss the role of macrophage-derived NO in the clearance of apoptotic cells. We were surprised that neither in the original paper nor in the accompanying interview was it discussed that these results rely entirely on data obtained in murine macrophages. It is, however, evident that there are differences in macrophage NO production and regulation among different species [3, 4]. Several years ago, we and others [5–7] described fundamental differences between macrophages from mice and humans regarding NO synthase (NOS) activity. Murine macrophages produce large amounts of NO and L-citrulline from L-arginine via induction of the inducible form of NOS (iNOS). In parallel, murine macrophages synthesize the obligatory cofactor tetrahydrobiopterin (BH4), essential for stabilization and function of the iNOS enzyme protein [8, 9]. Human as well as macrophages from other animal species, such as rabbits, goats, or Syrian hamsters, do not have NOS activity nor do they synthesize BH4 [7, 10, 11]. In addition, murine macrophages consume most of the L-arginine by another enzyme—arginase—and convert it into L-ornithine. This arginase is not active in human macrophages too [7, 9, 12]. A lot of research has since been done to demonstrate nitrite production in human macrophages. Recent reviews claim that human macrophages have iNOS activity, albeit induced by other stimuli rather than those inducing nitrite production in murine macrophages [13]. We evaluated several of these stimuli and could show that nitrite was not produced by iNOS activity in human macrophages. The nitrite measured came from other noncellular sources [14]. None of the articles cited as proof for iNOS activity in human macrophages provides sound biochemical data about L-arginine consumption, BH4 synthesis, and L-citrulline production [13]. Moreover, they do not control for nitrite generation from other nonmacrophage sources [14]. The only report that demonstrates nitrite production from NOS activity in human macrophage-like cells is an article by Bertholet et al. [15]. There, the U937 monoblastic leukemia cell line was transfected with a plasmid containing a functional iNOS gene. Nitrite production by these cells, however, totally depended on substitution of the cofactor BH4 and additional substrate [15]. As species differences are not confined to NOS activity and are fundamental in many aspects of immunology [3, 16, 17], we propose that the species involved should be mentioned in the title and abstract of articles dealing with common aspects of immunity.

Rapid Detection of Pathogenic Fungi from Clinical Specimens Using LightCycler Real-Time Fluorescence PCR

In the study presented here a LightCycler real-time PCR system was used for the diagnosis of fungal infections from clinical tissue samples. Nine specimens were investigated from six patients with suspected or proven invasive fungal infections. Seven of nine samples were positive in a broad-range fungal PCR assay. In four samples, Aspergillus fumigatus was detected both by a species-specific hybridization assay as well as by sequencing of amplification products. In addition, the broad-range fungal PCR assay and PCR sequencing detected and identified, respectively, the following organisms in the specimens noted: Candida albicans in a culture-negative liver biopsy, Histoplasma capsulatum in a bone marrow sample, and Conidiobolus coronatus in a facial soft tissue specimen. Real-time PCR is a promising tool for the diagnosis of invasive fungal infections in human tissue samples and offers some advantages over culture methods, such as rapid analysis and increased sensitivity.

Immunovascular communication : activation and deactivation of murine endothelial cell nitric oxide synthase by cytokines

A murine endothelial cell line, send1, was found to produce substantial amounts of nitric oxide, particularly after activation with cytokines. The endothelial cell activation paralleled that of macrophages. Macrophage deactivation opposing activation has recently been brought into focus. We therefore studied the cytokine-mediated deactivation of endothelial cells in send1 and vascular strips. Our observations document that activation of nitric oxide synthase of endothelial cells can be counterbalanced by deactivating cytokines such as interleukin-4, interleukin-8, interleukin-10 and transforming growth factor-beta. Deactivation of nitric oxide synthase in endothelial cells might be an essential mechanism for the control of immune-mediated vasodilatation or septic shock and represents a novel mechanism of communication between the immune and the vascular systems.

Constitutive and regulated expression of platelet basic protein in human monocytes

Platelet basic protein (PBP) and several of its derivatives are known for their broad range of functions as signaling molecules and cationic antimicrobial peptides and were considered hitherto megakaryocyte‐ and platelet‐specific. In search of glucocorticoid‐regulated antimicrobial systems of monocytes, we found a 15‐fold down‐regulation of PBP mRNA by differential display. Regulation was confirmed in vivo even at low prednisone doses. Quantitative mRNA analyses confirmed down‐regulation also for platelets. Western blotting and immunostains showed down‐regulation at the protein level. Pro‐PBP derivatives were in the size range of 7.5‐14 kD and in immunostains, gave granular cytoplasmatic patterns. Interleukin (IL)‐4 and IL‐10 induced a similar down‐regulation. Phagocytosis resulted in an increase of smaller derivatives in the range of 7.5 kD. Stimulation with interferon‐γ and lipopolysaccharide did decrease expression of PBP and affected derivatization. Expression of PBP and its derivatives is not restricted to the megakaryocytic cell lineage. PBP and some of its derivatives might contribute to the antimicrobial armamentarium of mononuclear phagocytes or have monokine functions. Our studies define PBPs as one among the many immunosuppressive targets of glucocorticoids.

Listeria species escape from the phagosomes of interleukin-4-deactivated human macrophages independent of listeriolysin

Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts. Human macrophages are able to phagocytose and digest L. monocytogenes but IL‐4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown. In the present study, we examined various listeria species and strains including wild‐type and deletion mutants in human macrophages pretreated with IL‐4. To analyse the IL‐4‐mediated deactivation process, we combined quantitative infection assays with various morphologic methods. IL‐4 facilitates survival and escape of the pathogenic L. monocytogenes wild‐type strain 10403S from the macrophage phagosomes. In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP‐L2161 was killed and did not escape from the phagolysosomes. However, after macrophage deactivation with IL‐4 DP‐L2161 survived and escaped from the phagosomes. This was also the case, but to a lesser extent, even for the naturally avirulent L. innocua. As detected by confocal laser‐scanning fluorescence microscopy and electron microscopy, IL‐4 permitted the escape of all listeria species tested, including DP‐L2161 and L. innocua from the phagosomal compartment of the macrophages. We conclude that escape from the phagosome and survival of the listeria species tested in IL‐4‐deactivated human macrophages is independent of the virulence factor listeriolysin.

Developing strategies for predicting hyperkalemia in potassium-increasing drug-drug interactions

Objective: To compare different strategies predicting hyperkalemia (serum potassium level ≥5.5 mEq/l) in hospitalized patients for whom medications triggering potassium-increasing drug-drug interactions (DDIs) were ordered. Materials and Methods: We investigated 5 strategies that combined prediction triggered at onset of DDI versus continuous monitoring and taking into account an increasing number of patient parameters. The considered patient parameters were identified using generalized additive models, and the thresholds of the prediction strategies were calculated by applying Youden’s J statistic to receiver operation characteristic curves. Half of the data served as the calibration set, half as the validation set. Results: We identified 132 incidences of hyperkalemia induced by 8413 potentially severe potassium-increasing DDIs among 76 467 patients. The positive predictive value (PPV) of those strategies predicting hyperkalemia at the onset of DDI ranged from 1.79% (undifferentiated anticipation of hyperkalemia due to the DDI) to 3.02% (additionally considering the baseline serum potassium) and 3.10% (including further patient parameters). Continuous monitoring significantly increased the PPV to 8.25% (considering the current serum potassium) and 9.34% (additional patient parameters). Conclusion: Continuous monitoring of the risk for hyperkalemia based on current potassium level shows a better predictive power than predictions triggered at the onset of DDI. This contrasts with efforts to improve DDI alerts by taking into account more patient parameters at the time of ordering.

Whipple's DNA Is Not Whipple's Disease

Sloan et al. report a real-time PCR method using the hsp65 gene of Tropheryma whipplei for a target sequence as a new and rapid diagnostic test for Whipples disease ([7][1]). We and others have already published real-time PCR-based methods for the detection of T. whipplei DNA using 16 rRNA or other

CME: Morbus Addison

Zusammenfassung. Beim Morbus Addison handelt es sich um eine primare Nebennierenrindeninsuffizienz mit Glukokortikoid- und Mineralokortikoidmangel. Die haufigste Ursache der primaren Nebennierenrindeninsuffizienz (NNRI) in westlichen Landern ist die Autoimmunadrenalitis (80 %). Weitere Ursachen sind Karzinommetastasen, besonders von Bronchialkarzinomen, malignen Melanomen und Nierenzellkarzinomen. In Entwicklungslandern gilt die Nierentuberkulose noch immer als die haufigste Ursache der primaren NNRI. Die Klinik besteht aus unspezifischen Allgemeinsymptomen wie Mudigkeit, Myalgien, Ubelkeit und Gewichtsverlust. Bei fehlender adaquater Substitution der Nebennierenhormone besteht die Gefahr einer Addison-Krise. Ausloser der Addison-Krise sind «Stresssituationen» mit erhohtem Cortisol-Bedarf (z.B. Infekte oder starke korperliche Belastungen). Die Therapie der Addison-Krise besteht in der Gabe von hochdosiertem Hydrocortison. Als Dauertherapie werden Hydrocortison und Fludrocortison verabreicht.

CME: Syndrom der inadäquaten ADH-Sekretion (SIADH).

Zusammenfassung. Als SIADH (Syndrom der inadaquaten ADH-Sekretion, Schwarz-Bartter-Syndrom) wird eine Elektrolytstorung aus dem Formenkreis der Hyponatriamie bezeichnet. Durch komplexe Mechanismen und verschiedene Atiologien wird das im Hypothalamus gebildete, und im Hypophysenhinterlappen freigesetzte ADH (antidiuretisches Hormon) in inadaquat hohem Ausmass in die systemische Zirkulation freigesetzt. Nach Erreichen der renalen Sammelrohre fuhrt das ADH zu einer der homoostatischen Situation unangemessenen Wasserretention, was konsekutiv eine hypoosmolare Hyponatriamie bewirkt. Mogliche Ausloser eines SIADH sind Medikamente, Neoplasien, Pneumopathien oder Pathologien des zentralen Nervensystems. Daneben konnen Operationen, Stress, Traumata, Schmerzen oder eine idiopathische Form zu diesem Syndrom fuhren. Die klinischen Symptome der Hyponatriamie sind mannigfaltig und haufig unspezifisch. Viele milde Formen verlaufen asymptomatisch. Bei schwererer Auspragung der Elektrolytstorung treten Appetitlosigkeit, N...

Too frequent low-dose methotrexate prescriptions: multicentre quality control and quality assurance with pre- and post-analysis.

INTRODUCTION Methotrexate is used to treat many medical conditions with medication schedules that differ widely in dosage and frequency. The high potential of erroneous too frequent low-dose methotrexate prescriptions leading to severe adverse reactions is well known; however, documentation is mainly limited to case reports. We reviewed all methotrexate prescriptions in a secondary and a tertiary care hospital to analyse the incidence of too frequent low-dose methotrexate prescriptions, and assessed the quality assurance concepts implemented. METHODS All nononcological low-dose methotrexate prescriptions issued for inpatients within 55 months were analysed to identify too frequent prescriptions potentially leading to harmful overdosing. Subsequently, clinical pharmacologists reviewed all new methotrexate prescriptions with resulting interventions at the physician level in the tertiary care hospital. The impact of an interruptive alert displayed at methotrexate order entry was assessed in the secondary care hospital. RESULTS The incidence of too frequent prescriptions at the tertiary hospital was 1.6% (five medication errors and nine near misses in 888 inpatients). After introducing checks by pharmacologists, two prescription errors were intercepted during the 8 month quality assurance period. At the secondary care hospital the incidence dropped from 2.5% (2/79, 20 months) to 0.8% (1/123, 35 months) after the alert was implemented. CONCLUSIONS The incidences of erroneous too frequent low-dose methotrexate prescriptions observed at both hospitals were considered too high due to the high potential for increased morbidity, mortality and costs. Therefore, quality assurance measures were implemented and the preliminary data show a positive impact on patient safety for both approaches.